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Instrumentation and development of a massspectrometry system for the study of gas-phasebiomolecular ion reactionsZiqing LinPurdue University

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Recommended CitationLin, Ziqing, "Instrumentation and development of a mass spectrometry system for the study of gas-phase biomolecular ion reactions"(2015). Open Access Dissertations. 503.https://docs.lib.purdue.edu/open_access_dissertations/503

INSTRUMENTATION AND DEVELOPMENT OF A MASS SPECTROMETRY

SYSTEM FOR THE STUDY OF GAS-PHASE BIOMOLECULAR ION REACTIONS

A Dissertation

Submitted to the Faculty

of

Purdue University

by

Ziqing Lin

In Partial Fulfillment of the

Requirements for the Degree

of

Doctor of Philosophy

May 2015

Purdue University

West Lafayette, Indiana

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ACKNOWLEDGEMENTS

I am deeply thankful to my PhD advisor, Prof. Zheng Ouyang. He is more than a

mentor or supervisor, but a kind friend, giving me a fantastic PhD experience at Purdue.

His passion, courage and extraordinary vision in scientific research makes him an

outstanding scientist and engineer. Prof. Ouyang works hard day and night, while on the

other side providing a free and comfortable environment for me and other colleagues in

the group to do research and raise opinions without pressure. These precious

characteristics no doubt affect me in my professional life. When we first met in Tsinghua,

he told me that PhD life is a best opportunity to test our boundary of capabilities. I learnt

a lot during my PhD study, not only in terms of technical knowledge but the

determination and belief in solving a problem. Five years is not a short period, I truly

appreciate his supervision and encouragement for me to explore the scientific world and

myself. It is my honor to know Zheng as a person and have the opportunity to work with

each other for both research and teaching experiences. I sincerely wish him good luck for

his future endeavors.

I would also like to extend my thanks to Prof. Yu Xia in Department of Chemistry. I

received a lot of valuable suggestions and comments from her for gas-phase ion

chemistry. Prof. Xia is very sophisticated and precise about her scientific findings. Her

dedication and persistence shows me how one could possibly be devoted to her career.

Moreover, Dr. Xia has a charismatic personality and always welcomes discussions at any

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time. She also allowed me to use the nanospray tip puller and the commercial mass

spectrometer in her laboratory, which was a great favor during my PhD study.

It is my privilege to have Prof. Kinam Park and Prof. Corey Neu in my thesis

committee, who are always trying to do everything to help. Prof. R. Graham Cooks,

although not in my committee, has offered tremendous guidance and support in different

perspectives of my graduate studies. I would also like to thank Prof. Mingji Dai, Dr.

Yang Yang from Purdue University, Prof. František Tureček from University of

Washington, Prof. Barney Ellison, Prof. Veronica M. Bierbaum from University of

Colorado, Prof. Michael L. Gross from Washington University, and Dr. Amber L. Russell

from Badger Technical Services for their kind help in organic synthesis, theoretical

calculation, and tips for the pyrolysis nozzle.

I am indebted to all the group members and alumni in Prof. Ouyang, Xia and Cooks’

research group. It has been a wonderful experience to have such a close relationship with

so many people. Dr. Tsung-Chi Chen, Dr. Wei Xu, Chien-Hsun Chen and Linfan Li are

the colleagues who helped me to build my instrument. Jason Duncan was a technician in

Cooks’ group, always delivering solid supports when I asked for different electronic

controls to achieve varies functions. Lei Tan is a close collaborator in Xia’s group with

whom I worked together on gas-phase ion chemistry. Besides, it is always inspiring and

pleasant to discuss with group members such as Dr. He Wang, Dr. Qian Yang, Dr.

Sandilya Garimella, Dr. Xiaoyu Zhou, Xiao Wang, Yue Ren, Melodie Du, Yuan Su, etc.

Without all your help, I would not have been able to finish my PhD program.

I would also like to take the time to acknowledge my parents who have been

supporting me mentally and financially throughout my student life in China and my PhD

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abroad. They have been extremely patient and understanding and I would like to thank

them for all the amazing opportunities they have given me over the years. Last but not

least, I owe my deepest gratitude to my lovely girlfriend and dance partner Esther Foo,

who shares with me tears and laughter, sadness and joy in my life and for being

supportive throughout this journey. Having blessed with a strong memory where I could

recall minute details in my everyday life, I’m glad that I would have the chance to

remember all the wonderful moments in my PhD life, and for this, I am eternally grateful.

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TABLE OF CONTENTS

Page

LIST OF TABLES ............................................................................................................ vii

LIST OF FIGURES ......................................................................................................... viii

ABBREVIATIONS .......................................................................................................... xv

ABSTRACT…. .............................................................................................................. xviii

CHAPTER 1. INTRODUCTION ..................................................................................... 1

1.1 Gas-phase biomolecular ion reactions and mass spectrometry

instrumentations .................................................................................................................. 1

1.1.1 Tandem mass spectrometry in gas-phase ion reactions .......................5

1.1.2 Gas-phase biomolecular ion/radical reactions at atmospheric pressure

7

1.1.3 Instrumentations for gas-phase ion reactions in vacuum ...................13

1.2 Conclusion ...............................................................................................38

1.3 References ................................................................................................40

CHAPTER 2. INSTRUMENTATION AND CHARACTERIZATION OF A HOME-

BUILT DAPI-RIT-DAPI MASS SPECTROMETER FOR GAS-PHASE

ION/MOLECULE AND ION/ION REACTIONS ........................................................... 50

2.1 Introduction ..............................................................................................50

2.2 Instrumentation ........................................................................................52

2.3 Materials ...................................................................................................54

2.4 Results and discussion .............................................................................54

2.4.1 System configuration .........................................................................54

2.4.2 Pressure effect on gas-phase ion reactions ........................................59

2.4.3 Dynamic gas flow effect on gas-phase ion reactions .........................62

2.5 Conclusion ...............................................................................................69

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Page

2.6 References ................................................................................................70

CHAPTER 3. Gas-Phase Reactions of C3H2 with Protonated Alkyl Amines: formation

of a c-n covalent bond ....................................................................................................... 73

3.1 Introduction ..............................................................................................73

3.2 Instrumentation ........................................................................................74

3.3 Materials ...................................................................................................78

3.4 Results and discussion .............................................................................79

3.4.1 Carbene reaction with protonated alkylamines ..................................79

3.4.2 Theoretical calculation of the reaction mechanism ...........................83

3.4.3 Experimental evidences of the reaction mechanism ..........................86

3.5 Conclusion ...............................................................................................91

3.6 Appendix ..................................................................................................92

3.7 References ..............................................................................................106

CHAPTER 4. Gas-Phase c-C3H2 carbene reactions with biomolecular ions ............... 109

4.1 Introduction ............................................................................................109

4.2 Experimental section ..............................................................................110

4.3 Results and discussion ...........................................................................110

4.3.1 Nucleobases and nucleosides ...........................................................110

4.3.2 Amino acids, peptides, proteins, and lipids .....................................117

4.4 Conclusion .............................................................................................131

4.5 References ..............................................................................................132

VITA…………. .............................................................................................................. 134

PUBLICATIONS ............................................................................................................ 136

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LIST OF TABLES

Table ..................................................................................................................... Page

Table 1.1 Tandem mass spectrometry dissociation techniques in proteomics ................... 3

Table 3.1 Ammonium reactions with various radical precursors ..................................... 92

Table 3.2 MS3-MS5 CID spectra of selected reaction products ........................................ 97

Table 3.3 GDEI spectra of radical precursors ................................................................. 101

Table 3.4 Ion/molecule reactions of c-C3H3+ and neutral amines .................................. 104

Table 4.1 Name, structure, and gas-phase basicity3 (GB) of nucleobases and nucleosides4

...................................................................................................................... 111

Table 4.2 Ion/carbene reactions of nucleobases, nucleosides and c-C3H2 ...................... 115

Table 4.3 Ion/carbene reactions of amino acids, peptides, protein and c-C3H2 .............. 126

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LIST OF FIGURES

Figure ..................................................................................................................... Page

Figure 1.1 Proton-transfer reaction in the gas-phase vs. the aqueous phase ....................... 2

Figure 1.2 Tandem mass spectrometry in space vs. in time ............................................... 5

Figure 1.3 Radical reactions in the nanoESI plume and bulk solution using low

temperature plasma (LTP)35 or ultraviolet (UV) lamp36. ................................. 7

Figure 1.4 Reactions of protonated disulfide peptide and oxygen-centered radicals5 to

form thiyl (-S•), sulfinyl (-SO•), and perthiyl (-SS•) radical ions in nanoESI

plume: (a) peptide chain CLPTR of HMAC-CLPTR, and (b) chain AGCK of

AGC(TFTSC)K.42 ............................................................................................ 9

Figure 1.5 Intramolecular cysteine sulfinyl radical (SO•Cys) transfer with (a) a free

thiol43 and (b) a disulfide bond44 .................................................................... 10

Figure 1.6 Coupling radical reactions in the ESI plume to cleave the disulfide bonds, with

a subsequent MS2 scan to improve sequence information of disulfide

peptides5, 35 ..................................................................................................... 11

Figure 1.7 Scheme of Paternò–Büchi (PB) reaction between ketone/aldehyde and olefin

together with possible retro P-B reactions.52 ................................................. 12

Figure 1.8 On-line coupling of P-B reactions of oleic acid and acetone with negative MS.

MS1 spectra of oleic acid (a) before P-B reaction (b) after P-B reaction; MS2

CID of the P-B reaction products at (c) m/z 339.3; and (d) structures of the

diagnostics ions (structures 3 and 5). The structures explain the origin of the

26-Da mass difference.52 ................................................................................ 13

Figure 1.9 Schematic of mass-analyzed ion-kinetic-energy spectrometry (MIKES) with

collision gas for chemical mixture analysis6a ................................................ 15

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Figure ..................................................................................................................... Page

Figure 1.10 Schematic of tandem time of flight (TOF-TOF) mass spectrometer56 .......... 16

Figure 1.11 Schematic of penta-quadrupole (QqQqQ) mass spectrometer59 ................... 17

Figure 1.12 Symbols and names given to QqQqQ MS experiments with 0, 1, 2, and 3

dimensions. A fixed mass (filled circle) or variable masses (open circle) can

be chosen for reactions.61 ............................................................................... 18

Figure 1.13 (a) Penta-quadrupole three-dimensional triple-stage (MS3) familial spectrum

for 3-octanone ions acquired by selecting ions of m/z 43 as the intermediates.

The angled Q1 axis displays the precursors of m/z 43 ions, whereas the

horizontal Q5 axis displays the ion/molecule products of these CID-generated

m/z 43 ions arising from reactions with isoprene. Triple-stage sequential

precursor spectra extracted from the 3D familial spectrum by selection in Q5

of the final products of (b) m/z 81 and (c) m/z 111.63 .................................... 19

Figure 1.14 Schematic of flowing-afterglow selected-ion flow-tube (FA-SIFT) coupled

with a radical source for ion/radical reactions69 ............................................ 21

Figure 1.15 Schematic of a basic ICR cell, consisting of two trapping plates (front and

back), two opposite excitation plates (right and left), and two detection plates

(top and bottom). Ions enter the cell through the front trapping plate. Mass

spectrum generated by fast Fourier transformation.74 .................................... 23

Figure 1.16 Schematic of the FT-ICR instrument, including the LIAD probe.78 ............. 24

Figure 1.17 Aminoketyl radicals and c and z• ions by ECD2, 84 ........................................ 24

Figure 1.18 Mass spectrum of ubiquitin ([M+9H]9+) by ECD FTICR with 9.4 T

instrument.89 ................................................................................................... 25

Figure 1.19 Schematic of a single neutral injection line (a)42 and a multiported pulsed

valve interface (b)90 of a linear quadrupole ion trap for ion/molecule reactions.

........................................................................................................................ 27

Figure 1.20 Schematic of the differentially pumped dual LQIT with ion optics and

differential pumping. LQIT1 and LQIT2 are the first and second linear

quadrupole ion traps (LQIT).91 ...................................................................... 28

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Figure ..................................................................................................................... Page

Figure 1.21 Schematic of a multi-source quadrupole ion trap instrument containing two

electrospray ionization (ESI) sources and one atmospheric sampling glow

discharge ionization (ASGDI) source interfaced.98b ...................................... 30

Figure 1.22 Schematic of a triple quadrupole/linear ion trap modified by addition of an

atmospheric sampling glow discharge ionization source on the side of Q3

linear ion trap for ion/ion reactions.100 ........................................................... 31

Figure 1.23 Schematic of (a) a 3D ion trap coupled with a fast atom bombardment (FAB)

gun for metastable atom-activation dissociation19 and (b) a laser coupled

linear ion trap for infrared multiple photon dissociation (IRMPD)102b and

ultraviolet photon dissociation (UVPD)103. ................................................... 32

Figure 1.24 Schematic of a modified quadrupole time-of-flight tandem mass spectrometer

for ion/ion reactions with dual ion source.104 ................................................. 34

Figure 1.25 Schematic of an Orbitrap Elite.107a ................................................................ 35

Figure 1.26 Schematic of (a) conventional linear ion trap mass spectrometer with

differential pumping system and ion optics and (b) miniature DAPI-RIT mass

spectrometers highlighted Mini 11 mass spectrometer of 9 pounds and Mini

12 of Point-of-care personal mass spectrometer.110, 113 .................................. 36

Figure 1.27 (a) Scan function for mass analysis using a DAPI mass spectrometer. (b)

Manifold pressure measured during scanning, with an open time of 20 ms and

a closed time of 850 ms for DAPI.110 ............................................................ 37

Figure 1.28 Schematic of a DAPI-RIT-DAPI mass spectrometer coupled with a pyrolysis

radical source for the study of gas-phase ion/radical reactions. .................... 39

Figure 2.1 (a) Schematic representation of the DAPI-RIT-DAPI system; (b) General

waveforms of the in-trap gas-phase reactions ................................................ 55

Figure 2.2 Ion/molecule reactions: thiyl radical cations react with allyl iodide ............... 56

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Figure ..................................................................................................................... Page

Figure 2.3 (a) Ion/ion reactions: Angiotensin I triply charged cations react with

deprotonated pentachlorophenol anions (insert: Isolation of the triply charged

species); (b) MS spectrum of Angiotensin I without opening DAPI-2; (c) MS

spectrum of isolated triply charged peptide ions with DAPI-2 opening but the

nanoESI source being turned off for anion production; and (d) aveforms of

the ion/ion reaction (dc was set to -10 V during mass analysis). ................... 58

Figure 2.4 CID spectra and MS/MS efficiency of protonated cocaine m/z 304 at different

pressures. Activation time 30 ms, amplitude 200 mV. .................................. 60

Figure 2.5 Ion-trap CID spectra of deprotonated 9-anthracenecarboxylic acid at (a) 6

mTorr 200 ms delay, (b) 2 mTorr 500 ms delay, (c) 1 mTorr 780 ms delay,

from the DAPI opening on DAPI-RIT-DAPI system. The yellow, red, and

blue columns illustrate the positions of ion introduction, dipolar ac excitation,

and MS scan, respectively; (d) Ion-trap CID spectrum of m/z 195 on DAPI-

RIT-DAPI system; (e) Ion-trap CID spectrum of deprotonated 9-

anthracenecarboxylic acid on QTRAP 4000; (f) Ion/molecule reactions of

deprotonated anthracene ions with water vapor on QTRAP 4000. The CID

activation time was set to 30 ms on DAPI-RIT-DAPI system. ..................... 62

Figure 2.6 Energy characterization of DAPI. (a) Calculated survival ion yields and (b)

Internal energy distributions of DAPI vs. continuous API for ion introduction;

Fragmentation spectra of thermometer ions after opening DAPI-2 for 20 ms

with capillary 3 (c) i.d. 0.5 mm and (d) i.d. 0.75 mm. *p-R represents the

fragment of p-R (R = OCH3, CH3, Cl, CN, NO2). ......................................... 64

Figure 2.7 Ion/molecule reactions of peptide thiyl radical ions and dimethyl disulfide. (a)

MS spectrum of S-nitrosoglutathione; (b) Thiyl radical (m/z 307) produced by

CID of protonated S-nitrosoglutathione; (c) Reactions between thiyl radicals

and dimethyl disulfide (Capillary 3 i.d. 0.125 mm; DAPI-2 opening time 25

ms; q=0.35 for thiyl radical ions); (d) Ion/molecule reactions between isolated

m/z 307 from panel (c) with dimethyl disulfide. ............................................ 66

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Figure ..................................................................................................................... Page

Figure 2.8 Thiyl radical reaction for different capillary inner diameters and opening times.

........................................................................................................................ 67

Figure 2.9 MS spectra of thiyl radical reaction for activation with ac amplitude (a) 0.06 V;

(b) 0.066 V; (c) 0.07 V; and (d) CID spectrum of thiyl radical ions. ............ 68

Figure 3.1 (a) Schematic of the home-built platform for the study of reactive radical

species and (b) the general MS scan function for ion/radical reactions (y-axis:

voltage). DAPI: discontinuous atmospheric pres-sure interface; RIT:

rectilinear ion trap; GDEI: glow discharge electron impact; rf: radio

frequency; ac: alternative current. .................................................................. 75

Figure 3.2 Carbene reaction spectra: (a) MS2 reaction of protonated n-heptylamine with

heated 1,3-dibromopropyne; (b) MS3 CID of the reaction product; (c) MS2

reaction of protonated n-heptylamine with heated d12-pentane; and (d) MS2

reaction of protonated n-heptylamine 18C6 complex with heated 1,3-

dibromopropyne. ............................................................................................ 79

Figure 3.3 MS2 reaction spectra of heptyl ammonium and heated (a) allylchloride, (b)

propargylbromide, (c) 1,5-hexadiene, and (d) pentane. ................................. 80

Figure 3.4 MS2 reaction spectra of protonated (a) dibutylamine, (b) tributylamine, and (c)

cetyltrimethyl ammonium with heated propargylbromide, and MS3 CID

spectra of the reaction products for (d) dibutylamine and (e) tributylamine. 81

Figure 3.5 MS2 reaction spectra of (a) protonated and (b) sodiated proline methyl ester

with heated allylchloride, and their corresponding product MS3 CID spectra

(c) and (d), respectively. ................................................................................ 82

Figure 3.6 Calculated PES of (a) CH3NH3+/c-C3H2 reaction to form a C-N covalent bond

from Brauman double-well complexes (numbers: CCSD(T)/aug-cc-pVTZ,

kJ/mol) and (b) the two Brauman intermediates as a function of the distance

of N-H---C, blue square: CCSD(T)/aug-cc-pVTZ single point energies on

M06-2X/6-311+G(2d,p) optimized geometries; open circle: M06-2X/6-

311+G(2d,p) optimized; filled circle: B3LYP/6-311+G(2d,p) optimized. .... 85

Figure 3.7 Calculated PES of N--H--C angle graph of Complex 1, 2 and product A ...... 86

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Figure ..................................................................................................................... Page

Figure 3.8 The breakdown curve of protonated butylamine/carbene reaction by collisional

activation: relative intensity of parent complex ([C], blue circle), covalent

product ([C-40], red diamond), proton-transfer product (m/z 39, green

triangle), and direct dissociation ([C-38], green square) as a function of

activation energy. ........................................................................................... 87

Figure 3.9 Selected MS3 reaction product CID spectra of (a) butylamine, (b)

ethylmethylamine, (c) dipropylamine, and (d) guanidine with c-C3H2 and

(e) %Covalent of protonated alkyl amines/c-C3H2 reactions as a function of

GBs of the corresponding alkyl amines. The %Covalent is calculated as I[c-

40]/(I[39]+I[c-40]+I[c-38]), where I[39], I[c-40], and I[c-38] correspond to the

intensity of m/z 39 as the proton-transfer product, neutral loss of 40 Da as the

covalent product, and neutral loss of 38 Da as the direct-dissociation product,

respectively, in the MS3 CID spectra based on 80-90% parent ions

fragmented. Error bars were based on 5 duplicates of an average of 3 spectra.

........................................................................................................................ 90

Figure 3.10 MS3 reaction product CID spectrum of C3H3+and neutral n-heptyl amine ... 91

Figure 4.1 MS2 carbene reaction spectra of (a) protonated adenine and (c) protonated

adenosine; and MS3 CID spectra (b) adenine carbene monoadduct and (d)

adenosine carbene monoadduct ................................................................... 113

Figure 4.2 MS2 carbene reaction spectra of (a) protonated uracil and (b) protonated

uridine; and MS3 CID spectrum (c) uridine carbene monoadduct ............... 114

Figure 4.3 (a) MS2 carbene reaction spectrum of protonated Lys, (b) MS3 CID spectrum

of Lys/carbene reaction product, and (c) proposed fragmentation pathway of

the reaction product. ..................................................................................... 119

Figure 4.4 MS2 carbene reaction spectra of (a) protonated Arg and (c) singly charged

MRFA; MS3 CID spectra of (b) Arg/carbene reaction product and (d)

MRFA/carbene reaction product. ................................................................. 120

Figure 4.5 MS2 reaction spectra of triply charged Angiotensin I with heated allylchoride

at (a) 750, (b) 800, and (c) 900 degree Celsius. ........................................... 122

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Figure ..................................................................................................................... Page

Figure 4.6 (a) MS1 spectrum of multiply-charged Ubiquitin, and (b) MS2 spectrum of its

reaction with heated allylchoride. ................................................................ 123

Figure 4.7 MS2 carbene reaction spectra of (a) protonated acetyl cysteine, (b) protonated

cystine, and (c) singly charged oxidized glutathione. .................................. 124

Figure 4.8 MS2 reaction spectra of (a) hydroxyl group containing choline and (b) double

bond containing LPC (18:2) sodium adduct. No reaction product was

observed. ...................................................................................................... 125

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ABBREVIATIONS

ac alternative-current

APCI atmospheric pressure chemical ionization

API atmospheric pressure interface

ASGDI atmospheric sampling glow discharge ionization

B magnetic field

BIRD blackbody infrared dissociation

c-C3H2 cyclopropenylidene

C3H2 vinylidene carbene

CAD collision-activated dissociation

CI chemical ionization

CID collision-induced dissociation

DAPI discontinuous atmospheric pressure interface

dc direct-current

E electric field

ECD electron capture dissociation

EDD electron detachment dissociation

EI electron impact

ESI electrospray ionization

ETD electron transfer dissociation

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FA flowing-afterglow

FAB fast atom bombardment

FT-ICR Fourier transfer ion-cyclotron-resonance

GB gas-phase basicity

GDEI glow discharge electron impact

GIB guided ion beam

H/D hydrogen/deuterium exchange

IR infrared

IRMPD infrared multiphoton dissociation

ISM interstellar medium

LIAD laser-induced acoustic desorption

LIT linear ion trap

LMCO low mass cut-off

LQIT linear quadrupole ion trap

LTP low temperature plasma

MAD metastable atom-activated dissociation

MALDI matrix-assisted laser desorption ionization

MIKES mass-analyzed ion-kinetic-energy spectrometry

MRM multiple reaction monitoring

MS mass spectrometry

MSn tandem mass spectrometry

OH hydroxyl radical

PD photon dissociation

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ppm parts per million

pptv parts per trillion volume

PTM posttranslational modification

QqQ triple-quadrupole

QqQqQ penta-quadrupole

rf radio frequency

RIT rectilinear ion trap

ROS reactive oxygen species

SIFT selected-ion flowing-tube

SORI sustained off-resonance irradiation

SRM selected reaction monitoring

TOF time-of-flight

UV ultraviolet

UVPD ultraviolet photon dissociation

VOC volatile organic compound

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ABSTRACT

Lin, Ziqing. Ph.D., Purdue University, May 2015. Instrumentation and Development of a

Mass Spectrometry System for the Study of Gas-Phase Biomolecular Ion Reactions.

Major Professor: Zheng Ouyang.

Gas-phase reactions of biomolecular ions are highly relevant to the understanding of

structures and functions of the biomolecules. Mass spectrometry is a powerful tool in

investigating gas-phase ion chemistry. Various mass spectrometers have been developed

to explore ion/molecule reactions, ion/ion reactions, ion/photon reactions, ion/radical

reactions etc., both at atmospheric pressure and in vacuum. In-vacuum reactions have an

advantage of involving pre-selecting the ions for the reactions using a mass analyzer.

Over the decades, a variety of mass analyzers have been employed in the research of ion

chemistry. Hybrid configurations, such as quadrupole ion trap with a time-of-flight and or

a quadrupole ion trap tandem with an Orbitrap, have been utilized to improve the

performances for both the reaction (in trapping mode) and the mass analysis (accurate

mass measurements).

Complicated instrument structures, including ion optics, multiple mass analyzers and

differential pumping for high vacuum, are typically required for the mass spectrometers

for gas phase ion chemistry study. An alternative approach is to simplify the

instrumentation by using pulsed discontinuous atmospheric pressure interfaces for

introducing ionic or neutral reactants and a single ion trap as both the reactor and the

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mass analyzer. Such a simple mass spectrometry system was set up and demonstrated

using two discontinuous atmospheric pressure interfaces in the study for this thesis. It

was capable of carrying out ion/molecule and ion/ion reactions at an elevated pressure

without the needs of ion optics or differential pumping system. Together with a pyrolysis

radical source, in-vacuum ion/radical reactions were performed and their associated

chemistry was studied. Radicals are important intermediates related to biochemical

processes and biological functions. There are very limited techniques to monitor the

reactive intermediates in-situ during a multi-step reaction in aqueous phase. On the other

hand, these intermediates can be “cooled down” and preserved into a single-step

procedure in gas-phase reactions since they only occur via collisions. Therefore, the

fundamental study of gas-phase radical ion chemistry will provide evidences of the

reactivity, energetics, and structural information of biological radicals, which has the

potential to solve puzzles of aging, disease biomarker identification, and enzymatic

activities.

Using the system described above, a new reaction between protonated alkyl amines

and pyrolysis formed cyclopropenylidene carbene was discovered, as the first

experimental evidence of the reactivity of cyclopropenylidene. Given the important role

of cyclopropenylidene in the combustion chemistry, organic synthesis, and interstellar

chemistry, it is highly desirable to establish a fundamental understanding of their physical

and chemical properties. The amine/cyclopropenylidene reactions were systematically

studied using both theoretical calculation and experimental evidences. A proton-bound

dimer reaction mechanism was proposed, with the amine and the carbene sharing a

proton to form a complex as the first step, which was closely related to the high gas-

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phase basicity of cyclopropenylidene. Subsequent unimolecular dissociation of the

complex yielded three possible reaction pathways, including proton-transfer to the

carbene, covalent product formation, and direct separation. These reactions were studied

with a variety of alkyl amines of different gas-phase basicities. For the covalent complex

formation, partial protonation on cyclopropenylidene within the dimer facilitates

subsequent nucleophilic attack to the carbene carbon by the amine nitrogen and leads to a

C-N bond formation. The highest yield of covalent complex was achieved with the gas-

phase basicity of the amine slightly lower but comparable to cyclopropenylidene. The

results demonstrated a new reaction pathway of cyclopropenylidene besides the

formation of cyclopropenium, which has long been considered as a “dead end” in

interstellar carbon chemistry.

Further reactivity study of cyclopropenylidene towards biomolecular ions was also

carried out for nucleobases, nucleosides, amino acids, peptides, proteins, and lipids. The

reaction to form proton-bound dimer for protonated biomolecular ions remained as the

dominant reaction pathway. Interestingly, other possible reaction pathways, such as

modifications of thiyl group or disulfide bonds, double bond addition, and single bond

insertion, were inhibited in gas-phase ion/carbene reactions. Such results inferred that the

reactivity of neutral species was not directly applicable to ion reactions, with the proton

involved in the gas-phase biomolecular ion reactions.

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CHAPTER 1. INTRODUCTION

1.1 Gas-phase biomolecular ion reactions and mass spectrometry instrumentations

Gas-phase ion reactions involve interactions between ions and various species

including molecules,1 electrons,2 ions,3 photons,4 reactive organic species,5 etc. Mass

spectrometry (MS), which analyzes charged particles by their mass-to-charge ratio (m/z),

is naturally involved with gas-phase ion chemistry and has been a powerful tool for

manipulating and analyzing gas-phase ions. Back in the 1950s’, researchers made great

efforts to decrease the pressure inside the vacuum chamber of mass spectrometers to

improve the resolution, accuracy and sensitivity. In the 1970s’, R. Graham Cooks at

Purdue University proposed and developed the early generations of tandem MS

instruments for complex mixture analysis using in-vacuum gas collisions.6 The most

common tandem MS technique is known as collision-induced dissociation (CID, a.k.a.

collision-activated dissociation, CAD), which utilizes collisions to break the selected ions

into fragments.7 These characteristic fragments, acting as fingerprints of the parent ion,

provide unambiguous structural identification of target analytes. The development of CID

promoted MS as a “gold standard” in chemical analysis in terms of both qualification and

quantification. The nature of CID is an energy-transfer ion/molecule reaction: the kinetic

energy of the ions accelerated by the electric/magnetic field through collisions between

the ions of interest and the inert buffer gas molecules, converts into internal energy,

further cleaving the weakest linkages of the ions to generate the fragments. The study of

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2

gas-phase reactions has been growing for both studies of basic chemistry and application

development in the past two decades. The advantage of gas-phase reactions, compared to

those in aqueous or liquid phase, is that the reaction occurs via direct collisions without

solvent clusters surrounded, where the energy barrier is lower, making it both kinetically

and thermodynamically favorable, e.g. proton-transfer reaction occurs more readily in

gas phase than in aqueous phase (Figure 1.1).8

Figure 1.1 Proton-transfer reaction in the gas-phase vs. the aqueous phase

Owing to the two revolutionary ionization techniques: electrospray ionization (ESI)9

and matrix assisted laser desorption/ionization (MALDI)10, which have been awarded the

Nobel Prize in Chemistry in 200211, intact ions of biomolecules such as peptides and

proteins can be produced from their condensed phase directly to the gas phase. MS was

then adopted for biomolecular identification and became the method of choice in

proteomics to study the structures and functions of proteins.12 The primary structure of a

peptide could be sequenced through the tandem MS methods.13 Two decades later, a

variety of dissociation methods have thrived in proteomics using different types of gas-

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3

phase reactions.13 Table 1.1 summarizes the typical dissociation techniques, their

corresponding gas-phase reaction types, and the mass spectrometers that were involved in

protein sequencing. It demonstrates how gas-phase ion reactions, together with the

development of MS instrumentations, are closely linked to identifying biomolecules and

searching for disease biomarkers.6 Today, almost all of the proteomic strategies are based

on MS because it allows high throughput and structural analysis with high sensitivity.13-14

Table 1.1 Tandem mass spectrometry dissociation techniques in proteomics

Tandem MS Gas-phase reaction type Instruments

Collision induced

dissociation (CID)

Ion/molecule

[M + nH]n+ + A → fragments

Triple

quadrupoles,

ion traps15

Electron capture

dissociation (ECD)

Ion/electron

[M + nH]n+ + e- → [M + H• + (n-1)H](n-1)+

→ fragments

Fourier transfer

ion-cyclotron-

resonance (FTI-

CR)16

Electron transfer

dissociation (ETD)

Ion/ion

[M + nH]n+ + A•- → [M + H• + (n-1)H](n-1)+

+ A → fragments

Ion traps17

Proton dissociation

(PD)

Ion/photon

[M + nH]n+ + hv → [(M + nH)n+]*→

fragments

Ion traps18

Metastable atom-

activated dissociation

(MAD)

Ion/metastable species

[M + nH]n+ + A* → [(M + nH)n+]* + A →

fragments

Ion traps19

Electron-detachment

dissociation

(EDD)

Ion/electron

[M - nH]n- + e- → [M -H• - (n-1)H](n+1)- →

fragments

Fourier transfer

ion cyclotron

resonance (FT-

ICR) 20

*Precursor peptide/protein ions shown in bold

Among all the gas-phase interactions in bioanalysis, reactions involving radicals

have been increasingly drawing attentions.5 Radical ions, which consist of at least one

unpaired electron are significantly more reactive than even-electron species, would

4

4

induce more gas-phase chemistry with two reactive functional groups within one species:

the charge and the radical sites. In living organisms, radicals are important intermediates

that are highly relevant to biochemical processes and biological functions. For instance,

radicals initiate modifications in proteins,21 DNAs,22 and lipids23 at the molecular level,

which are believed to be the cause of cell damage,24 cell death,25 aging,21a, 26

neurodegenerative diseases,25-26 and cancers27. Besides the damaging effects, radicals also

contribute to the catalytic activities of enzymes in several classes.28 Taking

ribonucleotide reductase as an example, a thiyl radical was used for catalysis,28b however,

the catalytic center is more than ten amino acid residues away from the radical site.28c

The puzzle of how the radical activity is controlled by the enzyme via such a long-

distance radical transfer process remains unsolved at the current stage. This is due to the

very limited techniques to monitor the reactive intermediates in-situ during a multi-step

reaction in the aqueous phase. On the other hand, these intermediates can be “cooled

down” and preserved into a single-step procedure since gas-phase reactions only occur

via collisions. Therefore, the fundamental study of gas-phase bio-radical reactions would

provide direct and/or indirect evidences of the reactivity, energetics, and structural

information of biological intermediates. The study might explain the intra- and inter-

molecular radical transfer in biological systems, and could potentially help to develop

drugs to cure serious diseases. Thus, developing a MS system suitable for gas-phase

reactions would be the very first step in achieving these goals. This review will start with

the concept of tandem mass spectrometry (MSn) and introduce gas-phase reactions at

different pressure stages of a tandem mass spectrometer: (1) the studies of gas-phase bio-

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5

radical chemistry at atmospheric pressure and (2) MS instrumentations for gas-phase

reactions in vacuum.

1.1.1 Tandem mass spectrometry in gas-phase ion reactions

Tandem mass spectrometry (MSn) is a stepwise mass spectrometry analysis.7a A

direct MS analysis of ions produced from the ion source according to their m/z values is

called an MS full scan, also known as MS1. Selected precursor ions can undergo reactions

or fragmentations between two MS analysis stages, and the resulting product ions can be

analyzed in the next MS stage to generate MS2 spectra. Using a variety of mass analyzers,

MSn has been achieved with i) tandem-in-space mode, using sectors,29 triple

quadrupoles,30 and time-of-fight (TOF)31; or ii) tandem-in-time, using quadrupole ion

traps32 and Fourier transform ion cyclotron resonance (FT-ICR)33. Figure 1.2 gives a brief

summary of these MSn techniques.

Figure 1.2 Tandem mass spectrometry in space vs. in time

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6

For the study of bio-radical (biomolecules that contain at least one unpaired electron)

chemistry, one additional reaction step must be applied to the biomolecules to create a

radical ion since soft ionization methods such as electrospray (ESI) and nano-

electrospray (nanoESI)34 usually generate lower-energy, even-electron species rather than

odd-electron radical ions. Bio-radical ions can be made directly at atmospheric pressure

through the interactions between the whole ion population and free radicals or

alternatively in vacuum through further reactions of the selected ionic species. Product

ions of ion/radical reactions at atmospheric pressure, for example in ESI plume, could

simply be introduced to the mass analyzer through atmospheric pressure interface (API)

and focusing lens. As a result, only MS1 is required to obtain a reaction spectrum.

Tandem mass spectrometry might be further applied to elucidate the molecular structure

of the product ions, as employed in almost all of the commercial MS systems. It normally

does not need modifications of the MS instruments; however, side reactions can occur

with multiple ionic reactants. Therefore, gas-phase ion reactions in vacuum with the pre-

selection of ions are preferred. Isolation of the precursor ions is performed before the

reaction and the reaction spectra are acquired in MS2. Yet, introducing a second flow of

neutral radicals or another ionic species in vacuum for reactions could be challenging and

usually requires extensive instrumentational modification. In the next two sections,

strategies for performing gas-phase ion reactions at atmospheric pressure and in vacuum

will be discussed.

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7

1.1.2 Gas-phase biomolecular ion/radical reactions at atmospheric pressure

The interaction of biomolecular ions and free radicals can be studied in the nanoESI

plume or in the spray bulk solution in atmosphere by adding a radical source such as a

low temperature plasma (LTP) generator35 (Figure 1.3a) and an ultraviolet (UV) lamp36

(Figure 1.3b) aside the ion source. The plasma or UV irradiation generates reactive

organic species (ROS) e.g. hydroxyl radicals (OHs), which subsequently react with the

ions and charged droplets in the nanoESI plume, or directly in the bulk solution. The high

number density and large cross-section at atmospheric pressure (760 Torr) facilitate the

reactions between two species. Rich chemistry can be observed through this simple

reaction approach for biomolecules such as disulfide-linked peptides and unsaturated

lipids. Note that although there is no restriction on MS instrumentation for these reactions

taking place before the product ions are introduced to the mass analyzer, MS systems

with versatile dissociation methods, e.g. beam-type CID or ion-trap slow-heating CID,37

could be essential to study the structure, reactivity, and further applications of the

reaction products.

Figure 1.3 Radical reactions in the nanoESI plume and bulk solution using low

temperature plasma (LTP)35 or ultraviolet (UV) lamp36.

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8

1.1.2.1 Peptides

Disulfide linkage (S-S) is a type of posttranslational modification (PTM) formed by

the oxidation reaction between two cysteine residues among intra- or inter- peptide chains.

It is responsible for the stabilization of the native structures of proteins.38 The disulfide

bonds are considered difficult to break using CID due to its bonding energy being

significantly higher than those of the peptide backbones. Therefore, intra-molecular

disulfide bonds increase the difficulty in sequencing using even-electron based

dissociation methods.39 The conventional method for analyzing disulfide peptides is to

cleave the disulfide bonds using wet chemistry before sequencing (reducing disulfide

bond by alkylation39a). However, this causes the loss of constructional information.

Alternative methods are to use odd-electron radical-based dissociation methods (CID,40

ECD,16 ETD,17 EDD,20a, 20b UVPD,41 MAD,19 and etc.) in vacuum through additional gas-

phase reactions. Fortunately, disulfide bonds are very sensitive to free OHs, producing

different types of radical products (such as carbon-centered versus heteroatom-centered

radicals) with highly specific radical sites. As an example (Figure 1.4), the cleavage of a

disulfide bond occurs with the attack by hydroxyl radicals which yields three sets of

products: thiyl (-S•), sulfinyl (-SO•), and perthiyl (-SS•) radical ions.42

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9

Figure 1.4 Reactions of protonated disulfide peptide and oxygen-centered radicals5 to

form thiyl (-S•), sulfinyl (-SO•), and perthiyl (-SS•) radical ions in nanoESI plume: (a)

peptide chain CLPTR of HMAC-CLPTR, and (b) chain AGCK of AGC(TFTSC)K.42

Among the three radical species generated by the reaction in the nanoESI plume, thiyl

radicals have the highest reactivity while sulfinyl radicals are the most inert.42 Although

the cysteine sulfinyl radicals have low reactivity in aqueous solvents or under gas-phase

bimolecular reaction conditions, additional energy could trigger intramolecular reactions.

The energy applied by collisional activation can be sufficient to conquer the reaction

energy barrier but barely enough to cause backbone cleavage. Two reaction pathways of

radical migration were observed for a cysteine sulfinyl radical (SO•Cys) with either a free

thiol (-SH)43 or a disulfide bond (S-S)44 inside one peptide sulfinyl ion (Figure 1.5). The

discovered reaction channels indicate: (1) the disulfide bond scrambling could happen in

a protein based on the radical migration; (2) protein conformation and structural change

would be possible by the attack of the cysteine sulfinyl radicals; and (3) an oxidative

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10

damage in terms of forming sulfinyl radicals, might potentially be cured with a nearby

free thiol group43.

Figure 1.5 Intramolecular cysteine sulfinyl radical (SO•Cys) transfer with (a) a free

thiol43 and (b) a disulfide bond44

Besides the reactivity study of bio-radical ions, ion/radical reactions in the ESI plume

enhance the backbone cleavage, thus improving the sequencing coverage.35 The

backbone cleavage for insulin, a protein with three disulfide bonds, occurred at an

efficiency rate of 26% obtained from even-electron based CID, but improved to about 60%

with odd-electron induced ECD or ETD methods.45 A favorable value of 75% backbone

cleavage has been achieved for cleaving disulfide bonds followed by applying a

conventional CID after the radical reaction in the ESI plume (Figure 1.6).35

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11

Figure 1.6 Coupling radical reactions in the ESI plume to cleave the disulfide bonds, with

a subsequent MS2 scan to improve sequence information of disulfide peptides5, 35

1.1.2.2 Lipids

Lipids are a group of biomolecules that function as the building blocks of cell

membranes, energy storage, and signaling transduction in living organisms.46 Individual

lipid47 or the lipid profile48 has been used as biomarkers for cancer diagnosis by ambient

ionization MS techniques, assuming that the lipid bio-synthesis pathways have been

changed in the tumor tissues.46 The degree of unsaturation in lipids (the number of C=C

bonds) and the location of double bonds determine the biomolecular structure and the

biological functions.49 Even-electron based dissociation methods are very helpful in

identifying the head group, the acyl chain composition, and the number of C=C bonds.50

However, the positions of C=C bonds, on the other hand, are difficult to be determined

since the bonding energy is much higher for C=C double bonds (~keV) than C-C single

bonds. This problem could not be solved with high energy collisions by sector or TOF-

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12

TOF instruments because the C-C bonds are cleaved before the C=C bonds in a lipid. In

order to differentiate the unsaturated lipid isomers (different double positions in the acyl

chain), radical chemistry has been applied in vacuum or at atmospheric pressure. Radical-

directed dissociation (RDD) of lipid ions in vacuum yields fragmentation patterns

revealing information of double bond positions: a photo-caged radical precursor releases

the radical by irradiation, which forms a complex with the unsaturated lipids and

fragments are subsequently generated through CID.51 Alternatively, a Paternò–Büchi (PB)

reaction can be performed in the bulk solution of the nanoESI spray tip by UV lamp

irradiation (Figure 1.3b).52 A four-member ring is first formed through the interaction of

a ketone/aldehyde with an olefin, thus creating a set of low energy covalent bonds which

are then cleaved in the retro P-B reaction as shown in Figure 1.7.

Figure 1.7 Scheme of Paternò–Büchi (PB) reaction between ketone/aldehyde and olefin

together with possible retro P-B reactions.52

In the analysis of oleic acid (Figure 1.8),52 a covalent adduct m/z 339 of P-B reaction

of deprotonated oleic acid and acetone was formed with the 254 nm UV irradiation

(Figure 1.8a, b). MS2 CID of the product clearly showed three peaks in Figure 1.8c: m/z

281 corresponding to the retro P-B reaction to produce the original reactants

(deprotonated oleic acid); and one set of diagnostic ions with a mass difference of 26 Da

13

13

resulting from the fragments (structures 3 and 5) of two possible arrangements of P-B

reaction (isomers 1 and 2) (Figure 1.8d). By this means, lipid isomers (fatty acids and

phospholipids) in tissue samples with different double bond locations have been

identified and potential lipid biomarkers of diseases are under investigation.

Figure 1.8 On-line coupling of P-B reactions of oleic acid and acetone with negative MS.

MS1 spectra of oleic acid (a) before P-B reaction (b) after P-B reaction; MS2 CID of the

P-B reaction products at (c) m/z 339.3; and (d) structures of the diagnostics ions

(structures 3 and 5). The structures explain the origin of the 26-Da mass difference.52

1.1.3 Instrumentations for gas-phase ion reactions in vacuum

Compared to the reactions occurring at atmospheric pressure, gas-phase ion reactions

can also be carried out in vacuum. It provides one huge advantage of controlling

variables by having a single ionic reactant selected from the ion population, which largely

decreases the uncertainty of side reactions by unknown ions generated by the ion source.

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14

The tradeoffs are however, the necessity of instrumentational modification and the

relatively low number density in vacuum (enough for reactions to occur) due to the low

pressures (usually 10-3 to 10-7 Torr). The first stage of tandem mass spectrometry (MS1)

can be used to isolate the precursor ions based on their m/z values. The subsequent gas-

phase reactions are then carried out in the following MS stages (MSn). Several types of

mass spectrometers have been modified to study gas-phase ion chemistry. For tandem-in-

space mass spectrometers such as triple-quadrupoles, penta-quadrupole, and selected-ion

flow-tube, a reaction chamber (designed for collisions) and at least one additional mass

analyzer for the following product analysis are required. On the other hand, the reactor

and mass analyzer can be integrated for trap-type instruments such as ion traps and

Fourier transform ion cyclotron resonance, which are able to store ions for a relatively

long reaction time and allow subsequent tandem MS. Hybrid instruments, involving a

trapping mode reaction chamber followed by a high accuracy mass analyzer for mass

analysis, are suitable for ion manipulation and reactions in the vessel, then providing a

high resolution product scan. Nevertheless, not all of these research prototype

instruments have been applied in gas-phase biomolecular ions reactions, especially for

bio-ion/radical reactions. This section will have an emphasis on the instrumentation: the

strategies to bring two species inside vacuum and the related reactions not limited to

biological ion interactions.

1.1.3.1 Sectors and time-of-flights (TOFs)

A sector mass spectrometer is a MS system using a static electric (E), a magnetic (B)

field, or a set of the combination of the two sectors to separate ions as a mass analyzer. In

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15

the case of mass-analyzed ion-kinetic-energy spectrometry (MIKES),6a a magnetic sector

followed by an electronic sector was originally designed to study kinetic energy release

associated with metastable ion fragmentation by measuring the kinetic energy of mass-

selected ions. This instrument is most famously recognized as the first generation of

tandem mass spectrometer,6b, 53 with collision gas introduced between the two sectors

(Figure 1.9). The collisional energy in MIKES tandem MS could be as high as ca. keV,

which is sufficient to obtain fragments from most ionic species during tandem MS.

Guided ion beam (GIB) tandem MS is another sector instrument involved in gas-phase

chemistry. The ion beams selected by a magnetic sector are introduced to the reaction cell

(octapole ion guide) with reactant neutral of choice, followed by a double focusing

sector54 or a quadrupole55 as mass analyzer. This instrument has been widely used for the

determination of thermodynamic information for bimolecular reactions and CID.55

Figure 1.9 Schematic of mass-analyzed ion-kinetic-energy spectrometry (MIKES) with

collision gas for chemical mixture analysis6a

As for a tandem time of flight56 (TOF-TOF, shown in Figure 1.10), the collisional

energy is able to reach keV level, similar to that of the sectors. Although TOF-TOF

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16

instruments have been barely used in research of gas-phase ion reactions, TOF-TOF

provides a good dissociation methods coupled with MALDI for peptide identification.57

Furthermore, high mass resolution capability of TOF analyzer serves as an excellent

product mass analyzer for in-vacuum gas-phase reaction in hybrid instruments, which

will be further discussed in section 0.

Figure 1.10 Schematic of tandem time of flight (TOF-TOF) mass spectrometer56

1.1.3.2 Tandem quadrupoles

The triple-quadrupole (QqQ) mass spectrometer was developed in late 1970s30 and

remains as an outstanding MS2 instrument for chemical quantification. Triple-

quadrupoles have demonstrated tremendous capabilities in performing a set of MS2

experiments in tandem-in-space mode in three steps: (1) isolating precursor ions in Q1 by

applying certain amplitudes of radio-frequency (rf) and direct-current (dc) to allow ion

transmission at a narrow m/z window; (2) dissociative or reactive collisions in rf-only

collisional quadrupole q2 with activation energy ranging from 0 to tens of eV when using

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17

inert or reactive buffer gas; and (3) product ion scan in Q3. The use of triple-quadrupoles

in studying ion/molecule reactions is valuable since the information of both precursors

and products are provided through different MS2 scan modes such as product ion scan,

precursor ion scan, neutral loss, and selected reaction monitoring (SRM) or multiple

reaction monitoring (MRM).58

Figure 1.11 Schematic of penta-quadrupole (QqQqQ) mass spectrometer59

The limitation inherent to triple-quadrupoles for ion/molecule reaction study is the

lack of structural analysis of the product ions with only two MS stages. Therefore, a

simple way to improve tandem-in-space instruments for ion/molecule reactions is to add

another MS stage, MS3. The penta-quadrupole (QqQqQ)60 (Figure 1.11) was designed

specifically for MS3 analysis for ion/molecule reactions. It employs two reaction rf-only

quadrupoles (q2 and q4) for reactions, and three rf/dc quadrupoles (Q1, Q3, and Q5) for

MS analysis. The tandem-in-space arrangement allows the study of ion/molecule

reactions involving mass-selected ions in two separate reaction regions (q2 and q4) by

using Q1, Q3, and Q5 for mass analysis. The first reaction region q2 can be set as

dissociative-collisional chamber for CID whereas q4 was filled with neutral reactants for

reactive collisions, or vice versa. A total of 15 different types of MS3 scan functions61 can

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18

be achieved with 0-3 dimensions as shown in Figure 1.12. Each circle represents the

operation of one of the three mass analyzers, with a filled circle for a mass selection and

an open circle for MS scanning. Note that with all three mass analyzers transmitting fixed

masses, the consecutive reaction monitoring (three filled circles, 0 dimension) provides

mass spectra of one dimension. A remarkable 4D mass spectra were therefore acquired

using the entire MS3 data domain.62

Figure 1.12 Symbols and names given to QqQqQ MS experiments with 0, 1, 2, and 3

dimensions. A fixed mass (filled circle) or variable masses (open circle) can be chosen for

reactions.61

An example of 3D familial scan is demonstrated in Figure 1.13.63 The ions produced

from 3-octanone by electron impact (EI) underwent CID in q2 via collisions with Argon

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19

gas and the CID products of m/z 43 were selected for reacting with isoprene in q4; finally

the ion/molecule reaction product ions were analyzed by Q5. This experiment was useful

for ion chemistry since it provided reactivity information of isobaric and isomeric ionic

populations. Both sequential product spectra (horizontal) and sequential precursor spectra

(angles) can be extracted. Figure 1.13b shows the sequential precursors generating m/z 43

reacting predominantly by proton transfer that led to the product ions of m/z 81, whereas

Figure 1.13c identifies the sequential precursors producing acetyl cations of m/z 43,

which reacted with isoprene to form [4+2+] cycloadduct of m/z 111.

Figure 1.13 (a) Penta-quadrupole three-dimensional triple-stage (MS3) familial spectrum

for 3-octanone ions acquired by selecting ions of m/z 43 as the intermediates. The angled

Q1 axis displays the precursors of m/z 43 ions, whereas the horizontal Q5 axis displays

the ion/molecule products of these CID-generated m/z 43 ions arising from reactions with

isoprene. Triple-stage sequential precursor spectra extracted from the 3D familial

spectrum by selection in Q5 of the final products of (b) m/z 81 and (c) m/z 111.63

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20

Penta-quadrupole MS system has been proven to be suitable to study various

ion/molecule reactions.60 However, other types of gas-phase reactions such as ion/ion and

ion/radical reactions could barely be done on the penta-quadrupole mass spectrometer

without further modifications.

1.1.3.3 Flowing-afterglow selected-ion flow-tube (FA-SIFT)

Selected-ion flow-tube mass spectrometer has been used to study ion/molecule

reactions64 and also for quantitative analysis of trace, volatile organic compounds (VOCs)

using chemical ionization (typically proton-transfer reactions) with the selected precursor

ionic species along a drift tube within a well-controlled period. The applications extend to

gas analysis,65 breath testing,66 VOC monitoring from natural products67 etc., with pptv

level of detection limit. Together with the flowing-afterglow (FA) techniques, selected-

ion flow-tube has been employed to measure numerous ion-molecule kinetics data

especially those related to interstellar chemistry at a low temperature.68 In an FA-SIFT

instrument, the precursor ions generated by EI ionizer in the source flow tube (FA) are

sequentially selected by a SIFT quadrupole mass filter. The mass-selected ions flow

through the reaction tube where ion/molecule reactions occur with the neutral reactant

being introduced into the flow tube. The resulting product ions are scanned in the

detection quadrupole mass filter to produce the MS2 spectra. With the well-defined flow

time inside the reaction tube, the ion/molecule reaction kinetics can be acquired by

introducing neutrals at different positions along the drift tube to set a series of reaction

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durations.68b FA-SIFT is also a tandem-in-space MS for ion/neutral reactions with the

reaction time in a well-defined fashion.

Figure 1.14 Schematic of flowing-afterglow selected-ion flow-tube (FA-SIFT) coupled

with a radical source for ion/radical reactions69

In 2004, this instrument was involved in the study of ion/radical reactions by adding

one additional radical source to the flow tube (Figure 1.14).69 The neutral radicals are

generated by a pulsed pyrolysis nozzle (also known as Chen nozzle70). Pulsed pyrolysis

has been an efficient means to produce gas phase radicals of high intensities in vacuum.71

It uses a silicon carbide tube for resistance-heating to dissociate organic precursors into

neutral radicals for the structural study of highly reactive species using infrared or

electronic spectra. Proton transfer reaction was observed between allyl radicals and

hydroniums, also associative detachment reaction between ortho-benzynes and

hydroxides, and radical recombination reaction between allyl radicals and benzyl radical

cations.69 The results firmly showed that FA-SIFT is capable of carrying out ion/radical

reactions with the additional radical source. No biological molecules have yet been

studied using this specific MS instrument.

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1.1.3.4 Fourier transform ion cyclotron resonance (FTICR)

Different from tandem-in-space mass spectrometers, mass analyzers such as Fourier

transform ion cyclotron resonance (FTICR, Penning trap33) and quadrupole ion traps

(Paul trap72) are able to trap ions for a certain period of time, while carrying out multiple

steps of reactions and mass analysis. The advantage of tandem-in-time mode is that the

instruments do not require extra mass analyzers for performing additional stages of MS

analysis or reactions. The tradeoffs, compared to tandem-in-space, are longer duty cycles

and potential interference by the leftover neutral reactants.

FTICR employs a cyclotron cell that operates at a high vacuum of 10-7 to 10-8 Torr

for trapping. It consists of three pairs of adjacent electrodes assembled into a cubic,

cylindrical, or hyperbolic designs.73 A typical setup of FTICR is illustrated in Figure

1.15.74 The front and back electrodes, perpendicular to the strong magnetic field, function

as trapping plates to trap the ions inside the cyclotron cell. A radio frequency (rf) is

applied on the side plates to excite the trapped ions to induce the image current. The

detection plates (top and bottom) are used for monitoring the current induced by the ions

motions. A chirp is applied by sweeping the rf sweep from several kHz to several mHz to

excite the trapped ions. The induced image current is collected in the time domain. Mass

spectrum in frequency domain is then obtained with a fast Fourier transformation. The

mass resolution on FTICR is correlated to the strength of the magnetic field so that

accurate mass (< 1 ppm) can be obtained with large magnetic field (10T).

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23

Figure 1.15 Schematic of a basic ICR cell, consisting of two trapping plates (front and

back), two opposite excitation plates (right and left), and two detection plates (top and

bottom). Ions enter the cell through the front trapping plate. Mass spectrum generated by

fast Fourier transformation.74

Similar to SIFT, FTICR was used in the study of interstellar chemistry at very low

temperatures68a and has been coupled with a pyrolysis nozzle.75 The reactions between

benzyl radical cations and pyrolysis-produced allyl radicals were studied, which formed a

carbon-carbon covalent bond. In another study, the protonated desR1-bradykinin ions

were mass-selectively isolated and underwent a hydrogen/deuterium (H/D) exchange

reaction with deuterium radicals generated by electron gun, which was one of the very

few reactions involving bio-ions and neutral radicals.76

Instead of carrying out reactions between bio-ions and neutral radicals, organic

radical ions can be generated in gas phase, trapped in FTICR, and then reacted with gas-

phase biomolecules. With laser-induced acoustic desorption (LIAD) techniques (Figure

1.16), neutral biomolecules, such as amino acids and peptide, can be made into gas phase

and interact with the trapped radical ions.77 Abstraction, addition, and proton-transfer

reactions have been observed for reactions between radical ions and biomolecules.

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24

Figure 1.16 Schematic of the FT-ICR instrument, including the LIAD probe.78

Besides ion/neutral (ion/molecule, ion/radical) reactions, FTICR is also well-used in

proteomic study, especially with the development of electron capture dissociation (ECD)

by McLafferty and coworkers in 1998.16, 79 To apply this technique, trapped positive

precursor bio-ions capture low energy electrons (<0.2 eV), forming odd-electron ions via

ion/electron reaction.80 Charge reduced ion [M+nH](n-1)+• is the main product of electron

capture, which causes peptide backbone cleavages.81 In contrast to CID, in which the

weakest bond is cleaved through an ergodic process, the major cleavage occurs at N-Cɑ

bond with ECD, resulting in c/z type of peptide ions.82 It is believed that an aminoketyl

radical is formed during the ECD, which subsequently produces c and z• fragments (or c•

and z) as shown in Figure 1.17.83 The reaction is fast, regarded as non-ergodic and a

perfect match with FTICR due to the low pressure in the cyclotron cell which allows

efficient electron capture.16

Figure 1.17 Aminoketyl radicals and c and z• ions by ECD2, 84

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25

The c/z pair of ions is unique in radical peptide ion fragmentation compared to even-

electron species. One important feature of ECD is that the cleavages randomly occur,

without a preferred site except for the proline amino cleavage.85 Thus, almost all the N-

Cɑ bonds throughout the backbone of peptides could cleave to generate fragments, which

produces meaningful information for peptide and protein sequencing (Figure 1.18).86

Together with slow heating ion activation techniques in FTICR, such as sustained off-

resonance irradiation collision-induced dissociation (SORI-CID), infrared multiphoton

dissociation (IRMPD), and blackbody infrared radiative dissociation (BIRD),87 more

internal energy is directed into the biological radical ions, and therefore more fragments

can be produced for proteins over 20 kDa. On the other hand, posttranslational

modifications can be preserved using ECD, which makes it possible to identify disulfide

linkage, glycosylation, phosphorylation and oxidation of proteins.2, 88

Figure 1.18 Mass spectrum of ubiquitin ([M+9H]9+) by ECD FTICR with 9.4 T

instrument.89

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26

A variety of gas-phase reactions, including ion/molecule, ion/radical, and

ion/electron reactions, have been investigated using FTICR-MS to study the biological

molecules. FTICR-MS instruments with a large magnetic field are of high cost for

ownership and maintenance,45 in addition, the number density of neutral reactants is

relatively low due to the high vacuum requirements, which is not in favor of ion/molecule

or ion/radical reactions.

1.1.3.5 Quadrupole ion traps

Quadrupole ion traps are operated with radio-frequency (rf), which creates a

quadrupole electric field to trap the ions.72 Based on different geometries, quadrupole ion

traps can be categorized into 3D ion traps, and linear or 2D ion traps.72 The linear ion

traps (LITs) have increased trapping capacity. Quadrupole ion traps are effective tools for

in-vacuum gas-phase ion reactions because of their capability to store ions for long

reaction time (as high as 10 seconds) and the relatively higher pressure reaction

environment (10-3-10-5 Torr). Since gas-phase reactions occur via collisions, which is

highly dependent on the number density, the pressure inside quadrupole ion traps is three

orders of magnitude higher than FTICR, making them highly preferable for carrying out

gas-phase ion reactions. With these features, quadrupole ion traps, especially linear ion

traps, have been widely employed for studies of gas-phase ion reactions.

Neutral reagent vapor can be simply mixed with the collision gas for ion/molecule

reactions in the ion trap. An example is shown in Figure 1.19a. The neutral reactants were

leaked into Q3 linear ion trap of a modified QTrap 4000 (AB Sciex) mass spectrometer to

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investigate the reactivity of thiyl, sulfinyl, and perthiyl radicals.42 To introduce multiple

neutral reagents subsequently into ion trap mass spectrometers, a multi-valve interface

consisting of three neutral lines was designed,90 where three different ion/molecule

reactions could be carried out at the same time for fast neutral reagent screening (Figure

1.19b).

Figure 1.19 Schematic of a single neutral injection line (a)42 and a multiported pulsed

valve interface (b)90 of a linear quadrupole ion trap for ion/molecule reactions.

As one of tandem-in-time mass spectrometers, ions are manipulated in one trap mass

analyzer to achieve multiple stages of mass analysis. For target analyte analysis, MSn

with CID can be easily carried out via isolating the precursor ions followed by multi-

collisional dissociation (ac activation induced rf heating). However, the structural

identification for the products of ion/molecule reactions can be tricky since neutral

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reactants remain inside the ion traps and can further react with the fragment ions. One

solution to this issue is to introduce two linear quadrupole ion traps in series as shown in

Figure 1.20,91 which borrows the idea of tandem-in-space MS systems yet keeps the

advantages of tandem-in-time MS systems. Ion/molecule reactions were carried out in the

first trap (LQIT1); the product ions of interest were then transferred into the second trap

(LQIT2) for structural determination; or vice versa, the CID products from LQIT1 could

be transferred to LQIT2 for ion/molecule reactions.

Figure 1.20 Schematic of the differentially pumped dual LQIT with ion optics and

differential pumping. LQIT1 and LQIT2 are the first and second linear quadrupole ion

traps (LQIT).91

Gas-phase ion/ion reactions usually require longer reaction time and high abundance

for both positive and negative ions, and one of them needs to be multiply charged for the

reaction products to be analyzed by MS.92 Ion/ion reactions can be readily applied to

peptides and proteins since multiply charged ionic species can be produced through

spray-based ionization methods (ESI or nanoESI). Electron transfer dissociation (ETD),

proven to be one of the most useful applications of ion/ion interactions, occurs between

peptide or protein cations and radical anions in ion traps. Ion/ion reactions take place at

the millisecond level, much faster than ECD, which is limited by the slow electron

transferring rate. Thereby, ETD can be coupled with liquid chromatography.93 Reagents

such as anthracene and azobenzene are used to generate radical anions by chemical

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ionization (CI) or atmospheric pressure chemical ionization (APCI).94 These anions act as

electron donors due to their low electron affinity. Through electron transfer, the odd-

electron ions of reduced charge are produced and fragmented into c/y pairs from peptide

ions. A competitive proton-transfer reaction also occurs in ETD,95 giving even-electron

ions by losing a proton similar to the products in ECD through hydrogen loss. The

advantages of ETD include its low cost, the use of quadrupole ion traps instead of FTICR,

and the flexibility in ion manipulation. Low energy CID of all the resulting ions from

electron transfer reaction (supplemental collisional activation) or one specific charge-

reduced species (charge-reduced species CID) can be carried out with high sequencing

coverage.45, 96 Instrumentation of ion/ion reactions is generally more complicated,

compared to those for ion/molecule reactions, since two different ion beams are required.

Unlike 2D ion traps, 3D ion traps can store ions of both polarities.92, 97 As shown in

Figure 1.21, a home-built 3D ion trap instrument was constructed with three ion sources:

a positive and a negative ESI sources to generate multiply charged peptide/protein ions,

and an atmospheric sampling glow discharge ionization (ASGDI) source to produce ions

of either polarity for reactions.97-98 The turning dc quadrupole was used for selecting the

ion beam into the 3D ion trap through the holes on the endcap, while the ASGDI source

was directly pointing at the hole of the ring electrode. Studies of both proton-transfer and

electron transfer reactions were carried out using this configuration.

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Figure 1.21 Schematic of a multi-source quadrupole ion trap instrument containing two

electrospray ionization (ESI) sources and one atmospheric sampling glow discharge

ionization (ASGDI) source interfaced.98b

Linear (2D) ion traps have inherent advantages over 3D ion traps, such as higher

trapping capacity and higher trapping efficiency for externally injected ions, mutual

storage of ions of opposite polarities is not effective in conventional instruments92. Two

approaches have been reported for mutually trapping cations and anions: (1) applying rf

directly to at the end electrodes of the linear ion trap to create rf trapping along z-axis,17

so that positive and negative ions can be axially trapped simultaneously for reaction and

(2) using an unbalanced trapping rf to create a trapping field along the axial direction.99

Ion/ion reactions can also be obtained in transmission mode, whereby ions of one polarity

are trapped in the linear ion trap, and ions of the other polarity are injected through the

ion trap for reaction. An example is shown in Figure 1.22,100 the negative ions generated

by ASGDI were injected through Q3 and accumulated in Q2 before multiply charged

positive ions were injected through Q2, and the final products were analyzed in Q3. The

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radial injection of negative reactant ions into Q3and subsequent trapping in Q2 is not

efficient, which limits the effectiveness of the transmission mode. A pulsed dual ion

source consisting of +ESI/-ESI or ESI/APCI has been utilized to generate and introduce

ions of opposite polarities subsequently into Q2 linear ion trap for reaction through the

same ion pathway but different potentials on the optics.101 Proton-transfer and ETD

reactions have been investigated by employing the pulsed dual ion source.

Figure 1.22 Schematic of a triple quadrupole/linear ion trap modified by addition of an

atmospheric sampling glow discharge ionization source on the side of Q3 linear ion trap

for ion/ion reactions.100

Quadrupole ion traps are also feasible for studying ion reactions with metastable

species and photons based on specific modifications. Two modified instruments used in

these studies are shown in Figure 1.23. A pulsed fast atom bombardment was placed next

to a hole on the ring electrode of a 3D ion trap to generate metastable atoms to react with

peptide ions for inducing dissociation;19 laser emission was directed into a linear ion trap

via optics for peptide ion/photon reactions. Metastable atom-activated dissociation

(MAD),19 infrared multiple photon dissociation (IRMPD),102 and ultraviolet photon

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dissociation (UVPD)102a, 103 have been developed using the interaction of peptide ions

with metastable species, infrared, and ultraviolet photons, respectively, to produce odd-

electron ions for peptide sequencing. Ion/neutral radical reactions should also be

employed in quadrupole ion trap mass spectrometers if additional radical sources are

installed. The application of gas-phase ion/radical reactions in an ion trap mass

spectrometer is still under investigation.

Figure 1.23 Schematic of (a) a 3D ion trap coupled with a fast atom bombardment (FAB)

gun for metastable atom-activation dissociation19 and (b) a laser coupled linear ion trap

for infrared multiple photon dissociation (IRMPD)102b and ultraviolet photon dissociation

(UVPD)103.

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1.1.3.6 Hybrid instruments

Linear quadrupole ion traps have large trapping capacity and allow axial ion

injection, which makes them particularly attractive reactors for gas-phase ion reactions in

hybrid instruments. High accuracy mass analyzers (FTICR, TOF and Orbitrap) can be

coupled with linear ion traps to study a wide variety of gas-phase ion reactions. In this

section, two hybrid instruments, one with a TOF analyzer and one with an Orbitrap

analyzer are presented as examples of hybrid instruments for gas-phase ion reactions.

Quadrupole tandem time-of-flight (QTOF)

Figure 1.24 shows a quadruople tandem time-of-flight (QTOF) mass spectrometer

modified for ion/ion reactions.104 This instrument contained three quadrupoles (ion guide

Q0, mass filter Q1, collision cell Q2 as a linear ion trap) and an orthogonal reflectron

TOF analyzer for product analysis. A pair of pulsed ion source, ±ESI or nano-ESI

(+)/APCI (-), were placed in front of the MS inlet to produce the ionic reactants of

opposite polarities to study proton transfer reactions or ETD reactions. The reflectron

TOF significantly had improved mass resolving power and accuracy, compared to

quadrupole mass filter or Paul traps. The mass resolving power is about 8000 over a wide

mass range up to m/z 6,600 with a mass accuracy better than 20 ppm.

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Figure 1.24 Schematic of a modified quadrupole time-of-flight tandem mass spectrometer

for ion/ion reactions with dual ion source.104

Orbitrap Instruments

Orbitrap is a mass analyzer for exact mass measurement, developed by Makarov and

coworkers based on a King trap.105 The ions are trapped by the electrostatic field with a

centrifugal forces. Image current is measured for the harmonic axial motions. Hybrid

linear ion trap tandem Orbitrap (LTQ-Orbitrap) was commercialized in 2005.106 The

recent Orbitrap Elite (Figure 1.25)107 consists of two linear ion traps with different

pressures,108 a quadrupole mass filter, a C-trap, a collision cell and an Orbitrap mass

analyzer with an optional ETD reagent generator. The high-pressure ion trap is used to

cool down the ions whereas the low-pressure cell is for mass analysis or isolation. Mass-

selected precursor ions can be transferred along the quadrupole mass filter to the C-trap.

C-trap is used to cool and transfer ions into the Orbitrap. In the early design, C-trap was

also used to carry out slow-heating CID by increasing the rf, but with the restriction of

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low-mass cutoff (LMCO) through the rf ramping.109 Later on, another linear ion trap

(HCD collision cell) is used for high energy CID. Negative ETD reagent ions can be

introduced through ETD cassette into the HCD collisional cell as well. Gas-phase ion

reaction products are then transferred back to the C-trap before entering Orbitrap for

accurate mass measurement. Hybrid Orbitrap mass spectrometers have been used

intensively for the analysis of protein, glycan and biopharmaceutical applications.

Figure 1.25 Schematic of an Orbitrap Elite.107a

1.1.3.7 DAPI mass spectrometers

For the study of gas-phase ion reactions in hybrid instruments, more delicate

compartments can be installed to achieve multi-functions of high performance and high

compatibility, while compartments can also be reduced and simplified as long as the

basic requirements of in-vacuum ion reactions are satisfied. In all MS instruments

mentioned in the previous sections with atmospheric ion sources such as ESI and APCI,

the continuous atmospheric pressure interface (API) requires differential pumping system

to support the high vacuum for mass analysis as well as ion optical tools to guide the ions

into the analyzers. Discontinuous atmospheric pressure interface (DAPI),110 on the other

hand, is another solution to substitute for the massive interface (removing all ion optics)

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and differential pumping (largely reducing pumping requirements) in the conventional

MS instruments as shown in Figure 1.26. The original purpose of using DAPI together

with a rectilinear ion trap (RIT)111 was to miniaturize the mass spectrometers.112

Figure 1.26 Schematic of (a) conventional linear ion trap mass spectrometer with

differential pumping system and ion optics and (b) miniature DAPI-RIT mass

spectrometers highlighted Mini 11 mass spectrometer of 9 pounds and Mini 12 of Point-

of-care personal mass spectrometer.110, 113

The operation of a DAPI-MS consists of four steps (Figure 1.27a):110 (1) ions are

generated in the atmosphere by nanoESI or APCI before the DAPI opens; (2) the pinch

valve in DAPI opens for 10 to 30 milliseconds to transfer the ions into the trap in vacuum

chamber. In this process, rf is applied to trap ions and the pressure inside chamber

increases to 10-2-10-1 Torr; (3) after ion introduction, the DAPI closes and the ions stored

in the trap cool down for hundreds of milliseconds while the pressure decreases to 10-3

Torr; (4) at this pressure, mass-selective instability scan is used for mass analysis. One

scan cycle of a DAPI mass spectrometer usually takes one second, as the tradeoff for

breaking the barrier of pumping speed to not include a differential pumping system.

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Figure 1.27 (a) Scan function for mass analysis using a DAPI mass spectrometer. (b)

Manifold pressure measured during scanning, with an open time of 20 ms and a closed

time of 850 ms for DAPI.110

Multiple DAPIs can be implemented to introduce charged or neutral species for gas

phase ion reactions with minimum modification to an instrument. Note that the pressure

during the ion or neutral introduction is different from that for mass analysis (Figure

1.27b). This represents a unique opportunity for using a single trap to carry out gas-phase

reactions at a much elevated pressure, with reaction products later analyzed at 10-3 Torr

in a single scan cycle. One previous study114 by our research group has shown that two

beams of ions or neutrals can be introduced into a LIT sequentially or simultaneously

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through two DAPIs for gas phase ion processing, including gas flow CID, gas flow

assisted desolvation, and one ion/ion reaction. Simplified home-built mass spectrometer

with multiple DAPIs could be potentially constructed to investigate a variety of gas-phase

ion reactions, especially the interactions between biological ion and organic radicals, with

additional radical sources like the pyrolysis valve.71

1.2 Conclusion

Gas-phase reactions of biological ions especially those producing odd-electron bio-

radical ions, have been applied for biomolecule structural identification, disease diagnosis,

and enzymatic reactivity evaluation. Gas-phase bio-ion interactions can be carried out in

atmosphere or in vacuum. Significant basic chemistry and applications have been shown

in the study of gas-phase biological ion/radical reactions in the ESI plume or bulk

solution. Minimum instrumentational modification is required for such reactions at

atmospheric pressure, however no single ionic reactant can be selected prior to the

reactions. Ion manipulation and mass-selectively isolation can be achieved for gas-phase

ion reactions in vacuum, which on the other hand, require multi-stages tandem mass

spectrometry. Various tandem mass spectrometers of both tandem-in-space and tandem-

in-time configurations, have been demonstrated as useful tools for studying in-vacuum

ion/molecule, ion/ion, ion/radical, and ion/photon reactions. Among all the mass

analyzers, ion traps are feasible for all kinds of reaction types with long ion storage time.

Quadrupole linear ion traps are ideal reaction vessels for gas-phase reactions due to the

convenience of introducing ionic or other reactants and the relatively high pressure for

reactions. Hybrid instruments employ linear ion traps as the reactor and accurate mass

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analyzers for product analysis, which represents an effective way of improving the

instrumentational performance in studying gas-phase reactions. On the other hand, a

multi-DAPI mass spectrometer presents an opposite approach that is “just good enough”

for the study, with the simplest means to introduce reactants in a controlled environment.

This thesis features a DAPI-RIT-DAPI mass spectrometer that has been developed

with a rectilinear ion trap as both the reactor and the mass analyzer, and two pulsed

interfaces to introduce ions and other species for reactions. Ion/molecule and ion/ion

reactions were used to characterize the established system at an elevated reaction pressure

and with the energetic gas flow by the DAPIs. With an additional radical source, a novel

ion/radical reaction between protonated alkyl amines and pyrolysis-generated C3H2

carbene bi-radicals to form a C-N covalent bond was discovered with the

instrumentational setup shown in Figure 1.28. This reaction was further applied to

biological ions such as nucleobases, nucleosides, amino acids, peptides, and proteins.

Figure 1.28 Schematic of a DAPI-RIT-DAPI mass spectrometer coupled with a pyrolysis

radical source for the study of gas-phase ion/radical reactions.

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1.3 References

1. (a) Green, M. K.; Lebrilla, C. B., Ion-molecule reactions as probes of gas-phase

structures of peptides and proteins. Mass Spectrom. Rev. 1997, 16 (2), 53-71; (b)

Brodbelt, J. S., Analytical applications of ion-molecule reactions. Mass Spectrom. Rev.

1997, 16 (2), 91-110.

2. Cooper, H. J.; Hakansson, K.; Marshall, A. G., The role of electron capture

dissociation in biomolecular analysis. Mass Spectrom. Rev. 2005, 24 (2), 201-222.

3. (a) Pitteri, S. J.; McLuckey, S. A., Recent developments in the ion/ion chemistry

of high-mass multiply charged ions. Mass Spectrom. Rev. 2005, 24 (6), 931-958; (b)

McLuckey, S. A.; Stephenson, J. L., Ion ion chemistry of high-mass multiply charged

ions. Mass Spectrom. Rev. 1998, 17 (6), 369-407.

4. Oomens, J.; Sartakov, B. G.; Meijer, G.; Von Helden, G., Gas-phase infrared

multiple photon dissociation spectroscopy of mass-selected molecular ions. Int. J. Mass

spectrom. 2006, 254 (1-2), 1-19.

5. Xia, Y.; Ma, X. X.; Yu, K. T., Radical Mass Spectrometry as a New Frontier for

Bioanalysis. Lc Gc N. Am. 2014, 32 (6), 410-+.

6. (a) Beynon, J. H.; Cooks, R. G.; Amy, J. W.; Baitinge.We; Ridley, T. Y.,

DESIGN AND PERFORMANCE OF A MASS ANALYZED ION KINETIC-ENERGY

(MIKE) SPECTROMETER. Anal. Chem. 1973, 45 (12), 1023-&; (b) Kondrat, R. W.;

Cooks, R. G., DIRECT ANALYSIS OF MIXTURES BY MASS-SPECTROMETRY.

Anal. Chem. 1978, 50 (1), A81-&; (c) Wright, L. G.; Schwartz, J. C.; Cooks, R. G.,

HYBRID MASS SPECTROMETERS - VERSATILE RESEARCH INSTRUMENTS.

Trac-Trends in Analytical Chemistry 1986, 5 (9), 236-240.

7. (a) McLafferty, F. W., Tandem Mass-Spectrometry. Science 1981, 214 (4518),

280-287; (b) Wells, J. M.; McLuckey, S. A., Collision-induced dissociation (CID) of

peptides and proteins. In Biol. Mass Spectrom., Elsevier Academic Press Inc: San Diego,

2005; Vol. 402, pp 148-185; (c) Sleno, L.; Volmer, D. A., Ion activation methods for

tandem mass spectrometry. J. Mass Spectrom. 2004, 39 (10), 1091-1112.

8. Stephenson, J. L.; McLuckey, S. A., Ion/ion reactions in the gas phase: Proton

transfer reactions involving multiply-charged proteins. J. Am. Chem. Soc. 1996, 118 (31),

7390-7397.

9. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M.,

Electrospray Ionization for Mass-Spectrometry of Large Biomolecules. Science 1989,

246 (4926), 64-71.

10. Karas, M.; Hillenkamp, F., Laser Desorption Ionization of Proteins with

Molecular Masses Exceeding 10000 Daltons. Anal. Chem. 1988, 60 (20), 2299-2301.

11. Nobelprize, The official website of the Nobel Prize.

http://nobelprize.org/nobel_prizes/chemistry/laureates/2002/ 2002.

12. (a) Hanash, S., Disease proteomics. Nature 2003, 422 (6928), 226-232; (b)

Blackstock, W. P.; Weir, M. P., Proteomics: quantitative and physical mapping of

cellular proteins. Trends Biotechnol. 1999, 17 (3), 121-127.

13. Domon, B.; Aebersold, R., Review - Mass spectrometry and protein analysis.

Science 2006, 312 (5771), 212-217.

41

41

14. (a) Steen, H.; Mann, M., The ABC's (and XYZ's) of peptide sequencing. Nature

Reviews Molecular Cell Biology 2004, 5 (9), 699-711; (b) Konermann, L.; Tong, X.; Pan,

Y., Protein structure and dynamics studied by mass spectrometry: H/D exchange,

hydroxyl radical labeling, and related approaches. J. Mass Spectrom. 2008, 43 (8), 1021-

1036.

15. Harrison, A. G.; Young, A. B.; Bleiholder, C.; Suhai, S.; Paizs, B., Scrambling of

sequence information in collision-induced dissociation of peptides. J. Am. Chem. Soc.

2006, 128 (32), 10364-10365.

16. Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W., Electron capture dissociation

of multiply charged protein cations. A nonergodic process. J. Am. Chem. Soc. 1998, 120

(13), 3265-3266.

17. Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F., Peptide

and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc.

Natl. Acad. Sci. U. S. A. 2004, 101 (26), 9528-9533.

18. (a) Morgan, J. W., Peptide sequencing by MALDI 193-nm photodissociation TOF

MS. In Biol. Mass Spectrom., Elsevier Academic Press Inc: San Diego, 2005; Vol. 402,

pp 186-209; (b) Little, D. P.; Speir, J. P.; Senko, M. W.; Oconnor, P. B.; McLafferty, F.

W., Infrared Multiphoton Dissociation of Large Multiply-Charged Ions for Biomolecule

Sequencing. Anal. Chem. 1994, 66 (18), 2809-2815; (c) Dunbar, R. C., BIRD (blackbody

infrared radiative dissociation): Evolution, principles, and applications. Mass Spectrom.

Rev. 2004, 23 (2), 127-158.

19. Cook, S. L.; Collin, O. L.; Jackson, G. P., Metastable atom-activated dissociation

mass spectrometry: leucine/isoleucine differentiation and ring cleavage of proline

residues. J. Mass Spectrom. 2009, 44 (8), 1211-1223.

20. (a) Kalli, A.; Hakansson, K., Preferential cleavage of S-S and C-S bonds in

electron detachment dissociation and infrared multiphoton dissociation of disulfide-

linked peptide anions. Int. J. Mass spectrom. 2007, 263 (1), 71-81; (b) Coon, J. J.;

Shabanowitz, J.; Hunt, D. F.; Syka, J. E. P., Electron transfer dissociation of peptide

anions. J. Am. Soc. Mass Spectrom. 2005, 16 (6), 880-882; (c) Budnik, B. A.; Haselmann,

K. F.; Zubarev, R. A., Electron detachment dissociation of peptide di-anions: an electron-

hole recombination phenomenon. Chem. Phys. Lett. 2001, 342 (3-4), 299-302.

21. (a) Stadtman, E. R., PROTEIN OXIDATION AND AGING. Science 1992, 257

(5074), 1220-1224; (b) Xu, G.; Chance, M. R., Hydroxyl radical-mediated modification

of proteins as probes for structural proteomics. Chem. Rev. 2007, 107 (8), 3514-3543.

22. (a) Valko, M.; Morris, H.; Cronin, M. T. D., Metals, toxicity and oxidative stress.

Curr. Med. Chem. 2005, 12 (10), 1161-1208; (b) Bjelland, S.; Seeberg, E., Mutagenicity,

toxicity and repair of DNA base damage induced by oxidation. Mutat. Res.-Fundam. Mol.

Mech. Mutag. 2003, 531 (1-2), 37-80; (c) Cooke, M. S.; Evans, M. D.; Dizdaroglu, M.;

Lunec, J., Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J. 2003,

17 (10), 1195-1214.

23. (a) Gutierrez, J.; Ballinger, S. W.; Darley-Usmar, V. M.; Landar, A., Free radicals,

mitochondria, and oxidized lipids - The emerging role in signal transduction in vascular

cells. Circul. Res. 2006, 99 (9), 924-932; (b) Pacher, P.; Beckman, J. S.; Liaudet, L.,

Nitric oxide and peroxynitrite in health and disease. Physiol. Rev. 2007, 87 (1), 315-424.

42

42

24. Lavie, L., Obstructive sleep apnoea syndrome - an oxidative stress disorder. Sleep

Med. Rev. 2003, 7 (1), 35-51.

25. Lin, M. T.; Beal, M. F., Mitochondrial dysfunction and oxidative stress in

neurodegenerative diseases. Nature 2006, 443 (7113), 787-795.

26. Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T. D.; Mazur, M.; Telser, J., Free

radicals and antioxidants in normal physiological functions and human disease. Int. J.

Biochem. Cell Biol. 2007, 39 (1), 44-84.

27. Valko, M.; Rhodes, C. J.; Moncol, J.; Izakovic, M.; Mazur, M., Free radicals,

metals and antioxidants in oxidative stress-induced cancer. Chem.-Biol. Interact. 2006,

160 (1), 1-40.

28. (a) Funk, C. D., Prostaglandins and leukotrienes: Advances in eicosanoid biology.

Science 2001, 294 (5548), 1871-1875; (b) Licht, S.; Gerfen, G. J.; Stubbe, J. A., Thiyl

radicals in ribonucleotide reductases. Science 1996, 271 (5248), 477-481; (c) Kolberg, M.;

Strand, K. R.; Graff, P.; Andersson, K. K., Structure, function, and mechanism of

ribonucleotide reductases. BBA-Proteins Proteomics 2004, 1699 (1-2), 1-34.

29. (a) Drewitz, H. J.; Taubert, R., DETERMINATION OF RELATIVE

IONIZATION CROSS-SECTIONS AND REACTION CONSTANTS OF ION-

MOLECULE REACTIONS IN INITIAL ENERGY DISCRIMINATION .1. FORMAL

DESCRIPTION OF INITIAL ENERGY DISCRIMINATION OF THERMAL IONS IN

MAGNETIC-SECTOR FIELD MASS SPECTROMETERS. Int. J. Mass Spectrom. Ion

Processes 1976, 19 (3), 293-312; (b) Yost, R. A.; Boyd, R. K., TANDEM MASS-

SPECTROMETRY - QUADRUPOLE AND HYBRID INSTRUMENTS. Methods

Enzymol. 1990, 193, 154-200.

30. Yost, R. A.; Enke, C. G., TRIPLE QUADRUPOLE MASS-SPECTROMETRY

FOR DIRECT MIXTURE ANALYSIS AND STRUCTURE ELUCIDATION. Anal.

Chem. 1979, 51 (12), 1251-&.

31. Wiley, W. C.; McLaren, I. H., TIME-OF-FLIGHT MASS SPECTROMETER

WITH IMPROVED RESOLUTION. Rev. Sci. Instrum. 1955, 26 (12), 1150-1157.

32. Douglas, D. J.; Frank, A. J.; Mao, D. M., Linear ion traps in mass spectrometry.

Mass Spectrom. Rev. 2005, 24 (1), 1-29.

33. Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S., Fourier transform ion

cyclotron resonance mass spectrometry: A primer. Mass Spectrom. Rev. 1998, 17 (1), 1-

35.

34. Korner, R.; Wilm, M.; Morand, K.; Schubert, M.; Mann, M., Nano electrospray

combined with a quadrupole ion trap for the analysis of peptides and protein digests. J.

Am. Soc. Mass Spectrom. 1996, 7 (2), 150-156.

35. Ma, X. X.; Love, C. B.; Zhang, X. R.; Xia, Y., Gas-Phase Fragmentation of [M +

nH + OH](n center dot+) Ions Formed from Peptides Containing Intra-Molecular

Disulfide Bonds. J. Am. Soc. Mass Spectrom. 2011, 22 (5), 922-930.

36. Stinson, C. A.; Xia, Y., Radical induced disulfide bond cleavage within peptides

via ultraviolet irradiation of an electrospray plume. Analyst 2013, 138 (10), 2840-2846.

37. Xia, Y.; Liang, X. R.; McLuckey, S. A., Ion trap versus low-energy beam-type

collision-induced dissociation of protonated ubiquitin ions. Anal. Chem. 2006, 78 (4),

1218-1227.

43

43

38. Thornton, J. M., DISULFIDE BRIDGES IN GLOBULAR-PROTEINS. J. Mol.

Biol. 1981, 151 (2), 261-287.

39. (a) Gorman, J. J.; Wallis, T. P.; Pitt, J. J., Protein disulfide bond determination by

mass spectrometry. Mass Spectrom. Rev. 2002, 21 (3), 183-216; (b) Lioe, H.; Duan, M.;

O'Hair, R. A. J., Can metal ions be used as gas-phase disulfide bond cleavage reagents?

A survey of coinage metal complexes of model peptides containing an intermolecular

disulfide bond. Rapid Commun. Mass Spectrom. 2007, 21 (16), 2727-2733.

40. (a) Ryzhov, V.; Lam, A. K. Y.; O'Hair, R. A. J., Gas-Phase Fragmentation of

Long-Lived Cysteine Radical Cations Formed Via NO Loss from Protonated S-

Nitrosocysteine. J. Am. Soc. Mass Spectrom. 2009, 20 (6), 985-995; (b) Hao, G.; Gross, S.

S., Electrospray tandem mass spectrometry analysis of S- and N-nitrosopeptides: Facile

loss of NO and radical-induced fragmentation. J. Am. Soc. Mass Spectrom. 2006, 17 (12),

1725-1730.

41. (a) Zhang, L. Y.; Reilly, J. P., Radical-Driven Dissociation of Odd-Electron

Peptide Radical Ions Produced in 157 nm Photodissociation. J. Am. Soc. Mass Spectrom.

2009, 20 (7), 1378-1390; (b) Sun, Q. Y.; Nelson, H.; Ly, T.; Stoltz, B. M.; Julian, R. R.,

Side Chain Chemistry Mediates Backbone Fragmentation in Hydrogen Deficient Peptide

Radicals. J. Proteome Res. 2009, 8 (2), 958-966.

42. Tan, L.; Xia, Y., Gas-Phase Reactivity of Peptide Thiyl (RS center dot),Perthiyl

(RSS center dot), and Sulfinyl (RSO center dot) Radical Ions Formed from Atmospheric

Pressure Ion/Radical Reactions. J. Am. Soc. Mass Spectrom. 2013, 24 (4), 534-542.

43. Durand, K. L.; Ma, X. X.; Xia, Y., Intra-molecular reactions as a new approach to

investigate bio-radical reactivity: a case study of cysteine sulfinyl radicals. Analyst 2014,

139 (6), 1327-1330.

44. Durand, K. L.; Ma, X.; Xia, Y., Intra-molecular reactions between cysteine

sulfinyl radical and a disulfide bond within peptide ions. Int. J. Mass spectrom. (In press).

45. Wiesner, J.; Premsler, T.; Sickmann, A., Application of electron transfer

dissociation (ETD) for the analysis of posttranslational modifications. Proteomics 2008, 8

(21), 4466-4483.

46. Piomelli, D.; Astarita, G.; Rapaka, R., A neuroscientist's guide to lipidomics.

Nature Reviews Neuroscience 2007, 8 (10), 743-754.

47. Dill, A. L.; Ifa, D. R.; Manicke, N. E.; Costa, A. B.; Ramos-Vara, J. A.; Knapp, D.

W.; Cooks, R. G., Lipid Profiles of Canine Invasive Transitional Cell Carcinoma of the

Urinary Bladder and Adjacent Normal Tissue by Desorption Electrospray Ionization

Imaging Mass Spectrometry. Anal. Chem. 2009, 81 (21), 8758-8764.

48. Eberlin, L. S.; Norton, I.; Dill, A. L.; Golby, A. J.; Ligon, K. L.; Santagata, S.;

Cooks, R. G.; Agar, N. Y. R., Classifying Human Brain Tumors by Lipid Imaging with

Mass Spectrometry. Cancer Res. 2012, 72 (3), 645-654.

49. Martinez-Seara, H.; Rog, T.; Pasenkiewicz-Gierula, M.; Vattulainen, I.; Karttunen,

M.; Reigada, R., Interplay of unsaturated phospholipids and cholesterol in membranes:

Effect of the double-bond position. Biophys. J. 2008, 95 (7), 3295-3305.

50. Marti, I.; Rubio, J.; Bolte, M.; Burguete, M. I.; Vicent, C.; Quesada, R.; Alfonso,

I.; Luis, S. V., Tuning Chloride Binding, Encapsulation, and Transport by Peripheral

Substitution of Pseudopeptidic Tripodal Small Cages. Chem.-Eur. J. 2012, 18 (52),

16728-16741.

44

44

51. Pham, H. T.; Ly, T.; Trevitt, A. J.; Mitchell, T. W.; Blanksby, S. J.,

Differentiation of Complex Lipid Isomers by Radical-Directed Dissociation Mass

Spectrometry. Anal. Chem. 2012, 84 (17), 7525-7532.

52. Ma, X. X.; Xia, Y., Pinpointing Double Bonds in Lipids by Paterno-Buchi

Reactions and Mass Spectrometry. Angew. Chem.-Int. Edit. 2014, 53 (10), 2592-2596.

53. Cooks, R. G.; Ifa, D. R.; Sharma, G.; Tadjimukhamedov, F. K.; Ouyang, Z.,

Perspectives and retrospectives in mass spectrometry: one view. Eur. J. Mass Spectrom.

2010, 16 (3), 283-300.

54. Basir, Y. J.; Christian, J. F.; Wan, Z. M.; Anderson, S. L., A triple sector, guided-

ion-beam mass spectrometer for cluster ion and fullerene scattering. Int. J. Mass

spectrom. 1997, 171 (1-3), 159-172.

55. Armentrout, P. B., Mass spectrometry - Not just a structural tool: The use of

guided ion beam tandem mass spectrometry to determine thermochemistry. J. Am. Soc.

Mass Spectrom. 2002, 13 (5), 419-434.

56. Medzihradszky, K. F.; Campbell, J. M.; Baldwin, M. A.; Falick, A. M.; Juhasz, P.;

Vestal, M. L.; Burlingame, A. L., The characteristics of peptide collision-induced

dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer.

Anal. Chem. 2000, 72 (3), 552-558.

57. Suckau, D.; Resemann, A.; Schuerenberg, M.; Hufnagel, P.; Franzen, J.; Holle, A.,

A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Anal. Bioanal.

Chem. 2003, 376 (7), 952-965.

58. (a) Hopfgartner, G.; Varesio, E.; Tschappat, V.; Grivet, C.; Bourgogne, E.;

Leuthold, L. A., Triple quadrupole linear ion trap mass spectrometer for the analysis of

small molecules and macromolecules. J. Mass Spectrom. 2004, 39 (8), 845-855; (b)

Lange, V.; Picotti, P.; Domon, B.; Aebersold, R., Selected reaction monitoring for

quantitative proteomics: a tutorial. Mol. Syst. Biol. 2008, 4.

59. Schwartz, J. C.; Schey, K. L.; Cooks, R. G., A PENTA-QUADRUPOLE

INSTRUMENT FOR REACTION INTERMEDIATE SCANS AND OTHER MS-MS-

MS EXPERIMENTS. Int. J. Mass Spectrom. Ion Processes 1990, 101 (1), 1-20.

60. Eberlin, M. N., Triple-stage pentaquadrupole (QqQqQ) mass spectrometry and

ion/molecule reactions. Mass Spectrom. Rev. 1997, 16 (3), 113-144.

61. Schwartz, J. C.; Wade, A. P.; Enke, C. G.; Cooks, R. G., SYSTEMATIC

DELINEATION OF SCAN MODES IN MULTIDIMENSIONAL MASS-

SPECTROMETRY. Anal. Chem. 1990, 62 (17), 1809-1818.

62. Juliano, V. F.; Gozzo, F. C.; Eberlin, M. N.; Kascheres, C.; doLago, C. L., Fast

multidimensional (3D and 4D) MS(2) and MS(3) scans in a high-transmission

pentaquadrupole mass spectrometer. Anal. Chem. 1996, 68 (8), 1328-1334.

63. Eberlin, M. N.; Majumdar, T. K.; Cooks, R. G., STRUCTURES AND

MECHANISMS OF REACTIONS OF ISOMERIC C2H3O+ AND C2H3S+ IONS

REVEALED THROUGH ION MOLECULE REACTIONS IN CONJUNCTION WITH

2D AND 3D MASS-SPECTROMETRY. J. Am. Chem. Soc. 1992, 114 (8), 2884-2896.

64. Adams, N. G.; Smith, D., SELECTED ION FLOW TUBE (SIFT) - TECHNIQUE

FOR STUDYING ION-NEUTRAL REACTIONS. Int. J. Mass Spectrom. Ion Processes

1976, 21 (3-4), 349-359.

45

45

65. (a) Smith, D.; Spanel, P., Selected ion flow tube mass spectrometry (SIFT-MS)

for on-line trace gas analysis. Mass Spectrom. Rev. 2005, 24 (5), 661-700; (b) Smith, D.;

Spanel, P., Ambient analysis of trace compounds in gaseous media by SIFT-MS. Analyst

2011, 136 (10), 2009-2032.

66. Spanel, P.; Smith, D., PROGRESS IN SIFT-MS: BREATH ANALYSIS AND

OTHER APPLICATIONS. Mass Spectrom. Rev. 2011, 30 (2), 236-267.

67. Amelynck, C.; Schoon, N.; Dhooghe, F., SIFT Ion Chemistry Studies

Underpinning the Measurement of Volatile Organic Compound Emissions by Vegetation.

Current Analytical Chemistry 2013, 9 (4), 540-549.

68. (a) Petrie, S.; Bohme, D. K., Mass spectrometric approaches to interstellar

chemistry. In Modern Mass Spectrometry, Schalley, C. A., Ed. 2003; Vol. 225, pp 37-75;

(b) Snow, T. P.; Bierbaum, V. M., Ion Chemistry in the Interstellar Medium. In Annual

Review of Analytical Chemistry, 2008; Vol. 1, pp 229-259.

69. Zhang, X.; Kato, S.; Bierbaum, V. M.; Nimlos, M. R.; Ellison, G. B., Use of a

flowing afterglow SIFT apparatus to study the reactions of ions with organic radicals. J.

Phys. Chem. A 2004, 108 (45), 9733-9741.

70. Kohn, D. W.; Clauberg, H.; Chen, P., FLASH PYROLYSIS NOZZLE FOR

GENERATION OF RADICALS IN A SUPERSONIC JET EXPANSION. Rev. Sci.

Instrum. 1992, 63 (8), 4003-4005.

71. Zhang, X.; Friderichsen, A. V.; Nandi, S.; Ellison, G. B.; David, D. E.; McKinnon,

J. T.; Lindeman, T. G.; Dayton, D. C.; Nimlos, M. R., Intense, hyperthermal source of

organic radicals for matrix-isolation spectroscopy. Rev. Sci. Instrum. 2003, 74 (6), 3077-

3086.

72. March, R. E., Quadrupole ion trap mass spectrometry: a view at the turn of the

century. Int. J. Mass spectrom. 2000, 200 (1-3), 285-312.

73. Anderson, J. S.; Vartanian, V. H.; Laude, D. A., EVOLUTION OF TRAPPED

ION CELLS IN FOURIER-TRANSFORM ION-CYCLOTRON RESONANCE MASS-

SPECTROMETRY. Trac-Trends in Analytical Chemistry 1994, 13 (6), 234-239.

74. Schrader, W.; Klein, H. W., Liquid chromatography Fourier transform ion

cyclotron resonance mass spectrometry (LC-FTICR MS): an early overview. Anal.

Bioanal. Chem. 2004, 379 (7-8), 1013-1024.

75. Russell, A. L.; Rohrs, H. W.; Read, D.; Giblin, D. E.; Gaspar, P. P.; Gross, M. L.,

Radical cation/radical reactions: A Fourier transform ion cyclotron resonance study of

allyl radical reacting with aromatic radical cations. Int. J. Mass Spectrom. 2009, 287 (1-3),

8-15.

76. Demirev, P. A., Generation of hydrogen radicals for reactivity studies in Fourier

transform ion cyclotron resonance mass spectrometry. Rapid Commun. Mass Spectrom.

2000, 14 (9), 777-781.

77. Fu, M. K.; Li, S.; Archibold, E.; Yurkovich, M. J.; Nash, J. J.; Kenttamaa, H. T.,

Reactions of an Aromatic sigma,sigma-Biradical with Amino Acids and Dipeptides in the

Gas Phase. J. Am. Soc. Mass Spectrom. 2010, 21 (10), 1737-1752.

78. Campbell, J. L.; Crawford, K. E.; Kenttamaa, H. I., Analysis of saturated

hydrocarbons by using chemical ionization combined with laser-induced acoustic

desorption/Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem.

2004, 76 (4), 959-963.

46

46

79. Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.;

Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W., Electron capture dissociation for

structural characterization of multiply charged protein cations. Anal. Chem. 2000, 72 (3),

563-573.

80. Horn, D. M.; Ge, Y.; McLafferty, F. W., Activated ion electron capture

dissociation for mass spectral sequencing of larger (42 kDa) proteins. Anal. Chem. 2000,

72 (20), 4778-4784.

81. (a) Turecek, F., N-C(alpha) bond dissociation energies and kinetics in amide and

peptide radicals. Is the dissociation a non-ergodic process? J. Am. Chem. Soc. 2003, 125

(19), 5954-5963; (b) Turecek, F.; Syrstad, E. A., Mechanism and energetics of

intramolecular hydrogen transfer in amide and peptide radicals and cation-radicals. J. Am.

Chem. Soc. 2003, 125 (11), 3353-3369.

82. Syrstad, E. A.; Stephens, D. D.; Turecek, F., Hydrogen atom adducts to the amide

bond. Generation and energetics of amide radicals in the gas phase. J. Phys. Chem. A

2003, 107 (1), 115-126.

83. Zubarev, R. A.; Haselmann, K. F.; Budnik, B.; Kjeldsen, F.; Jensen, F., Towards

an understanding of the mechanism of electron-capture dissociation: a historical

perspective and modern ideas. Eur. J. Mass Spectrom. 2002, 8 (5), 337-349.

84. Moore, B. N.; Ly, T.; Julian, R. R., Radical Conversion and Migration in Electron

Capture Dissociation. J. Am. Chem. Soc. 2011, 133 (18), 6997-7006.

85. Cooper, H. J.; Hudgins, R. R.; Hakansson, K.; Marshall, A. G., Secondary

fragmentation of linear peptides in electron capture dissociation. Int. J. Mass spectrom.

2003, 228 (2-3), 723-728.

86. (a) Strupat, K.; Zeller, M.; Delanghe, B.; Metelmann-Strupat, W.; Muenster, H.,

Analysis of post-translationally modified peptides and proteins by high resolution

electron capture dissociation (ECD) FTICR-MS. Mol. Cell. Proteomics 2005, 4 (8),

S310-S310; (b) Cooper, H. J.; Hudgins, R. R.; Marshall, A. G., Electron capture

dissociation fourier transform ion cyclotron resonance mass spectrometry of

cyclodepsipeptides, branched peptides, and epsilon-peptides. Int. J. Mass spectrom. 2004,

234 (1-3), 23-35.

87. Ge, Y.; Lawhorn, B. G.; ElNaggar, M.; Strauss, E.; Park, J. H.; Begley, T. P.;

McLafferty, F. W., Top down characterization of larger proteins (45 kDa) by electron

capture dissociation mass spectrometry. J. Am. Chem. Soc. 2002, 124 (4), 672-678.

47

47

88. (a) Crane, R.; Gadea, B.; Littlepage, L.; Wu, H.; Ruderman, J. V., Aurora A,

meiosis and mitosis. Biol. Cell 2004, 96 (3), 215-229; (b) Cieniewski-Bernard, C.; Acosta,

A.; Dubois, E.; Lamblin, N.; Beseme, O.; Chwastyniak, M.; Amouyel, P.; Bauters, C.;

Pinet, F., Proteomic analysis in cardiovascular diseases. Clin. Exp. Pharmacol. Physiol.

2008, 35 (4), 362-366; (c) Dias, W. B.; Hart, G. W., O-GlcNAc modification in diabetes

and Alzheimer's disease. Molecular Biosystems 2007, 3 (11), 766-772; (d) Kelleher, N. L.;

Zubarev, R. A.; Bush, K.; Furie, B.; Furie, B. C.; McLafferty, F. W.; Walsh, C. T.,

Localization of labile posttranslational modifications by electron capture dissociation: the

case of gamma-carboxyglutamic acid. Anal. Chem. 1999, 71 (19), 4250-3; (e) Shi, S. D.

H.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W.,

Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass

spectrometry. Anal. Chem. 2001, 73 (1), 19-22; (f) Hakansson, K.; Cooper, H. J.; Emmett,

M. R.; Costello, C. E.; Marshall, A. G.; Nilsson, C. L., Electron capture dissociation and

infrared multiphoton dissociation MS/MS of an N-glycosylated tryptic peptide to yield

complementary sequence information. Anal. Chem. 2001, 73 (18), 4530-4536; (g) Guan,

Z. Q.; Yates, N. A.; Bakhtiar, R., Detection and characterization of methionine oxidation

in peptides by collision-induced dissociation and electron capture dissociation. J. Am. Soc.

Mass Spectrom. 2003, 14 (6), 605-613.

89. Hakansson, K.; Chalmers, M. J.; Quinn, J. P.; McFarland, M. A.; Hendrickson, C.

L.; Marshall, A. G., Combined electron capture and infrared multiphoton dissociation for

multistage MS/MS in a Fourier transform ion cyclotron resonance mass spectrometer.

Anal. Chem. 2003, 75 (13), 3256-3262.

90. Jarrell, T.; Riedeman, J.; Carlsen, M.; Replogle, R.; Selby, T.; Kenttamaa, H.,

Multiported Pulsed Valve Interface for a Linear Quadrupole Ion Trap Mass Spectrometer

to Enable Rapid Screening of Multiple Functional-Group Selective Ion-Molecule

Reactions. Anal. Chem. 2014, 86 (13), 6533-6539.

91. Owen, B. C.; Jarrell, T. M.; Schwartz, J. C.; Oglesbee, R.; Carlsen, M.; Archibold,

E. F.; Kenttamaa, H. I., A Differentially Pumped Dual Linear Quadrupole Ion Trap

(DLQIT) Mass Spectrometer: A Mass Spectrometer Capable of MSn Experiments Free

From Interfering Reactions. Anal. Chem. 2013, 85 (23), 11284-11290.

92. Xia, Y.; McLuckey, S. A., Evolution of instrumentation for the study of gas-phase

ion/ion chemistry via mass spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19 (2), 173-

189.

93. Udeshi, N. D.; Shabanowitz, J.; Hunt, D. F.; Rose, K. L., Analysis of proteins and

peptides on a chromatographic timescale by electron-transfer dissociation MS. FEBS J.

2007, 274 (24), 6269-6276.

94. Mikesh, L. M.; Ueberheide, B.; Chi, A.; Coon, J. J.; Syka, J. E. P.; Shabanowitz,

J.; Hunt, D. F., The utility of ETD mass spectrometry in proteomic analysis. BBA-

Proteins Proteomics 2006, 1764 (12), 1811-1822.

95. Gunawardena, H. P.; He, M.; Chrisman, P. A.; Pitteri, S. J.; Hogan, J. M.; Hodges,

B. D. M.; McLuckey, S. A., Electron transfer versus proton transfer in gas-phase ion/ion

reactions of polyprotonated peptides. J. Am. Chem. Soc. 2005, 127 (36), 12627-12639.

48

48

96. (a) Swaney, D. L.; McAlister, G. C.; Wirtala, M.; Schwartz, J. C.; Syka, J. E. P.;

Coon, J. J., Supplemental activation method for high-efficiency electron-transfer

dissociation of doubly protonated peptide precursors. Anal. Chem. 2007, 79 (2), 477-485;

(b) Wu, S.-L.; Huehmer, A. F. R.; Hao, Z.; Karger, B. L., On-line LC-MS approach

combining collision-induced dissociation (CID), electron-transfer dissociation (ETD),

and CID of an isolated charge-reduced species for the trace-level characterization of

proteins with post-translational modifications. J. Proteome Res. 2007, 6 (11), 4230-4244.

97. Stephenson, J. L.; McLuckey, S. A., Adaptation of the Paul Trap for study of the

reaction of multiply charged cations with singly charged anions. Int. J. Mass Spectrom.

Ion Processes 1997, 162 (1-3), 89-106.

98. (a) Wells, J. M.; Chrisman, P. A.; McLuckey, S. A., "Dueling" ESI:

Instrumentation to study ion/ion reactions of electrospray-generated cations and anions. J.

Am. Soc. Mass Spectrom. 2002, 13 (6), 614-622; (b) Badman, E. R.; Chrisman, P. A.;

McLuckey, S. A., A quadrupole ion trap mass spectrometer with three independent ion

sources for the study of gas-phase ion/ion reactions. Anal. Chem. 2002, 74 (24), 6237-

6243.

99. Yu, X.; Jin, W.; McLuckey, S. A.; Londry, F. A.; Hager, J. W., Mutual storage

mode ion/ion reactions in a hybrid linear ion trap. J. Am. Soc. Mass Spectrom. 2005, 16

(1), 71-81.

100. Wu, J.; Hager, J. W.; Xia, Y.; Londry, F. A.; McLuckey, S. A., Positive ion

transmission mode ion/ion reactions in a hybrid linear ion trap. Anal. Chem. 2004, 76

(17), 5006-5015.

101. (a) Xia, Y.; Liang, X. R.; McLuckey, S. A., Pulsed dual electrospray ionization

for ion/ion reactions. J. Am. Soc. Mass Spectrom. 2005, 16 (11), 1750-1756; (b) Liang, X.

R.; Xia, Y.; McLuckey, S. A., Alternately pulsed nanoelectrospray

ionization/atmospheric pressure chemical ionization for ion/ion reactions in an

electrodynamic ion trap. Anal. Chem. 2006, 78 (9), 3208-3212; (c) Huang, T. Y.; Emory,

J. F.; O'Hair, R. A. J.; McLuckey, S. A., Electron-transfer reagent anion formation via

electrospray ionization and collision-induced dissociation. Anal. Chem. 2006, 78 (21),

7387-7391.

102. (a) Brodbelt, J. S., Photodissociation mass spectrometry: new tools for

characterization of biological molecules. Chem. Soc. Rev. 2014, 43 (8), 2757-2783; (b)

Crowe, M. C.; Brodbelt, J. S., Infrared multiphoton dissociation (IRMPD) and

collisionally activated dissociation of peptides in a quadrupole ion trap with selective

IRMPD of phosphopeptides. J. Am. Soc. Mass Spectrom. 2004, 15 (11), 1581-1592.

103. Madsen, J. A.; Boutz, D. R.; Brodbelt, J. S., Ultrafast Ultraviolet

Photodissociation at 193 nm and its Applicability to Proteomic Workflows. J. Proteome

Res. 2010, 9 (8), 4205-4214.

104. Xia, Y.; Chrisman, P. A.; Erickson, D. E.; Liu, J.; Liang, X. R.; Londry, F. A.;

Yang, M. J.; McLuckey, S. A., Implementation of ion/ion reactions in a quadrupole/time-

of-flight tandem mass spectrometer. Anal. Chem. 2006, 78 (12), 4146-4154.

105. Makarov, A., Electrostatic axially harmonic orbital trapping: A high-performance

technique of mass analysis. Anal. Chem. 2000, 72 (6), 1156-1162.

49

49

106. (a) Hu, Q. Z.; Noll, R. J.; Li, H. Y.; Makarov, A.; Hardman, M.; Cooks, R. G.,

The Orbitrap: a new mass spectrometer. J. Mass Spectrom. 2005, 40 (4), 430-443; (b)

Makarov, A.; Denisov, E.; Kholomeev, A.; Baischun, W.; Lange, O.; Strupat, K.;

Horning, S., Performance evaluation of a hybrid linear ion trap/orbitrap mass

spectrometer. Anal. Chem. 2006, 78 (7), 2113-2120.

107. (a) Michalski, A.; Damoc, E.; Lange, O.; Denisov, E.; Nolting, D.; Muller, M.;

Viner, R.; Schwartz, J.; Remes, P.; Belford, M.; Dunyach, J. J.; Cox, J.; Horning, S.;

Mann, M.; Makarov, A., Ultra High Resolution Linear Ion Trap Orbitrap Mass

Spectrometer (Orbitrap Elite) Facilitates Top Down LC MS/MS and Versatile Peptide

Fragmentation Modes. Mol. Cell. Proteomics 2012, 11 (3); (b) Denisov, E.; Damoc, E.;

Lange, O.; Makarov, A., Orbitrap mass spectrometry with resolving powers above

1,000,000. Int. J. Mass spectrom. 2012, 325, 80-85.

108. Olsen, J. V.; Schwartz, J. C.; Griep-Raming, J.; Nielsen, M. L.; Damoc, E.;

Denisov, E.; Lange, O.; Remes, P.; Taylor, D.; Splendore, M.; Wouters, E. R.; Senko, M.;

Makarov, A.; Mann, M.; Horning, S., A Dual Pressure Linear Ion Trap Orbitrap

Instrument with Very High Sequencing Speed. Mol. Cell. Proteomics 2009, 8 (12), 2759-

2769.

109. Olsen, J. V.; Macek, B.; Lange, O.; Makarov, A.; Horning, S.; Mann, M., Higher-

energy C-trap dissociation for peptide modification analysis. Nat. Methods 2007, 4 (9),

709-712.

110. Gao, L.; Cooks, R. G.; Ouyang, Z., Breaking the pumping speed barrier in mass

spectrometry: Discontinuous atmospheric pressure interface. Anal. Chem. 2008, 80 (11),

4026-4032.

111. Ouyang, Z.; Wu, G. X.; Song, Y. S.; Li, H. Y.; Plass, W. R.; Cooks, R. G.,

Rectilinear ion trap: Concepts, calculations, and analytical performance of a new mass

analyzer. Anal. Chem. 2004, 76 (16), 4595-4605.

112. Ouyang, Z.; Cooks, R. G., Miniature Mass Spectrometers. Annual Review of

Analytical Chemistry 2009, 2, 187-214.

113. (a) Gao, L.; Sugiarto, A.; Harper, J. D.; Cooks, R. G.; Ouyang, Z., Design and

characterization of a multisource hand-held tandem mass spectrometer. Anal. Chem. 2008,

80 (19), 7198-205; (b) Li, L. F.; Chen, T. C.; Ren, Y.; Hendricks, P. I.; Cooks, R. G.;

Ouyang, Z., Mini 12, Miniature Mass Spectrometer for Clinical and Other Applications-

Introduction and Characterization. Anal. Chem. 2014, 86 (6), 2909-2916.

114. Xu, W.; Charipar, N.; Kirleis, M. A.; Xia, Y.; Ouyang, Z., Study of Discontinuous

Atmospheric Pressure Interfaces for Mass Spectrometry Instrumentation Development.

Anal. Chem. 2010, 82 (15), 6584-6592.

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CHAPTER 2. INSTRUMENTATION AND CHARACTERIZATION OF A HOME-

BUILT DAPI-RIT-DAPI MASS SPECTROMETER FOR GAS-PHASE

ION/MOLECULE AND ION/ION REACTIONS

2.1 Introduction

Mass spectrometry (MS) has been widely used to study gas-phase ion chemistry via

inducing interactions or reactions between ions and matter in vacuo, such as neutrals,1

radicals,2 electrons,1c, 3 ions,4 photons,5 surfaces,6 metastable species,7 etc. Besides being

used as a mass analyzer, a quadruple ion trap can serve as a reactor for studying gas-

phase reactions with capabilities of manipulating the trapped ions in a controlled-time

fashion at relatively high pressure for reactions. Owing to the development of MS

systems with atmospheric pressure ionization sources such as electrospray ionization

(ESI)8 and atmospheric pressure chemical ionization (APCI)9, ions of intact molecules

can be generated directly from condensed phase at atmospheric pressure. The

atmospheric pressure interface (API) is an essential component in these mass

spectrometers, through which the ions are transferred from atmosphere to the mass

analyzer in the vacuum.

The APIs adopted in commercial mass spectrometers typically perform at

continuous ionization and ion introduction mode. The continuous APIs require a

differential pumping system with a large pumping capacity to support multiple vacuum

regions at different pressures. Delicate ion optics is used to transfer the ions through

different regions.10 Instrumentational modification is always needed to conduct gas-

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phase reactions on a mass spectrometer consisting of a single API. For instance, neutral

reactant needs to be leaked into the vacuum for ion/molecule reactions,11 which is

practically done for quadrupole ion trap or filter instruments through the collision gas

introduction line. To implement ion/ion reactions, a different ionization interface is

typically required to introduce an ion beam of the other charge polarity,12 which can

demand significant efforts in the instrument modification. An alternative solution is to

use a discontinuous atmospheric pressure interface (DAPI) for introduction of ions or

neutrals, which was initially developed for miniature mass spectrometers and has been

demonstrated to be effective with small pumping systems.13 In a DAPI, a pinch valve is

used to control the opening of the interface (10 to 30 ms) and the ions are transferred

from the atmosphere directly to the ion trap mass analyzer in the vacuum. During the ion

introduction period, the pressure inside the vacuum manifold increases to 10-2 to 10-1 Torr.

The ions are trapped at the elevated pressure and mass analyzed after a delay of several

hundred milliseconds to allow the pressure dropping back to 10-3 Torr.13b DAPI can be

implemented even with bent capillaries without losing ion intensity significantly,14 which

provides flexibility for the instrument modification. It has been demonstrated in a

previous study15 that multiple beams of ions or neutrals can be introduced into a

rectilinear ion trap (RIT)16 sequentially or simultaneously through two DAPIs for gas

phase processes including collisional induced dissociation, gas flow assisted desolvation,

and ion/molecule or ion/ion reactions.

In this study, we adopted the simple DAPI-RIT-DAPI configuration previously

proposed15 to set up an instrument that could potentially be used for gas-phase ion

reactions with multiple beams of ions or neutrals conveniently introduced through the

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two DAPIs. A distinct feature of the MS systems with DAPI is that the in-vacuum

pressure varies during a scan cycle and the ions trapped in vacuum experience stronger

and more energetic gas flows. This represents a unique opportunity for using a single trap

to carry out gas-phase reactions at a condition different from MS analysis of the reaction

products in a single scan cycle. The pressure changes and gas flow impact can be critical

to gas-phase reactions and need to be characterized for studying complicated reactions.

Herein, the gas dynamic effects on ion internal energy and ion reactivity as well as the

pressure effect on the reaction rate have been investigated. Two types of ion reactions

were used to test this system, including ion/molecule and ion/ion reactions. Ion/ion

reactions have been widely applied in proteomics1c and they have been made available on

several types of commercial instruments. We are interested to test the performance of our

instrument with extremely simplified ion optics for ion/ion reaction applications.

Ion/molecule reactions, on the other hand, offer a great means for studying gas-phase ion

structures and energetics. Many research labs have modified their instruments to facilitate

this type of fundamental studies. In this work, we chose ion/molecule reactions involving

peptide radical ions because the structures and reactivity of radical ions are very sensitive

to instrument conditions, e.g. energy deposition associated with gas collisions during the

ion transfer and storage.17

2.2 Instrumentation

The home-built DAPI-RIT-DAPI mass spectrometer was developed using the

vacuum chamber with inside dimensions of 35 × 25 × 25 cm. The manifold was pumped

by a 210 L/s turbo pump (THH 262 P, Pfeiffer Vacuum Inc, New Hampshire) and a 30

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m3/h rotary vane pump (UNO-030M, Pfeiffer Vacuum Inc., New Hampshire). A nanoESI

emitter consisting of a tip-pulled (i.d. < 20 m) boron silica glass capillary (0.85 mm i.d.

and 1.5 mm o.d.) was used to generate ions in front of DAPI-1 (the left DAPI in Figure

2.1a). Other reagents were introduced by the gas flow through DAPI-2. A pinch valve

(ASCO 390NC24330, ASCO Scientific, Florham Park, NJ) with a semi-conductive

silicone tubing (3 cm long, ∼350 Ω resistance, with 1.6 mm i.d. and 3.2 mm o.d.) was used

for each DAPI. Capillary 1 (10 cm long and 0.5 mm i.d.) was attached to DAPI-1;

Capillary 2 (25 cm long and 0.75 mm i.d.) and Capillary 3 (5 cm long with various i.d.s

tested) were attached to DAPI-2. All the capillaries were grounded. The DAPI-1 and 2

were controlled separately each by a 0-24 V pulsed dc signal from the control electronics.

An RIT with dimensions x0 = 5 mm, y0 = 4 mm, and z = 40 mm was operated with a

single-phase radiofrequency (rf) at 850 kHz for ion storage and MS analysis. The end

electrodes were made from electrogalvanized steel mesh electrodes (0.009” Wire,

McMaster-Carr, Aurora, OH) approximately 2 mm away from the capillaries of each

DAPI to allow ion/neutral injection. A previous study with simulations18 has shown that a

mesh electrode, in comparison with a plate electrode with a center hole, can minimize the

disturbance to the trapped ions by the gas flow from the DAPI while allowing high

throughput of the ions. A set of control electronics, from a Griffin Minotaur 300 (FLIR

Systems, Inc., Wilsonville, OR), was used for the instrument control. A 30 V dc was

applied on both end electrodes to create a dc trapping potential well along the z direction.

Resonance ejection was implemented for MS scan with a dipolar ac signal at 300 kHz (q

= 0.81). The conversion dynode of the detector was set to 0 V for positive ion mode and

+4000 V for negative ion mode. The isolation of the trapped ions was achieved by a

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notched SWIFT (stored waveform inverse Fourier transform). Collision-induced

dissociation (CID) was implemented by dipolar excitation with a single-frequency ac

applied between the x electrodes of the RIT. The typical nanoESI spray voltage was set to

+/-1600 V in positive/negative ionization mode. Multiple triggers were provided from the

control electronics for synchronizing the operations of nanoESI, DAPIs, and MS analysis.

All the spectra presented are the average of three continuous scans.

2.3 Materials

S-nitrosoglutathione (γ-E(NOC)G), dimethyl disulfide, allyl iodide, angiotensin I

(single letter sequence: DRVYIHPFHL), pentachlorophenol (PCP), 9-anthracene

carboxylic acid, cocaine, and benzylpyridinium thermometer ions were purchased from

Sigma Chemical Co. (Sigma-Aldrich, St. Louis, MO). Different concentrations (5-100

μM) of the samples were prepared with methanol/water (1:1, v/v) with 0.2 % acetic acid

added to increase the intensity of protonated species if necessary.

2.4 Results and discussion

2.4.1 System configuration

The DAPI-RIT-DAPI system (Figure 2.1a) consists of two DAPIs, one RIT, and one

detector. The general scan function for gas-phase reactions shown in Figure 2.1b was set

as following. DAPI-1 was opened for 10-30 ms to introduce ions into the RIT; the ions of

interest for reaction were isolated using SWIFT, and CID was performed and followed by

another isolation if a fragment ion was used for reactions; the second DAPI, DAPI-2 was

opened to introduce other reagents into the RIT, which can be neutral molecules for

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ion/molecule reactions or ions for ion/ion reactions. Capillary 3 could be easily switched

with capillaries of different inner diameters from 0.125 to 0.75 mm, which was selected

based on the desirable pressure or reaction time (for neutrals) for the reaction. An

opening time as long as 250 ms could be applied with a 5 cm capillary of id 0.125 mm

and multiple introductions could also be implemented.15 The species introduced from

both DAPIs interact with each other inside the RIT during the cooling period after DAPI-

2 was closed. The final product ions were analyzed by rf scan with resonance ejection at

q = 0.81. CID could also be performed to the product ions.

Figure 2.1 (a) Schematic representation of the DAPI-RIT-DAPI system; (b) General

waveforms of the in-trap gas-phase reactions

Two types of reactions, ion/molecule and ion/ion reactions, were performed using

the DAPI-RIT-DAPI system to show the feasibility of this configuration. The product

spectra are shown in Figure 2.2 and Figure 2.3a, respectively. For ion/molecule reactions,

peptide thiyl radical ions [γ-E(S•C)G+H]+ (m/z 307) were generated by CID19 of the

protonated S-nitrosoglutathione [γ-E(NOC)G+H]+ (m/z 337) introduced through DAPI-1

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(Scheme 2.1) and reacted with the allyl iodide vapor introduced through DAPI-2

(capillary 3: i.d. 0.25 mm, opening time 25 ms). The reaction time was set to 750 ms after

closing DAPI-2 and before rf scan. The thiyl radical located on the cysteine side-chain

attacked the C-I bond within allyl iodide, leading to the formation of allyl adduct at m/z

348 (+•C3H5, +41 Da) and iodine adduct at m/z 434 (+•I, +127 Da). The reactions

between the thiyl radical with dimethyl disulfide was also observed with product ions at

m/z 354 (+•SCH3, +47 Da). The reaction phenomenon is consistent with literature reports

on peptide thiyl radical species (Scheme 2.2).20 The details of the thiyl radical reaction

will be further discussed later to characterize the gas flow effect on the radical reactivity.

Figure 2.2 Ion/molecule reactions: thiyl radical cations react with allyl iodide

Scheme 2.1

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Scheme 2.2

For ion/ion reactions, protonated angiotensin I (DRVYIHPFHL) generated via

positive mode nanoESI (77 μM) was introduced into the RIT through DAPI-1 with a

pulsed open time of 30 ms (Figure 2.3b). The triply charged ions (m/z 433) were isolated

at q = 0.80 after 250 ms of cooling time (Figure 2.3a inset). The anion reagent was

generated with negative mode nanoESI of 37 μM pentachlorophenol (PCP, MW: 266.34)

and introduced through DAPI-2 with capillary 3 of 0.25 mm i.d. for 60 ms. The single-

phase rf applied on the RIT allowed efficient trapping of both cations and anions with the

dc voltage on the end electrodes set to zero. A low-mass cutoff at m/z 220 and a reaction

time of 260 ms were used (scan function shown in Figure 2.3d). As shown in Figure 2.3a,

product ions resulted from proton transfer reactions, i.e., doubly charged angiotensin I at

m/z 649, can be well detected. Noticing that fragment ions such as b4+ and y4

+ 21 were

observed, which was not common in ion/ion reactions. This phenomenon is probably due

to an impact of the second gas flow (collisional activation) on the triply charged peptide

ions. Same fragments were also observed when using the same scan function (DAPI-2

opened), however, with the nanoESI source turned off for anion production (Figure 2.3c).

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Figure 2.3 (a) Ion/ion reactions: Angiotensin I triply charged cations react with

deprotonated pentachlorophenol anions (insert: Isolation of the triply charged species); (b)

MS spectrum of Angiotensin I without opening DAPI-2; (c) MS spectrum of isolated

triply charged peptide ions with DAPI-2 opening but the nanoESI source being turned off

for anion production; and (d) aveforms of the ion/ion reaction (dc was set to -10 V during

mass analysis).

Based on the results of Figure 2.2 and Figure 2.3, both ion/molecule and ion/ion

reactions could be performed on the DAPI-RIT-DAPI system, while the pressure change

and the energy deposition by the gas flow occur during the DAPI opening. As discussed

above, these two features could play important roles in gas-phase reactions. In the

following studies, we focused on the characterization of these two factors for carrying on

the gas-phase ion reactions using the DAPI-RIT-DAPI system.

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2.4.2 Pressure effect on gas-phase ion reactions

With the DAPI MS systems, the pressure in the ion trap can rise to 10-1 Torr or

higher after the opening of DAPIs, which is much higher than the pressure for

conventional MS analysis using quadrupole ion traps (mTorr or lower). After the DAPI is

closed, the pressure is gradually pumped down. By varying the delay time for performing

the CID, the dissociation reactions can be carried at different pressures. As shown in a

series of CID experiments (q = 0.30) of cocaine ([M+H]+, m/z 304, 3 μM, Scheme 2.3) at

different trap pressures, the optimal fragmentation efficiency (defined as the fraction of

fragment ion intensity over total ion intensity) occurs at ~8 x 10-4 Torr as shown in Figure

2.4. The activation time was set to 30 ms and the amplitude of the excitation AC was 200

mV. The relatively lower fragmentation efficiency at pressures lower than 5 x 10-4 Torr is

likely due to the lower collision rate at lower pressures. The decrease in fragmentation

efficiencies at pressures higher than 3 mTorr could be due to the significantly enhanced

damping effect that cools the ions to the center of the trap.22

Scheme 2.3

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Figure 2.4 CID spectra and MS/MS efficiency of protonated cocaine m/z 304 at different

pressures. Activation time 30 ms, amplitude 200 mV.

CID was also performed (q = 0.30) for deprotonated 9-anthracene carboxylic acid

(m/z 221, 5 μM) at different pressures (Figure 2.5). When the CID was performed at

about 6 mTorr (200 ms after DAPI-1 opening), only product ion m/z 195 was observed

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(Figure 2.5a); At pressure of about 2 mTorr (500 ms delay), another product ion m/z 177

was observed in addition to m/z 195 (Figure 2.5b); Product ion m/z 177 became the only

product when CID was carried out at 1 mTorr or lower (780 ms delay) (Figure 2.5c). The

ions m/z 177 could be obtained also by isolating and activating m/z 195 (Figure 2.5d).

The CID activation time was 30 ms. In-trap CID experiments were performed on a

commercial triple quadrupole/linear ion trap (QTRAP 4000, AB Sciex, Toronto, Canada)

for product verification. The precursor ions at m/z 221 were isolated in Q1 mass filter and

fragmented in Q3 linear ion trap via on-resonance CID. As shown in Figure 2.5e, the only

fragment pathway observed was CO2 loss, giving rise to the anthracene anion at m/z 177,

which has been demonstrated as an ETD reagent.23 This result only matches the CID

results obtained at low pressure when using the DAPI instrument (Figure 2.5c). Given the

high reactivity of anthracene radical anions, m/z 195 observed under higher pressures

(Figure 2.5a and b) might result from sequential ion/molecule reactions of m/z 177 with

trace H2O in the trap. To test this hypothesis, water vapor was mixed into the collision

gas on QTRAP 4000. The anthracene anions (m/z 177) were stored in Q3 linear ion trap

for various reaction times with H2O at the trap pressure of 4 × 10-5 Torr. Figure 2.5f

shows the data after 3 s reaction time, in which m/z 195 can be clearly detected. A peak at

m/z 221 was also observed, which is probably due to reaction of the anthracene anions

with trace CO2 in the water vapor. Note that the reaction time on QTRAP 4000 was long

(3000 ms), yet anthracene anions at m/z 177 remained as the base peak, while 100% yield

of water adduct was observed with the DAPI instrument at only 600 ms reaction time

(Figure 2.5a).

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Figure 2.5 Ion-trap CID spectra of deprotonated 9-anthracenecarboxylic acid at (a) 6

mTorr 200 ms delay, (b) 2 mTorr 500 ms delay, (c) 1 mTorr 780 ms delay, from the

DAPI opening on DAPI-RIT-DAPI system. The yellow, red, and blue columns illustrate

the positions of ion introduction, dipolar ac excitation, and MS scan, respectively; (d)

Ion-trap CID spectrum of m/z 195 on DAPI-RIT-DAPI system; (e) Ion-trap CID

spectrum of deprotonated 9-anthracenecarboxylic acid on QTRAP 4000; (f) Ion/molecule

reactions of deprotonated anthracene ions with water vapor on QTRAP 4000. The CID

activation time was set to 30 ms on DAPI-RIT-DAPI system.

As a summary, an optimal CID efficiency was obtained at about 0.8 mTorr and

ion/molecule reactions happened at much faster rates at higher pressures (6 mTorr) using

the DAPI-RIT-DAPI instrument and therefore the reaction products can be obtained with

short reaction time.

2.4.3 Dynamic gas flow effect on gas-phase ion reactions

According to the previous simulation study,18 the gas expansion after a DAPI is

significantly larger and the gas velocity is much higher than those for a continuous API.

To characterize the internal energy distributions of the ions via DAPI introduction, the

“survival yield” method, which has been developed to determine ion internal energies,24

was applied in our study. A comparison was made between the DAPI using the home-

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built instrument and a continuous API using the QTRAP 4000. A methanol/water (1:1,

v/v) solution containing the following five compounds at 10-4 M each, p-

chlorobenzylpyridinium chloride (p-Cl), p-methylbenzylpyridinium bromide (p-CH3), p-

methoxybenzylpyridinium tetrafluoroborate (p-OCH3), p-nitrobenzylpyridinium bromide

(p-NO2) and p-cyanobenzylpyridinium chloride (p-CN), was used to generate the

thermometer ions by nanoESI. The ions were introduced into the RIT directly through

DAPI-1 (DAPI opening time of 15 ms with a low-mass cutoff m/z 60) in the home-built

instrument, or through the continuous API into Q3 linear ion trap of QTRAP 4000 with

declustering potential of 10 V, collisional energy of 5 V, and Q3 entry barrier of 2 V (the

minimum energy setup on the commercial instrument), respectively. Figure 2.6a shows

the breakdown curves that were calculated and plotted using the methods described

previously.24b In detail, the critical dissociation energies (E) of the five benzylpyridinium

ions (p-OCH3, p-CH3, p-Cl, p-CN, and p-NO2) are 1.51, 1.77, 1.90, 2.10, and 2.35 eV,

respectively. The survival yield (SY) was calculated as I(parent ion)/[ I(parent ion) + Σ

I(fragment ion)], where I(parent ion) and I(fragment ion) represent the intensities of

precursor and fragment ions, respectively. The corresponding internal energy distribution

P(E) (Figure 2.6b) was then calculated by taking the derivative of the breakdown curve in

Figure 2.6a. The internal energy distribution associated with DAPI is about 0.4 eV higher

than the continuous API.

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Figure 2.6 Energy characterization of DAPI. (a) Calculated survival ion yields and (b)

Internal energy distributions of DAPI vs. continuous API for ion introduction;

Fragmentation spectra of thermometer ions after opening DAPI-2 for 20 ms with

capillary 3 (c) i.d. 0.5 mm and (d) i.d. 0.75 mm. *p-R represents the fragment of p-R (R =

OCH3, CH3, Cl, CN, NO2).

To conduct reactions on the DAPI-RIT-DAPI instrument, a second reagent species

was introduced with a gas flow from DAPI-2 after the ions injected through DAPI-1 had

been trapped and cooled. It was observed that the second gas flow could cause the

dissociation of the ions of small organic compounds from our previous studies15 and

triply charged peptide ions. Therefore, the ion internal energy change due to the second

gas flow was investigated using the thermometer ions. After the thermometer ions were

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introduced through DAPI-1, their corresponding molecular ions were isolated and the

DAPI-2 was opened while an rf voltage corresponding to a low-mass cutoff of m/z 60

was used. No fragmentation of the thermometer ions was observed with capillary 3 of

0.125 mm or 0.25 mm i.d., when DAPI-2 was open for up to 250 ms. The most fragile p-

OCH3 ions fragmented with a capillary 3 of 0.5 mm i.d. and an opening time of 20 ms

(Figure 2.6c), while p-OCH3, p-CH3, and p-Cl started to fragment with capillary 3 of 0.75

mm i.d. DAPI-2 opening time 20 ms (Figure 2.6d). The disturbance and the energy

deposition to the trapped ions could be controlled by selecting capillary 3 of proper

conductance, i.e. capillary i.d. smaller than 0.5 mm. However, for introduction of ions for

a reaction, the transfer efficiency decreases significantly when the capillary i.d. is smaller

than 0.25 mm.13b These results indicate that the second gas flow led to an energy

deposition to the trapped ions. Its influence on the reactivity of the radical ions was

further studied with the reactions between glutathione thiyl radical ions and dimethyl

disulfide.

As discussed earlier, glutathione thiyl radical ions could be produced from

protonated S-nitrosoglutathione (30 μM) through CID by a facile NO loss (Scheme 1).19,

20b Figure 2.7a shows a MS spectrum with a DAPI-1 opening time of 15 ms using a scan

without applying excitation AC for CID. A fragment ion at m/z 307 was observed,

corresponding to the loss of a NO from the precursor (m/z 337). This ion presumably is

due to additional internal energy deposition to the precursor during ion introduction

through DAPI-1, for which the average internal energy distribution is 0.4 eV higher than

that from continuous API as shown in Figure 2.7b. For the ion/molecule reaction, the

protonated S-nitrosoglutathione (m/z 337) was isolated and excited to produce thiyl

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radical ions (m/z 307) through CID (Figure 2.7b) for subsequent ion/molecule reactions.

The head-space vapor of dimethyl disulfide (placed in a glass tube with a plastic

Swagelok connected to capillary 3) was introduced into the vacuum chamber for reaction

through DAPI-2. Dissociative addition of the dimethyl disulfide to thiyl radical was

observed to occur (Figure 2.7c), leading to the formation of glutathione methyl disulfide

(Scheme 2) at m/z 354. The reaction yield, defined as the fraction of the product intensity

(m/z 354) over the total ion intensity (sum of m/z 307 and m/z 354), was 70% under this

condition.

Figure 2.7 Ion/molecule reactions of peptide thiyl radical ions and dimethyl disulfide. (a)

MS spectrum of S-nitrosoglutathione; (b) Thiyl radical (m/z 307) produced by CID of

protonated S-nitrosoglutathione; (c) Reactions between thiyl radicals and dimethyl

disulfide (Capillary 3 i.d. 0.125 mm; DAPI-2 opening time 25 ms; q=0.35 for thiyl

radical ions); (d) Ion/molecule reactions between isolated m/z 307 from panel (c) with

dimethyl disulfide.

Scheme 2.2

HOOCNH

OS

HN

NH2 O

COOH + H

+

m/z 354+ ●SCH3 (47 Da)

m/z 307

CH3SSCH3

●

HOOCNH

OS

HN

NH2 O

COOH

S

+ H

+

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Multiple introductions of the reactant species within a single scan through DAPI-2

could be implemented using the method described previously.13d Higher reaction yield

should be expected with longer DAPI opening time and/or higher number of neutral

introduced; however, no significant difference in the reaction yield was observed for the

different scan functions with different DAPI-2 opening times and various numbers of

injections (Figure 2.8).

Figure 2.8 Thiyl radical reaction for different capillary inner diameters and opening times.

A new scan function was designed and implemented to isolate the thiyl radical ions

m/z 307 (Figure 2.7c) after one reaction cycle and then allowed to react with the dimethyl

disulfide introduced again from DAPI-2 (capillary 3 i.d. 0.125 mm; DAPI-2 opening time

25 ms). Surprisingly, no product ions were observed (Figure 2.7d). This indicates that a

portion of thiyl radical ions had been converted into nonreactive species, presumably Cα-

radical ions,17, 19 during the first reaction cycle. In fact, the energy barrier of thiyl radical

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isomerization is relatively low (7.4 kcal/mol).17 It has been demonstrated that in-source

fragmentation or in-trap CID can promote this process and lead to the loss of thiyl radical

reactivity. In order to confirm this hypothesis, an ac excitation was used to activate the

thiyl radicals immediately after they were produced and isolated; dimethyl disulfide was

then introduced for reactions. The reaction yield decreased as the ac amplitude for

excitation increased (Figure 2.9). Specifically, the reaction yield was only 15% with an ac

excitation voltage of 0.07 V and the fragments of the thiyl radical ions, m/z 289 due to

loss of water and m/z 232 ([b2-H]+•) were also observed (Figure 2.9c).19

Figure 2.9 MS spectra of thiyl radical reaction for activation with ac amplitude (a) 0.06 V;

(b) 0.066 V; (c) 0.07 V; and (d) CID spectrum of thiyl radical ions.

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These results suggest that more stable Cα-radical ions could be formed with energy

deposition through an excitation, either by ac activation or gas flow. The side reactions

might occur due to the gas flow with DAPI opening.

2.5 Conclusion

Use of simple instrument configuration of multiple DAPIs for gas-phase ion

chemistry study was explored with a home built DAPI-RIT-DAPI system. The

discontinuous atmospheric pressure interfaces enable introduction of multiple reactants

with simple instrument configuration. The in-trap pressure varies during a scan, which

also allows a reaction to be carried out at a high pressure while MS analysis of products

at a much lower one. The gas dynamic effect on the internal energy deposition to the ions

has been studied with thermometer ions. The ion/molecule reaction between glutathione

thiyl radical and dimethyl disulfide was investigated to demonstrate the feasibilities of the

DAPI-RIT-DAPI system, where a radical immigration reaction occurred at the same time

of the dissociative addition reaction. With the simplicity of the configuration and the

easiness in instrumentation modification, a variety of sources for ions and radicals, such

as synchronized discharge ionization,25 low temperature plasma ionization,26 UV light

irradiation,27 and pyrolysis nozzle,28 can also be coupled with this system in the future for

investigation of the gas-phase reactions.

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2.6 References

1. (a) Green, M. K.; Lebrilla, C. B., Ion-molecule reactions as probes of gas-phase

structures of peptides and proteins. Mass Spectrom. Rev. 1997, 16 (2), 53-71; (b) Gronert,

S., Mass spectrometric studies of organic ion/molecule reactions. Chem. Rev. 2001, 101

(2), 329-360; (c) Reid, G. E.; McLuckey, S. A., 'Top down' protein characterization via

tandem mass spectrometry. J. Mass Spectrom. 2002, 37 (7), 663-675; (d) Osburn, S.;

Ryzhov, V., Ion-Molecule Reactions: Analytical and Structural Tool. Anal. Chem. 2013,

85 (2), 769-778.

2. Zhang, X.; Maccarone, A. T.; Nimlos, M. R.; Kato, S.; Bierbaum, V. M.; Ellison,

G. B.; Ruscic, B.; Simmonett, A. C.; Allen, W. D.; Schaefer, H. F., Unimolecular thermal

fragmentation of ortho-benzyne. J. Chem. Phys. 2007, 126 (4).

3. (a) Zubarev, R. A., Reactions of polypeptide ions with electrons in the gas phase.

Mass Spectrom. Rev. 2003, 22 (1), 57-77; (b) Zubarev, R. A., Electron-capture

dissociation tandem mass spectrometry. Curr. Opin. Biotechnol. 2004, 15 (1), 12-16; (c)

Cooper, H. J.; Hakansson, K.; Marshall, A. G., The role of electron capture dissociation

in biomolecular analysis. Mass Spectrom. Rev. 2005, 24 (2), 201-222.

4. (a) Pitteri, S. J.; McLuckey, S. A., Recent developments in the ion/ion chemistry

of high-mass multiply charged ions. Mass Spectrom. Rev. 2005, 24 (6), 931-958; (b)

Good, D. M.; Coon, J. J., Advancing proteomics with ion/ion chemistry. BioTechniques

2006, 40 (6), 783-789; (c) Wiesner, J.; Premsler, T.; Sickmann, A., Application of

electron transfer dissociation (ETD) for the analysis of posttranslational modifications.

Proteomics 2008, 8 (21), 4466-4483.

5. (a) Oomens, J.; Sartakov, B. G.; Meijer, G.; Von Helden, G., Gas-phase infrared

multiple photon dissociation spectroscopy of mass-selected molecular ions. Int. J. Mass

Spectrom. 2006, 254 (1-2), 1-19; (b) Reilly, J. P., Ultraviolet Photofragmentation of

Biomolecular Ions. Mass Spectrom. Rev. 2009, 28 (3), 425-447.

6. Wysocki, V. H.; Joyce, K. E.; Jones, C. M.; Beardsley, R. L., Surface-induced

dissociation of small molecules, peptides,and non-covalent protein complexes. J. Am. Soc.

Mass Spectrom. 2008, 19 (2), 190-208.

7. (a) Benedikt, J.; Hecimovic, A.; Ellerweg, D.; von Keudell, A., Quadrupole mass

spectrometry of reactive plasmas. Journal of Physics D-Applied Physics 2012, 45 (40); (b)

Cook, S. L.; Collin, O. L.; Jackson, G. P., Metastable atom-activated dissociation mass

spectrometry: leucine/isoleucine differentiation and ring cleavage of proline residues. J.

Mass Spectrom. 2009, 44 (8), 1211-1223.

8. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M.,

Electrospray Ionization for Mass-Spectrometry of Large Biomolecules. Science 1989,

246 (4926), 64-71.

9. Horning, E. C.; Horning, M. G.; Carroll, D. I.; Dzidic, I.; Stillwel.Rn, New

Picogram Detection System Based on a Mass-Spectrometer with an External Ionization

Source at Atmospheric-Pressure. Anal. Chem. 1973, 45 (6), 936-943.

10. (a) Hager, J. W., A new linear ion trap mass spectrometer. Rapid Commun. Mass

Spectrom. 2002, 16 (6), 512-526; (b) Schwartz, J. C.; Senko, M. W.; Syka, J. E. P., A

two-dimensional quadrupole ion trap mass spectrometer. J. Am. Soc. Mass Spectrom.

2002, 13 (6), 659-669.

71

71

11. (a) Pyatkivskyy, Y.; Ryzhov, V., Coupling of ion-molecule reactions with liquid

chromatography on a quadrupole ion trap mass spectrometer. Rapid Commun. Mass

Spectrom. 2008, 22 (8), 1288-1294; (b) Poad, B. L. J.; Pham, H. T.; Thomas, M. C.;

Nealon, J. R.; Campbell, J. L.; Mitchell, T. W.; Blanksby, S. J., Ozone-Induced

Dissociation on a Modified Tandem Linear Ion-Trap: Observations of Different

Reactivity for Isomeric Lipids. J. Am. Soc. Mass Spectrom. 2010, 21 (12), 1989-1999.

12. (a) Badman, E. R.; Chrisman, P. A.; McLuckey, S. A., A quadrupole ion trap

mass spectrometer with three independent ion sources for the study of gas-phase ion/ion

reactions. Anal. Chem. 2002, 74 (24), 6237-6243; (b) Xia, Y.; McLuckey, S. A.,

Evolution of instrumentation for the study of gas-phase ion/ion chemistry via mass

spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19 (2), 173-189.

13. (a) Gao, L.; Sugiarto, A.; Harper, J. D.; Cooks, R. G.; Ouyang, Z., Design and

characterization of a multisource hand-held tandem mass spectrometer. Anal. Chem. 2008,

80 (19), 7198-205; (b) Gao, L.; Cooks, R. G.; Ouyang, Z., Breaking the pumping speed

barrier in mass spectrometry: Discontinuous atmospheric pressure interface. Anal. Chem.

2008, 80 (11), 4026-4032; (c) Huang, G.; Xu, W.; Visbal-Onufrak, M. A.; Ouyang, Z.;

Cooks, R. G., Direct analysis of melamine in complex matrices using a handheld mass

spectrometer. Analyst 2010, 135 (4), 705-711; (d) Gao, L.; Li, G.; Nie, Z.; Duncan, J.;

Ouyang, Z.; Cooks, R. G., Characterization of a discontinuous atmospheric pressure

interface. Multiple ion introduction pulses for improved performance. Int. J. Mass

Spectrom. 2009, 283 (1-3), 30-34.

14. Chen, T.-C.; Xu, W.; Garimella, S.; Ouyang, Z., Study of the efficiency for ion

transfer through bent capillaries. J. Mass Spectrom. 2012, 47 (11), 1466-1472.

15. Xu, W.; Charipar, N.; Kirleis, M. A.; Xia, Y.; Ouyang, Z., Study of Discontinuous

Atmospheric Pressure Interfaces for Mass Spectrometry Instrumentation Development.

Anal. Chem. 2010, 82 (15), 6584-6592.

16. Ouyang, Z.; Wu, G. X.; Song, Y. S.; Li, H. Y.; Plass, W. R.; Cooks, R. G.,

Rectilinear ion trap: Concepts, calculations, and analytical performance of a new mass

analyzer. Anal. Chem. 2004, 76 (16), 4595-4605.

17. Rauk, A.; Armstrong, D. A.; Berges, J., Glutathione radical: Intramolecular H

abstraction by the thiyl radical. Can. J. Chem. 2001, 79 (4), 405-417.

18. Garimella, S.; Xu, W.; Ouyang, Z., Simulation of Transient Rarefied Gas

Expansion for Discontinuous Atmospheric Pressure Interface (DAPI) Using Direct

Simulation Monte Carlo (DSMC). American Society for Mass Spectrometry 60th Annual

Conference Proceedings: Ion Manipulation, Analysis and Detection: New Developments

2012, ThOF (PM), 349.

19. Zhao, J.; Siu, K. W. M.; Hopkinson, A. C., Glutathione radical cation in the gas

phase; generation, structure and fragmentation. Org. Biomol. Chem. 2011, 9 (21), 7384-

7392.

20. (a) Osburn, S.; Berden, G.; Oomens, J.; O'Hair, R. A. J.; Ryzhov, V., Structure

and Reactivity of the N-Acetyl-Cysteine Radical Cation and Anion: Does Radical

Migration Occur? J. Am. Soc. Mass Spectrom. 2011, 22 (10), 1794-1803; (b) Tan, L.; Xia,

Y., Gas-Phase Reactivity of Peptide Thiyl (RS center dot),Perthiyl (RSS center dot), and

Sulfinyl (RSO center dot) Radical Ions Formed from Atmospheric Pressure Ion/Radical

Reactions. J. Am. Soc. Mass Spectrom. 2013, 24 (4), 534-542.

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21. (a) Coon, J. J.; Syka, J. E. P.; Schwartz, J. C.; Shabanowitz, J.; Hunt, D. F., Anion

dependence in the partitioning between proton and electron transfer in ion/ion reactions.

Int. J. Mass Spectrom. 2004, 236 (1-3), 33-42; (b) Tang, X. J.; Thibault, P.; Boyd, R. K.,

Fragmentation Reactions of Multiply-Protonated Peptides and Implications for

Sequencing by Tandem Mass-Spectrometry with Low-Energy Collision-Induced

Dissociation. Anal. Chem. 1993, 65 (20), 2824-2834.

22. Tolmachev, A. V.; Vilkov, A. N.; Bogdanov, B.; Pasa-Tolic, L.; Masselon, C. D.;

Smith, R. D., Collisional activation of ions in RF ion traps and ion guides: The effective

ion temperature treatment. J. Am. Soc. Mass Spectrom. 2004, 15 (11), 1616-1628.

23. Huang, T. Y.; Emory, J. F.; O'Hair, R. A. J.; McLuckey, S. A., Electron-transfer

reagent anion formation via electrospray ionization and collision-induced dissociation.

Anal. Chem. 2006, 78 (21), 7387-7391.

24. (a) Gabelica, V.; De Pauw, E., Internal energy and fragmentation of ions

produced in electrospray sources. Mass Spectrom. Rev. 2005, 24 (4), 566-587; (b) Nefliu,

M.; Smith, J. N.; Venter, A.; Cooks, R. G., Internal energy distributions in desorption

electrospray ionization (DESI). J. Am. Soc. Mass Spectrom. 2008, 19 (3), 420-427; (c)

Wang, H.; Liu, J. J.; Cooks, R. G.; Ouyang, Z., Paper Spray for Direct Analysis of

Complex Mixtures Using Mass Spectrometry. Angew. Chem.-Int. Edit. 2010, 49 (5), 877-

880.

25. Chen, T.-C.; Ouyang, Z., Synchronized Discharge Ionization for Analysis of

Volatile Organic Compounds Using a Hand-Held Ion Trap Mass Spectrometer. Anal.

Chem. 2013, 85 (3), 1767-1772.

26. Ma, X. X.; Love, C. B.; Zhang, X. R.; Xia, Y., Gas-Phase Fragmentation of [M +

nH + OH](n center dot+) Ions Formed from Peptides Containing Intra-Molecular

Disulfide Bonds. J. Am. Soc. Mass Spectrom. 2011, 22 (5), 922-930.

27. Stinson, C. A.; Xia, Y., Radical induced disulfide bond cleavage within peptides

via ultraviolet irradiation of an electrospray plume. Analyst 2013, 138 (10), 2840-2846.

28. Zhang, X.; Friderichsen, A. V.; Nandi, S.; Ellison, G. B.; David, D. E.; McKinnon,

J. T.; Lindeman, T. G.; Dayton, D. C.; Nimlos, M. R., Intense, hyperthermal source of

organic radicals for matrix-isolation spectroscopy. Rev. Sci. Instrum. 2003, 74 (6), 3077-

3086.

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CHAPTER 3. GAS-PHASE REACTIONS OF C3H2 WITH PROTONATED ALKYL

AMINES: FORMATION OF A C-N COVALENT BOND

“The reaction between alkyl amines and C3H2 carbenes was a surprise in the study

of bio-ion/radical reactions. However, science is sometimes not about how to follow the

design and prediction, but discovering new things others never had.”

3.1 Introduction

Vinylidene carbenes (C3H2) are unsaturated divalent carbon species displaying two

non-bonding electrons in a singlet or triplet configuration. There are three structural

isomers of C3H2: cyclopropenylidene, propadienylidene, and propynylidene. Vinylidene

carbenes overall are highly reactive and not stable in the condensed phase. However, they

have been detected in hydrocarbon flames and are considered as the key intermediates for

soot formation;1 also, cyclopropenylidene (c-C3H2), a singlet carbene, is the most

abundant cyclic hydrocarbons observed in the interstellar space.2 A significant amount of

effort has been made to synthesize relatively stable c-C3H2 derivatives.3 These derivatives

have been widely utilized as ligands of transition metal catalysts in cross-coupling

reactions.4

Given the important role of C3H2 species in the combustion chemistry, organic

synthesis, and interstellar chemistry, it is highly desirable to establish a fundamental

understanding of their physical and chemical properties. The relative energies,

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geometries, and the isomerization among the three isomers of C3H2 have been well

mapped with the experimental gas-phase studies and theoretical calculations.5

Cyclopropenylidene is the most stable among the three isomers. Driven toward forming a

stable aromatic system (c-C3H3+) with 2 π-electrons, c-C3H2 has a gas-phase basicity (GB)

of 916 kJ/mol from experimental measurements,6 which is significantly higher than the

GB of ammonia (819 kJ/mol).7 Investigation of c-C3H2 reactivity toward organic

molecules so far has been rarely achieved by experimental methods because of its high

instability in the condense phase.3 Instead, theoretical calculations have been used to

study several types of reactions of c-C3H2 with organic molecules, including double bond

addition,8 single bond insertion,9 and skeletal rearrangement.10 In this study, we

experimentally studied gas-phase reactions of c-C3H2 with protonated alkyl amines for

the first time.

3.2 Instrumentation

Our platform (Figure 2.1a) was constructed with a DAPI-RIT-DAPI configuration

(DAPI: discontinuous atmospheric pressure interface11; RIT: rectilinear ion trap12), which

has been previously shown to provide high pressure and energetic gas flow for gas-phase

ion/molecule reactions13. The ion trap served as both reactor and mass analyzer with a

pyrolysis nozzle to generate radical species.

For general ion/radical reactions (MS scan function shown in Figure 2.1b), DAPI-1

was used to introduce alkyl ammoniums generated by nanoelectrospray (nanoESI) while

DAPI-2 connected to the pyrolysis nozzle was used to introduce vapor of carbene

precursors (placed in a glass tube with a plastic Swagelok connected to capillary 3 of 0.25

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mm i.d.). Ions of interest introduced through DAPI-1 were mass-selectively isolated and

then interacting with carbenes from the dissociated precursors by pyrolysis from DAPI-2.

A glow discharge electron impact (GDEI)14 source was installed to emit electrons into the

RIT to ionize the neutral radicals or radical precursors that are subsequently characterized

by MS analysis. This system represented a simple and intuitionistic approach to carry out

and study gas-phase reactions and show the first experimental evidence of a gas-phase

reaction between c-C3H2 and protonated ammoniums forming a C-N covalent bond.

Figure 3.1 (a) Schematic of the home-built platform for the study of reactive radical

species and (b) the general MS scan function for ion/radical reactions (y-axis: voltage).

DAPI: discontinuous atmospheric pres-sure interface; RIT: rectilinear ion trap; GDEI:

glow discharge electron impact; rf: radio frequency; ac: alternative current.

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A single-phase radio frequency (rf) was applied to trap ions at 920 kHz or 1490 kHz

for mass range of m/z 80-600 or 20-200, respectively. The control electronics was

directly adopted from a Griffin Minotaur 300 (FLIR Systems, Inc., Wilsonville, OR). The

front endcap (close to DAPI-1) was a flat electrode with a center hole of 3 mm diameter

for ion introduction, while a mesh electrode (18 x 18 mesh size, 0.015” wire diameter;

McMaster-Carr, Aurora, OH, USA) was used for the back endcap (close to DAPI-2). A

dc of 10-30 V was applied on both end electrodes to create a dc trapping potential well

along the z direction. Resonance ejection (q = 0.81), was implemented for MS scan with

a dipolar ac signal at 324 and 524 kHz for rf frequency of 920 and 1490 kHz, respectively.

The frequency of rf 1490 kHz was used for m/z 20-180 to observe low mass ions while rf

920 kHz was used for m/z 100-600. The inductance and capacitance of the rf coil were

changed to accomplish the two frequencies. The conversion dynode of the detector was

grounded. The isolation of the trapped ions was achieved by forward/backward scan.

Collision-induced dissociation (CID) was implemented by dipolar excitation with a

single-frequency ac applied between the x electrodes of the RIT. In the mechanism study,

the low mass cutoff (LMCO) of CID was set to m/z 25 for rf 1490 kHz to trap low mass

CID fragments (eg. C3H3+, m/z 39).

The pyrolysis nozzle was first developed by Peter Chen to generate intense radical

species,15 which has been an efficient means to produce and transfer intense radicals to

gas phase16. Reactive species such as ethylny, viny, allyl, phenyl radical, and carbenes

etc. were generated by resistance-heated SiC nozzle from corresponding precursors15,

whose structures were then analyzed by infrared and electronic spectra. Flowing-

afterglow selected-ion flow tube (SIFT)17 and Fourier transform ion cyclotron resonance

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(FTICR)18 mass spectrometers were the two instruments reported to study ion/radical

reactions by pyrolysis nozzle. Proton transfer, associative detachment, and radical

recombination reactions were observed when using allyl radicals with hydroniums, ortho-

benzynes with hydroxides, and allyl radicals with benzyl radical cations, respectively. In

our setup, a silicon carbide (SiC) tube of 1 cm long, 1 mm i.d., 2 mm o.d. (Hexoloy SA,

Niagara Falls, NY) was employed as the resistance heater with an alumina tube

(8746K12, McMaster-Carr, Aurora, OH, USA) for insulation. The SiC and the Al2O3

tube were nested and cemented with ceramic adhesive (903HP, Cotronics, Brooklyn, NY)

into the machinable Al2O3 flange (8484K78, McMaster-Carr, Aurora, OH, USA).

Stainless steel capillary 2 inside the pinch valve of DAPI-2 was directly inserted into the

Al2O3 tube without further sealing. Electrical contact was provided by graphite disks

(AXF-FQ Poco Graphite, Decatur, TX) and 0.127-mm tantalum-foil clips (Alfa Aesar,

Ward Hill, MA). Two light bulbs of 100 W and 200 W, parallel to each other and in

series with the nozzle, were used as the ballast. Both nanoESI and radical precursors were

protected in Helium environment ca 760 Torr to prevent the heated SiC from oxidation.

The major differences for the pyrolysis nozzle in our platform compared to those in

flowing-afterglow selected ion flow tube or Fourier transform ion cyclotron resonance is

1) the high pressure ca 10-100 mTorr in the vacuum chamber during precursor injection

and 2) injection duration of 50 ms continuously instead of 200 µs with 20-40 Hz

frequency. Therefore, the radicals produced through the pyrolysis nozzle were expected

to have 103 times more collisions in our platform than that in high vacuum (10-5 Torr) and

pyrolysis temperature was dropping during the opening. The high pressure and energetic

gas flow are the two factors contributing to the formation of nonspecific C3H2 carbene

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from various precursors. Among three structures of C3H2 carbene species, the most stable

c-C3H2 is more likely to survive under the given environment.

Multiple triggers provided by the control electronics were used to synchronize the

operations of DAPIs, GDEI and MS analysis. All the spectra presented are the average of

5-9 continuous scans.

3.3 Materials

Ionic reactants of alkyl amines and amino acids were purchased from Sigma

Chemical Co. (Sigma-Aldrich, St. Louis, MO, USA). Different concentrations (5-100 µM)

of the samples were prepared with methanol for nanoESI with 0.2 % acetic acid added to

increase the intensity of protonated species if necessary.

Radical precursors including allylchloride, 1,5-hexadiene, pentane,

propargylchloride were purchased from Sigma Chemical Co. (Sigma-Aldrich, St. Louis,

MO, USA). Propargylbromide was purchased from TCI America (Portland, OR, USA),

and d12-pentane was purchased from Alfa Aesar (Ward Hill, MA, USA). The head-vapor

of radical precursors in Helium buffer was injected into the platform to generate radical

species. 1,3-dibromopropyne was synthesized as C3H2 carbene precursors according to

the previous papers.19 In short, 1,3-dibromopropyne was prepared in two steps: Propargyl

alcohol was converted to 1-bromopropyn-3-ol by triphenyl phosphite methiodide.20 PBr3

was used in benzene and then pyridine to replace the hydroxyl group with Br.19 The final

product was distilled under reduced pressure with Argon.

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3.4 Results and discussion

3.4.1 Carbene reaction with protonated alkylamines

We used 1,3-dibromopropyne (BrC≡CCH2Br) as a precursor for C3H2 formation by

pyrolysis.19 Figure 3.2a shows a typical reaction spectrum of protonated n-heptyl amine

(m/z 116) and the pyrolysis products of 1,3-dibromopropyne. Three new peaks, m/z 114,

154 and 192, were observed after the reaction. The ions of m/z 154 and 192 correspond to

a mass increase of 38 Da and 2 × 38 Da, respectively, from the precursor ion (m/z 116).

The peak at m/z 114 likely resulted from the dissociation of ion m/z 154 due to excitation

by hot gas flow when opening DAPI-2 for introducing pyrolysis products.13 Collisional

activation of ion m/z 154 (Figure 3.2b) yielded ion m/z 114 through a neutral loss of 40

Da (C3H4), which supported C3H2 adduct formation.

Figure 3.2 Carbene reaction spectra: (a) MS2 reaction of protonated n-heptylamine with

heated 1,3-dibromopropyne; (b) MS3 CID of the reaction product; (c) MS2 reaction of

protonated n-heptylamine with heated d12-pentane; and (d) MS2 reaction of protonated n-

heptylamine 18C6 complex with heated 1,3-dibromopropyne.

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Besides 1,3-dibromopropyne, we found that analogous reaction products, viz. the

mass increases of 38 Da, 2 × 38 Da, or even 3 × 38 Da to the protonated n-heptyl amine

ions, were obtained when using other hydrocarbons as precursors for pyrolysis, including

allylchloride, propargyl-bromide, 1,5-hexadiene, and pentane (Figure 3.3). The last

reaction allowed us to use fully deuterated pentane (d12-pentane) as a pyrolysis precursor

to confirm the chemical identity of the 38 Da adduct. The reaction spectrum is shown in

Figure 3.2c. Reaction products at m/z 156 and 196 correspond to mass increases of 40 Da

(C3D2) and 2 × 40 Da, respectively, from the amine ions (m/z 116), consistent with our

conclusion that the addition indeed results from C3H2. All the reaction and their

corresponding CID spectra of the test amines and pyrolysis precursors can be found in

Table 3.1 and Table 3.2.

Figure 3.3 MS2 reaction spectra of heptyl ammonium and heated (a) allylchloride, (b)

propargylbromide, (c) 1,5-hexadiene, and (d) pentane.

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We also investigated reactions of secondary, tertiary, and quaternary

alkylammonium ions with C3H2. Similar reaction phenomena were observed for all the

amine ions except for the quaternary alkylammonium ion, for which no reaction product

was observed (Figure 3.4). This series of experiments indicated that an amine nitrogen

and an ammonium proton were involved in the reaction. We further tested reactions of

C3H2 with the non-covalent complex of 18-crown-6 ether (18C6) and protonated n-heptyl

amine. In such a complex, three strong hydrogen bonds are formed between the

protonated primary amine and three oxygen atoms of 18C6, making neither the proton

nor the nitrogen available for further reaction or binding. Under the same reaction

condition previously used for the n-heptyl amine, the addition of C3H2 to the complex ion

m/z 380 ([M+18C6+H]+) was not observed (Figure 3.2d).

Figure 3.4 MS2 reaction spectra of protonated (a) dibutylamine, (b) tributylamine, and (c)

cetyltrimethyl ammonium with heated propargylbromide, and MS3 CID spectra of the

reaction products for (d) dibutylamine and (e) tributylamine.

The studies reported above suggest that protonation is an important factor for

observing the C3H2 addition reaction. In order to gain further evidence, we compared the

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C3H2 reactions with proline methyl ester ions in protonated vs. sodium adduct forms. The

most favorable protonation site of proline methyl ester is at the pyrrolidine nitrogen,

while for the sodium adduct, the sodium ion is chelated by the carbonyl oxygen and the

amine nitrogen atoms.21 The reaction course for these two ions was drastically different.

While an efficient C3H2 addition was observed for the protonated ions, the reaction yield

was extremely low for the sodium adduct ions (Figure 3.5). This observation is consistent

with the results for the alkyl amines, for which only the protonated amines undergo

efficient C3H2 additions.

Figure 3.5 MS2 reaction spectra of (a) protonated and (b) sodiated proline methyl ester

with heated allylchloride, and their corresponding product MS3 CID spectra (c) and (d),

respectively.

C3H2 has three known isomeric structures, viz. cyclopropenylidene (c-C3H2),

propadienylidene, and propynylidene which is a 3A” triplet in the ground electronic

state.5d To explain the course of the ammonium ion addition, it is important to identify

which structure(s) are involved in the observed reactions. When GDEI was used to

identify the pyrolysis product(s), the different MS patterns with heating on and off (Table

3.3) prove that new species were generated through pyrolysis. The GDEI data is not

conclusive but indicative for the identity of C3H2 since extensive ion/molecule reactions

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would occur after the initial electron impact at the high pressure (10 mTorr). The fact that

a variety of hydrocarbons could all produce C3H2 suggests that the C3H2 formation might

not be closely tied to the pyrolysis chemistry; instead, it is most likely determined by the

energetics and reactions after the pyrolysis. Note that the ion trap pressure was relatively

high (10-100 mTorr) when the pyrolysis valve was open, and the free sonic expansion

was shorter than 2 cm, while the distance between the nozzle and the center of the RIT is

5 cm. The first generation pyrolysis products can undergo extensive and energetic

collisions as they are entering the ion trap,13, 22 be converted to C3H2, which subsequently

reacts with the trapped ions. Since the reaction chemistry involves the protons on the

ammonium ion, we suspect that c-C3H2, which has a relatively high GB, 916 kJ/mol at

298 K for the 1A1 state, as compared to 860 kJ/mol for (1A1) propadienylidene, and 687

kJ/mol for the 3A” state of propynylidene, might be the dominant species formed by

pyrolysis. In order to clarify the reaction mechanism, further evidence was sought from

theoretical calculations and additional experiments.

3.4.2 Theoretical calculation of the reaction mechanism

Since C3H2 has three identified isomeric structures, i.e., cyclopropenylidene (c-

C3H2), propadienylidene (l-C3H2), and propynylidene (l-C3H2), it is of importance to

know which structure(s) are involved in the observed reactions. We tested a variety of

hydrocarbons including allylchloride, 1,5-hexadiene, propargyl-bromide, and pentane as

C3H2 precursors and observed the same reaction phenomenon as shown in Figure 3.2a,

where 1,3-dibromopropyne was used as the C3H2 precursor and reacted with protonated

alkyl amine. These results suggest that the C3H2 formation is not closely tied to the

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pyrolysis chemistry or process, instead mostly determined by the energetics and reactions

after pyrolysis. Note that the ion trap pressure is relatively high (10-100 mTorr) upon the

opening of the pyrolysis valve of the current instrument design, which gives only less

than 2 cm free sonic expansion of the radical species after the initially formed from

pyrolysis in comparison with 4 cm between the nozzle and the center of the RIT. These

radical species undergo extensive and energetic collisions as they enter the trap13, 22 and

are further converted into other products. We suspect that c-C3H2 might be the dominant

species formed from such process since c-C3H2 has lower energy by about 42–59 and 59–

92 kJ/mol than that of the propadienylidene and propynylidene.1, 5a, 23 This hypothesis is

also supported by the reaction chemistry which is mainly driven by proton. Indeed, c-

C3H2 has a significant GB of 916 kJ/mol as mentioned above. A reaction mechanism of

proton-bound dimer formation between c-C3H2 and protonated alkyl amine as the first

step followed by C-N covalent bond formation was proposed to explain the observed

reaction phenomenon in Figure 3.2a. This mechanism was further investigated and

evaluated by theoretical calculations and experimental results.

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Figure 3.6 Calculated PES of (a) CH3NH3+/c-C3H2 reaction to form a C-N covalent bond

from Brauman double-well complexes (numbers: CCSD(T)/aug-cc-pVTZ, kJ/mol) and (b)

the two Brauman intermediates as a function of the distance of N-H---C, blue square:

CCSD(T)/aug-cc-pVTZ single point energies on M06-2X/6-311+G(2d,p) optimized

geometries; open circle: M06-2X/6-311+G(2d,p) optimized; filled circle: B3LYP/6-

311+G(2d,p) optimized.

The reaction between c-C3H2 and protonated methyl amine (CH3NH3+) was used as

a simplified model for theoretical calculations at the CCSD(T)/aug-cc-pVTZ//M06-2X/6-

311+G(2d,p) level of theory and including zero point vibrational energies. The key

structures involved in the reaction together with their 0 K enthalpies (relative to the

enthalpy of the reactants) are illustrated in Figure 3.6a. The first step of the reaction is the

formation of a proton-bound dimer between c-C3H2 and CH3NH3+. This step is

exothermic, producing two Brauman intermediates, Complex 1 (-127 kJ/mol) and

Complex 2 (-121 kJ/mol) that were found as the structures with the lowest energies

(Figure 3.6b). Complex 2 has a structure with a bonding distance between the proton and

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the carbon of the c-C3H2 (N-H---C = 1.16 Å), as compared to Complex 1 (N-H---C =

1.72 Å), which indicates that the proton moved from the amine nitrogen to the c-C3H2

moiety. This shift places a partial positive charge on the C1 of c-C3H2 and facilities a

subsequent nucleophilic attack by the amine nitrogen, leading to the formation of a C-N

bond in product A (-216 kJ/mol) through a low energy barrier. Product A might quickly

go through rearrangements, such as ring opening and hydrogen atom migration (TS1 in

Figure 3.6a), to form a more stable product B (-372 kJ/mol). Product B is shown with a

cis-syn geometry that can isomerize to a slightly more stable trans-isomer. In either

isomer, two of the original amine hydrogens are transferred to the C3H4 moiety. It is

worth noting that Complex 1 has the proton more tightly bound to the amine nitrogen

and, as a result, the N-C covalent bond formation has a high energy barrier (Figure 3.7).

Figure 3.7 Calculated PES of N--H--C angle graph of Complex 1, 2 and product A

3.4.3 Experimental evidences of the reaction mechanism

The mechanism proposed in Figure 3.6 shows that the formation of a proton-bound

dimer between c-C3H2 and protonated alkyl amine is the key step for the overall reaction.

The proton-bound dimer can stay as it is (Complex 1) or undergo further rearrangement

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to form a covalent product. Although the proton-bound dimer and the covalent product

has the same m/z (indicated as [C]), they should be distinguished via unique unimolecular

dissociation channels upon collisional activation. For instance, loss of 40 Da (m/z [C-40])

is the signature fragmentation channel from the covalent product as shown in Figure 3.2b.

CID of proton-bound dimer, however, leads to the separation of c-C3H2 and alkyl amine.

Proton transfer product, C3H3+ (m/z 39), and protonated amines (m/z [C-38]) should be

the main products, and their relative abundances are dependent on the gas-phase

basicities of the corresponding neutrals. A breakdown curve of protonated

butylamine/carbene reaction as an example is illustrated in Figure 3.8 to show the

relatively abundance of these three CID products together with the precursor complex as

a function of the resonance activation ac amplitude. The yields of three products are

relatively stable throughout the range of the activation energies, which implies that the

three products have similar energy barriers for dissociation.

Figure 3.8 The breakdown curve of protonated butylamine/carbene reaction by collisional

activation: relative intensity of parent complex ([C], blue circle), covalent product ([C-

40], red diamond), proton-transfer product (m/z 39, green triangle), and direct

dissociation ([C-38], green square) as a function of activation energy.

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The relative intensity of [C-40] among all fragment ions can be used to indicate the

yield of the covalent product (% Covalent Complex). If the mechanism proposed in

Figure 3.6 truly reflects the nature of the reaction, % Covalent Complex could be varied

by changing the gas-phase basicity of the amines. Using the calculated GB of c-C3H2 920

kJ/mol as a bench mark (CCSD(T)/aug-cc-pVTZ single-point energies on CCSD/6-

311+G(2d,p) optimized geometries), we investigated reactions of c-C3H2 produced by

pyrolysis of allylchloride with a series of protonated alkyl amines and guanidine, with

their GBs ranging from 884 to 949 kJ/mol.7 The complexes ([C]) formed by these

reactions were subjected to CID to achieve 80-90% of dissociation.

The CID spectra of complexes formed by c-C3H2 addition to protonated n-butyl

amine, ethylmethyl amine, di-n-propylamine, and guanidine are shown in Figure 3.9a-d.

They represent cases that amines have GBs lower, comparable, higher, and significant

higher than that of c-C3H2, respectively. CID of the butylamine-C3H2 complex (m/z 112)

generates [C-40] (m/z 72) and m/z 39 at about equal relative intensity while the intensity

of [C-38] (m/z 74) is very low (Figure 3.9a). This is because the GB of n-butylamine

(887 kJ/mol) is smaller than that of c-C3H2 which is therefore more favorably protonated.

Based on the data shown in Figure 3.9a, the yield of the covalent complex is about 52%.

The CID spectrum of the ethylmethylamine-C3H2 complex (Figure 3.9b) shows the loss

of 40 Da (m/z 58) as the main fragmentation, leading to 68 % Covalent Complex. Given

the similar GBs of ethylmethylamine (909 kJ/mol) and c-C3H2, the ion intensities of

C3H3+ (m/z 39) and protonated amine (m/z 60) are comparable. When the GB is further

increased, as in the case of di-n-propylamine, (GB= 929 kJ/mol), only two fragment

channels are observed, [C-40] and [C-38], while m/z 39 intensity diminishes as a result of

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a lower GB of c-C3H2 relative to the amine. The % Covalent Complex is 49%. Guanidine

has the highest GB (949 kJ/mol)24 among all the species tested. CID of the complex

shows a loss of neutral carbene ([C-38], m/z 60) as the only fragmentation pathway.

Figure 3.9e summarizes the correlation between the % Covalent Complex and the

GB values of various alkyl amines and guanidine. The corresponding CID spectra are

shown in Table 3.2. The highest yield (82%) for the formation of a covalent product is

obtained for cyclohexylamine, which has a GB value slightly lower than c-C3H2. The

yield decreases as the GB difference becomes larger. The fact that covalent complex

formation is most favored with a minimal GB difference25 between the amines and c-

C3H2 strongly supports the formation of the proton-bound dimer is the critical step, as

pointed out by the theoretical calculations (Figure 3). Furthermore, it also supports that c-

C3H2 instead of other two isomers is mainly responsible for the reaction. The 1A1 ground

state of propadienylidene is both substantially less stable (+53 kJ/mol) and less basic (GB

= 860 kJ/mol) than c-C3H2. Propynylidene exists as a high-energy triplet state (3A”, +47

kJ/mol) of low basicity (GB = 687 kJ/mol) when forming a linear triplet C3H3+ cation.

90

90

Figure 3.9 Selected MS3 reaction product CID spectra of (a) butylamine, (b)

ethylmethylamine, (c) dipropylamine, and (d) guanidine with c-C3H2 and (e) %Covalent

of protonated alkyl amines/c-C3H2 reactions as a function of GBs of the corresponding

alkyl amines. The %Covalent is calculated as I[c-40]/(I[39]+I[c-40]+I[c-38]), where I[39], I[c-40],

and I[c-38] correspond to the intensity of m/z 39 as the proton-transfer product, neutral loss

of 40 Da as the covalent product, and neutral loss of 38 Da as the direct-dissociation

product, respectively, in the MS3 CID spectra based on 80-90% parent ions fragmented.

Error bars were based on 5 duplicates of an average of 3 spectra.

We also formed amine-C3H2 proton-bound dimer from reactions of neutral amines

(i.e. heptylamine) and C3H3+ ions. Ethyl, propyl, butyl, hexyl and heptyl amines were

investigated for ion/molecule reaction with c-C3H3+ ions. Table 1.1 summarized the

ion/molecule reaction spectra and their corresponding MS3 CID spectra. In comparison

with corresponding ion/carbene reactions, complexes of exactly the same m/z values were

observed for the reactions with neutral amines. Collisional activation of the heptylamine-

91

91

C3H3+ complex (Figure 3.10) produced a very similar fragmentation pattern to that of the

C3H2-protonted heptyl amine product shown in Figure 3.2b. The above result also

supports the reaction mechanism with the proton-bound dimer intermediate suggested for

the ion-carbene reactions.

Figure 3.10 MS3 reaction product CID spectrum of C3H3+and neutral n-heptyl amine

3.5 Conclusion

In summary, the home-developed DAPI-ion trap-pyrolysis MS platform facilitated

studies of reactions of c-C3H2 with protonated alkyl amines in the gas phase. Addition of

C3H2 to the protonated amine was observed as the only reaction channel. Mechanistic

studies demonstrate that non-covalent and covalent (via C-N formation) complexes can

be produced and they both stem from the formation of proton-bound dimers as the key

step. The covalent product formation is in favor of alkyl amines with similar GB values

as that of the c-C3H2. In the interstellar carbon chemistry, it has been long believed that

the major pathway for the formation of c-C3H2 comes from electron attachment to c-

C3H3+,26 while the major pathway for the destruction of c-C3H2 goes through the

protonation of c-C3H2 to form c-C3H3+, which makes c-C3H2 a “dead end” in this carbon

cycle.26-27 This study therefore provides a new perspective to other possible reaction

92

92

pathways of c-C3H2 in the interstellar medium. In the presence of protonated amines or

even ammonium, c-C3H2 can form covalent adducts to seed the formation of larger

organic molecules/ions consisting of C, N, and H atoms.

3.6 Appendix

Table 3.1 summarizes MS2 reaction spectra of all alkyl ammoniums investigated

with C3H2 generated by the pyrolysis of various organic precursors. The number of +38

Da adducts was corresponding to the number of protons on the charged amine site. The

nonspecific generation of C3H2 carbenes was observed from precursors with three or

more hydrocarbons. Therefore, cyclopropenylidene was most-likely the carbene species

produced during pyrolysis because it is more stable compared to the other two C3H2

carbenes. The series of adducts could be both covalent products and simple complexes,

thus CIDs were then used for structure elucidation on the selected carbene reaction

products.

Table 3.1 Ammonium reactions with various radical precursors

Reactant Radical

Precursor MS Spectrum No.

Heptylamine

[M+H]+

m/z 116

BrC≡CCH2Br

1,3-

Dibromopropyne

1

Allylchloride

2

NH2

Cl

93

93

Allylbromide

3

Allyliodide

4

Propargylbromide

5

1, 5-Hexadiene

6

Pentane

7

Pentane-D12

8

Heptylamine

18-Crown-6

[M+18C6+H]+

m/z 380

Propargylbromide

9

Heptylamine

12-Crown-4

[M+18C6+H]+

m/z 292

Propargylbromide

10

Br

I

Br

D3C

D2C

CD2

D2C

CD3

NH2

Br

NH2

Br

94

94

Heptylamine H/D

[M-2H+3D]+

m/z 119

1, 5-Hexadiene

11

Methylamine

[M+H]+

m/z 32

Propargylbromide

12

Ethylamine

[M+H]+

m/z 46

1, 5-Hexadiene

13

Propylamine

[M+H]+

m/z 60

Allylchloride

14

Butylamine

[M+H]+

m/z 74

Allylchloride

15

Isobutylamine

[M+H]+

m/z 74

Allylchloride

16

Hexylamine

[M+H]+

m/z 102

Allylchloride

17

Cyclohexylamine

[M+H]+

m/z 100

Allylchloride

18

ND

DD

Br

Cl

Cl

Cl

Cl

Cl

95

95

Ethylmethylamine

[M+H]+

m/z 60

Allylchloride

19

Diethylamine

[M+H]+

m/z 74

Allylchloride

20

Dipropylamine

[M+H]+

m/z 102

Allylchloride

21

Dibutylamine

[M+H]+

m/z 130

Allylchloride

22

Propargylbromide

23

1, 5-Hexadiene

24

Dibutylamine H/D

[M-H+2D]+

m/z 132

Propargylbromide

25

Diisopropylamine

[M+H]+

m/z 102

Allylchloride

26

Cl

Cl

Cl

HN

Cl

Br

NDD

Br

Cl

96

96

Guanidine

[M+H]+

m/z 60

Allylchloride

27

Tributylamine

[M+H]+

m/z 186

Allylchloride

28

Propargylbromide

29

1, 5-Hexadiene

30

Tributylamine H/D

[M+D]+

m/z 187

Propargylbromide

31

Cetyltrimethyl

ammonium

M+

m/z 284

Propargylbromide

32

1, 5-Hexadiene

33

Ion-trap CID for the carbene adducts was performed (shown in Table 3.2) to

illustrate three reaction channels discussed in 3.4.3. For the data points in Figure 3.9e of

%Covalent complex vs. GB curve, a LMCO of m/z 25 was used for all MS2 reactions and

Cl

N

Cl

Br

N

D

Br

N

CH3

CH3

CH3

H3C(H2C)15

Br

97

97

MS3 CIDs with the rf frequency of 1490 kHz. The rf frequency of 920 kHz was used to

trap ions larger than m/z 100 and to record the rest reaction and CID spectra. The final

stable product shown in Figure 3.6 had two of the original hydrogens of the carbene side,

so that deuterium scrambling was expected in MS3 CID spectra when using d12-pentane

as the carbene precursor (CID No.44) and H/D exchanged heptyl ammonium (CID

No.45).

Table 3.2 MS3-MS5 CID spectra of selected reaction products

Reaction No. Selected CID

precursor m/z MS Spectrum CID No.

1 MS3

116-154

34

2 MS3

116-154

35

2 MS3

116-192

36

2-36 MS4

116-192-154

37

2 MS3

116-230

38

98

98

2-38 MS4

116-230-192

39

2-38-39

MS5

116-230-192-

154

40

5 MS3

116-154

41

6 MS3

116-154

42

7 MS3

116-154

43

8 MS3

116-156

44

11 MS3

116-157

45

13 MS3

46-84

46

99

99

14 MS3

60-98

47

15 MS3

74-112

48

16 MS3

74-112

49

17 MS3

102-140

50

18 MS3

100-138

51

19 MS3

60-98

52

20 MS3

74-112

53

21 MS3

102-140

54

100

100

22 MS3

130-168

55

22 MS3

130-206

56

22-56 MS4

130-206-168

57

26 MS3

102-140

58

27 MS3

60-98

59

28 MS3

186-224

60

The internal ion source GDEI was designed to show the MS characterization with

pyrolysis on or off in Table 3.3. However, the radical precursors are usually easy to

generate reactive ion species (eg. C3H3+)28 by electron impact ionization. Based on the

pressure of 10-100 mTorr after DAPI opening, further reactions would occur between

ions and neutral precursors (also the impurity in the precursor) at the number density of

101

101

3.3-33x1014 cm-3. Therefore, the GDEI spectra could not directly reflect the radical

species generated by pyrolysis. Since the reactions between alkyl ammoniums and

radicals from various precursors to form +38 covalent adducts have strongly supported

the generation of C3H2 carbene species by the pyrolysis nozzle, the purpose of GDEI

characterization was just to observe the pattern changing when the pyrolysis nozzle was

turned on or off. The changing of GDEI pattern between room temperature and heated

precursors indicated the formation of new species by pyrolysis for all the precursors,

which was considered an indirectly evidence of C3H2 carbene reaction.

Table 3.3 GDEI spectra of radical precursors Precursor Pyrolysis MS Spectrum

BrC≡CCH2Br

1,3-Dibromopropyne

Off

On

Allylchloride

Off

On

Cl

102

102

Allylbromide

Off

On

Allyliodide

Off

On

Propargylbromide

Off

On

1, 5-Hexadiene

Off

On

Br

I

Br

103

103

Pentane

Off

On

C3H3+ ion/molecule reactions have been previously studied with multiple reaction

channels observed such as interactions with unsaturated hydrocarbons,28-29 proton transfer

and complexes.30 However, no CID results were reported to study the final structure of

the products, probably due to the instrumentational setup. In this section, we used

propargylchloride to generate a majority of c-C3H3+ ions by GDEI as comparison to the

proposed c-C3H2 carbene reactions. It was previously shown that propargylchloride

produced 85-90% of non-reactive cyclic C3H3+ ions by electron impact or chemical

ionization.28 No effort was made to further separate linear (reactive) and cyclic (non-

reactive) C3H3+ species. After isolating C3H3

+ m/z 39 ions with rf 1490 kHz at a LMCO of

m/z 10, neutral amines were injected through DAPI-1 with an additional capillary. The

reaction took place at LMCO of m/z 25, which allowed the trapping of m/z 39-180. CIDs

were performed at LMCO of m/z 25 as well. The MS3 CID spectra of ion/molecule

reactions in Table 1.1 were very similar to those of ion/carbene reactions in Table 3.2,

which proved they shared common reaction mechanism by forming a proton-bound

dimer.

104

104

Table 3.4 Ion/molecule reactions of c-C3H3+ and neutral amines

Reactants MS type MS Spectrum

c-C3H3+

+

Ethylamine

MS2

39-

MS3 CID

39-84-

c-C3H3+

+

Prpylamine

MS2

39-

MS3 CID

39-98-

c-C3H3+

+

Butylamine

MS2

39-

MS3 CID

39-112-

c-C3H3+

+

Hexylamine

MS2

39-

MS3 CID

39-140-

105

105

c-C3H3+

+

Heptylamine

MS2

39-

MS3 CID

39-154-

106

106

3.7 References

1. Taatjes, C. A.; Klippenstein, S. J.; Hansen, N.; Miller, J. A.; Cool, T. A.; Wang, J.;

Law, M. E.; Westmoreland, P. R., Synchrotron photoionization measurements of

combustion intermediates: Photoionization efficiency and identification of C3H2 isomers.

Phys. Chem. Chem. Phys. 2005, 7 (5), 806-813.

2. (a) Thaddeus, P.; Vrtilek, J. M.; Gottlieb, C. A., LABORATORY AND

ASTRONOMICAL IDENTIFICATION OF CYCLOPROPENYLIDENE, C3H2.

Astrophys. J. 1985, 299 (1), L63-L66; (b) Madden, S. C.; Irvine, W. M.; Matthews, H. E.;

Friberg, P.; Swade, D. A., A SURVEY OF CYCLOPROPENYLIDENE (C3H2) IN

GALACTIC SOURCES. Astron. J. 1989, 97 (5), 1403-1422.

3. Lavallo, V.; Canac, Y.; Donnadieu, B.; Schoeller, W. W.; Bertrand, G.,

Cyclopropenylidenes: From interstellar space to an isolated derivative in the laboratory.

Science 2006, 312 (5774), 722-724.

4. (a) Wass, D. F.; Haddow, M. F.; Hey, T. W.; Orpen, A. G.; Russell, C. A.;

Wingad, R. L.; Green, M., Cyclopropenylidene carbene ligands in palladium C-C

coupling catalysis. Chem. Commun. 2007, (26), 2704-2706; (b) Kuchenbeiser, G.;

Donnadieu, B.; Bertrand, G., Stable bis(diisopropylamino)cyclopropenylidene (BAC) as

ligand for transition metal complexes. J. Organomet. Chem. 2008, 693 (5), 899-904; (c)

Wilde, M. M. D.; Gravel, M., Bis(amino)cyclopropenylidenes as Organocatalysts for

Acyl Anion and Extended Umpolung Reactions. Angew. Chem., Int. Ed. 2013, 52 (48),

12651-12654.

5. (a) Seburg, R. A.; Patterson, E. V.; Stanton, J. F.; McMahon, R. J., Structures,

automerizations, and isomerizations of C3H2 isomers. J. Am. Chem. Soc. 1997, 119 (25),

5847-5856; (b) Mebel, A. M.; Jackson, W. M.; Chang, A. H. H.; Lin, S. H.,

Photodissociation dynamics of propyne and allene: A view from ab initio calculations of

the C3Hn (n=1-4) species and the isomerization mechanism for C3H2. J. Am. Chem. Soc.

1998, 120 (23), 5751-5763; (c) Vazquez, J.; Harding, M. E.; Gauss, J.; Stanton, J. F.,

High-Accuracy Extrapolated ab Initio Thermochemistry of the Propargyl Radical and the

Singlet C3H2 Carbenes. J. Phys. Chem. A 2009, 113 (45), 12447-12453; (d) Wu, Q.;

Cheng, Q.; Yamaguchi, Y.; Li, Q.; Schaefer, H. F., III, Triplet states of

cyclopropenylidene and its isomers. J. Chem. Phys. 2010, 132 (4).

6. Chyall, L. J.; Squires, R. R., DETERMINATION OF THE PROTON AFFINITY

AND ABSOLUTE HEAT OF FORMATION OF CYCLOPROPENYLIDENE. Int. J.

Mass Spectrom. Ion Processes 1995, 149, 257-266.

7. Hunter, E. P. L.; Lias, S. G., Evaluated gas phase basicities and proton affinities

of molecules: An update. J. Phys. Chem. Ref. Data 1998, 27 (3), 413-656.

8. (a) Tan, X. J.; Li, Z.; Sun, Q.; Li, P.; Wang, W. H., Theoretical Study on the

Mechanism of the Addition Reaction between Cyclopropenylidene and Formaldehyde.

Bull. Korean Chem. Soc. 2012, 33 (6), 1934-1938; (b) Tan, X. J.; Li, Z.; Sun, Q.; Li, P.;

Wang, W. H.; Wang, G. R., A Theoretical Study on the Mechanism of the Addition

Reaction between Cyclopropenylidene and Ethylene. J. Chil. Chem. Soc. 2012, 57 (2),

1174-1177; (c) Li, Q. L.; Sun, Q.; Gu, J. S.; Tan, X. J., A computational study of the

addition reaction of cyclopropenylidene with methyleneimine. Russ. J. Phys. Chem. A

2013, 87 (5), 806-812.

107

107

9. (a) Jing, Y.; Liu, H.; Wang, H. L.; Yu, Y.; Tan, X. J.; Chen, Y. G.; Zhang, Y. L.;

Gu, J. S., Quantum Mechanical Study of the Insertion Reaction between

Cyclopropenylidene with R-H (R=F, Oh, Nh2, Ch3). J. Chil. Chem. Soc. 2013, 58 (4),

2218-2221; (b) Tan, X. J.; Li, Z.; Sun, Q.; Li, P.; Wang, W. H.; Wang, M. Y.; Chen, Y.

G., Theoretical study on the reaction mechanisms between propadienylidene and R-H

(R=F, OH, NH2, CH3): an alternative approach to the formation of alkyne. Struct. Chem.

2013, 24 (1), 33-38.

10. (a) Tan, X. J.; Wang, W. H.; Jing, Y.; Wang, F.; Li, P., Theoretical study on the

reaction mechanism of cyclopropenylidene with azacyclopropane: ring expansion process.

Monatsh. Chem. 2014, 145 (7), 1109-1115; (b) Tan, X. J.; Wang, W. H.; Sun, Q.; Jing, Y.;

Li, P., Theoretical study of the ring expansion reaction mechanism of cyclopropenylidene

with azetidine. J. Mol. Model. 2014, 20 (3).

11. Gao, L.; Cooks, R. G.; Ouyang, Z., Breaking the pumping speed barrier in mass

spectrometry: Discontinuous atmospheric pressure interface. Anal. Chem. 2008, 80 (11),

4026-4032.

12. Ouyang, Z.; Wu, G. X.; Song, Y. S.; Li, H. Y.; Plass, W. R.; Cooks, R. G.,

Rectilinear ion trap: Concepts, calculations, and analytical performance of a new mass

analyzer. Anal. Chem. 2004, 76 (16), 4595-4605.

13. Lin, Z.; Tan, L.; Garimella, S.; Li, L.; Chen, T.-C.; Xu, W.; Xia, Y.; Ouyang, Z.,

Characterization of a DAPI-RIT-DAPI System for Gas-Phase Ion/Molecule and Ion/Ion

Reactions. J. Am. Soc. Mass Spectrom. 2014, 25 (1), 48-56.

14. Gao, L.; Song, Q.; Noll, R. J.; Duncan, J.; Cooks, R. G.; Zheng, O., Glow

discharge electron impact ionization source for miniature mass spectrometers. J. Mass

Spectrom. 2007, 42 (5), 675-680.

15. Kohn, D. W.; Clauberg, H.; Chen, P., FLASH PYROLYSIS NOZZLE FOR

GENERATION OF RADICALS IN A SUPERSONIC JET EXPANSION. Rev. Sci.

Instrum. 1992, 63 (8), 4003-4005.

16. Zhang, X.; Friderichsen, A. V.; Nandi, S.; Ellison, G. B.; David, D. E.; McKinnon,

J. T.; Lindeman, T. G.; Dayton, D. C.; Nimlos, M. R., Intense, hyperthermal source of

organic radicals for matrix-isolation spectroscopy. Rev. Sci. Instrum. 2003, 74 (6), 3077-

3086.

17. Zhang, X.; Kato, S.; Bierbaum, V. M.; Nimlos, M. R.; Ellison, G. B., Use of a

flowing afterglow SIFT apparatus to study the reactions of ions with organic radicals. J.

Phys. Chem. A 2004, 108 (45), 9733-9741.

18. Russell, A. L.; Rohrs, H. W.; Read, D.; Giblin, D. E.; Gaspar, P. P.; Gross, M. L.,

Radical cation/radical reactions: A Fourier transform ion cyclotron resonance study of

allyl radical reacting with aromatic radical cations. Int. J. Mass Spectrom. 2009, 287 (1-3),

8-15.

19. Clauberg, H.; Minsek, D. W.; Chen, P., Mass and Photoelectron-Spectroscopy of

C3h2 - Delta-Hf of Singlet Carbenes Deviate from Additivity by Their Singlet Triplet

Gaps. J. Am. Chem. Soc. 1992, 114 (1), 99-107.

20. Greaves, P. M.; Kalli, M.; Landor, P. D.; Landor, S. R., Allenes .21. Preparation

of 1,1-Dialkyl-3-Iodoallenes and 1,1-Dihalogenoallenes. Journal of the Chemical Society

C-Organic 1971, (4), 667-&.

108

108

21. Talley, J. M.; Cerda, B. A.; Ohanessian, G.; Wesdemiotis, C., Alkali metal ion

binding to amino acids versus their methyl esters: Affinity trends and structural changes

in the gas phase. Chem.-Eur. J. 2002, 8 (6), 1377-1388.

22. Garimella, S.; Zhou, X.; Ouyang, Z., Simulation of Rarefied Gas Flows in

Atmospheric Pressure Interfaces for Mass Spectrometry Systems. J. Am. Soc. Mass

Spectrom. 2013, 24 (12), 1890-1899.

23. Maier, G.; Preiss, T.; Reisenauer, H. P.; Hess, B. A.; Schaad, L. J., Small

Rings .82. Chlorinated Cyclopropenylidenes, Vinylidenecarbenes, and Propargylenes -

Identification by Matrix-Isolation Spectroscopy. J. Am. Chem. Soc. 1994, 116 (5), 2014-

2018.

24. Yao, C.; Syrstad, E. A.; Tureček, F., Electron transfer to protonated beta-alanine

N-methylamide in the gas phase: An experimental and computational study of

dissociation energetics and mechanisms. J. Phys. Chem. A 2007, 111 (20), 4167-4180.

25. (a) Davidson, W. R.; Sunner, J.; Kebarle, P., HYDROGEN-BONDING OF

WATER TO ONIUM IONS - HYDRATION OF SUBSTITUTED PYRIDINIUM IONS

AND RELATED SYSTEMS. J. Am. Chem. Soc. 1979, 101 (7), 1675-1680; (b) Larson, J.

W.; McMahon, T. B., FORMATION, THERMOCHEMISTRY, AND RELATIVE

STABILITIES OF PROTON-BOUND DIMERS OF OXYGEN N-DONOR BASES

FROM ION-CYCLOTRON RESONANCE SOLVENT-EXCHANGE EQUILIBRIA

MEASUREMENTS. J. Am. Chem. Soc. 1982, 104 (23), 6255-6261; (c) Bian, L., Proton

donor is more important than proton acceptor in hydrogen bond formation: A universal

equation for calculation of hydrogen bond strength. J. Phys. Chem. A 2003, 107 (51),

11517-11524.

26. Maluendes, S. A.; McLean, A. D.; Herbst, E., Calculations Concerning

Interstellar Isomeric Abundance Ratios for C3h and C3h2. Astrophys. J. 1993, 417 (1),

181-186.

27. Millar, T. J.; Farquhar, P. R. A.; Willacy, K., The UMIST database for

astrochemistry 1995. Astron. Astrophys. Suppl. Ser. 1997, 121 (1), 139-185.

28. Ozturk, F.; Baykut, G.; Moini, M.; Eyler, J. R., REACTIONS OF C3H3+ WITH

ACETYLENE AND DIACETYLENE IN THE GAS-PHASE. J. Phys. Chem. 1987, 91

(16), 4360-4364.

29. Smyth, K. C.; Lias, S. G.; Ausloos, P., THE ION-MOLECULE CHEMISTRY OF

C3H3+ AND THE IMPLICATIONS FOR SOOT FORMATION. Combust. Sci. Technol.

1982, 28 (3-4), 147-154.

30. Prodnuk, S. D.; Gronert, S.; Bierbaum, V. M.; Depuy, C. H., GAS-PHASE

REACTIONS OF C3HN+ IONS. Org. Mass Spectrom. 1992, 27 (4), 416-422.

109

109

CHAPTER 4. GAS-PHASE C-C3H2 CARBENE REACTIONS WITH

BIOMOLECULAR IONS

4.1 Introduction

It has been discussed in the previous chapter that c-C3H2 can be generated by non-

specific pyrolysis under the conditions in DAPI-RIT-DAPI mass spectrometer with 10-2

Torr pyrolysis pressure and energetics gas flows. The reactions between protonated alkyl

amines and c-C3H2 have been studied and covalent products have been observed to form

through a proton-bound dimer mechanism with further rearrangements.

Biomolecules such as nucleobases, nucleosides, amino acids, peptides, and proteins

are more complicated than alkyl amines, which also contain amine functional groups. The

protonated biomolecules are expected to be modified by c-C3H2 carbene through proton-

bound dimer, possibly to form covalent reaction products. Besides, c-C3H2 carbene have

been theoretically calculated to react with a single R-H (R=F, O, N) bond for insertion

and also with C=O and C=C bonds for addition.1 Furthermore, reactive functional groups

including thiyl group, disulfide linkages are present in amino acids and peptides. These

functional groups were reported to be reactive with free radicals like hydroxyl radicals

and sulfinyl radicals.2 It is still unknown if the bi-radical species c-C3H2 could modify

these functional groups in their gas-phase ionic forms. In this chapter, the reactivity of c-

C3H2 carbene was studied by the interaction with protonated biomolecules mentioned

above for proton-bound reactions as well as other possible gas-phase reactions.

110

110

4.2 Experimental section

Materials

All nucleobases, nucleosides, amino acids, peptides, proteins and lipids were

purchased from Sigma Chemical Co. (Sigma-Aldrich, St. Louis, MO). Different

concentrations (5-100 μM) of the samples were prepared with methanol/water (9:1, v/v)

with 1 % acetic acid added to increase the intensity of protonated species if necessary.

Radical precursors including allylchloride and 1,5-hexadiene were also purchased from

Sigma. Allylchloride was chosen as the major C3H2 carbene precursor unless otherwise

specified.

MS System

All conditions were directly adapted from Chapter 3 except for the rf frequency. Two

rf frequencies were utilized: the frequency of 950 kHz was employed for m/z 100-500 to

observe reactions of amino acids, small peptides, nucleobases and nucleosides, whereas

the frequency of 650 kHz allowed high mass range to m/z 1500 to analyze ions of large

peptides and proteins. Resonance ejection (q = 0.81) was implemented for MS scan with

a dipolar ac signal at 334 and 229 kHz for rf frequency of 950 and 650 kHz, respectively.

4.3 Results and discussion

4.3.1 Nucleobases and nucleosides

The interactions of protonated nucleobases and nucleosides (structures and gas-phase

basicities shown in Table 4.1) with c-C3H2 carbene generated by pyrolysis of

allylchloride have been investigated to study the carbene modification of DNA/RNA

related biomolecules in gas phase.

111

111

Table 4.1 Name, structure, and gas-phase basicity3 (GB) of nucleobases and nucleosides4

Nucleobase Structure

Gas-

phase

basicity

(kJ/mol)

Nucleoside Structure

Gas-

phase

basicity

(kJ/mol)

Adenine

912 Adenosine

957

Guanine

928 Guanosine

961

Cytosine

918 Cytidine

950

Uracil

842 Uridine

917

Thymine

850 Thymidine

916

For nucleobases of adenine, guanine, and cytosine, their GBs are comparable to that

of c-C3H2 carbene.5 Thus, the MS2 carbene reaction spectra were very similar for these

three nucleobases and a significant amount of +38 Da adducts were observed. Taking

adenine reaction as an example shown in Figure 4.1a, mass increases from 1 × 38 Da to 3

× 38 Da; which corresponded to mono-, di-, and tri- carbene adducts were observed.

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112

Despite adenine having multiple protonation sites,6 the 3 × 38 Da mass increase of m/z

250 implied a primary amine was responsible for protonation in this specific reaction. In

the MS3 CID spectrum of Figure 4.1b, a direct loss of 38 Da was obtained when

activating the mono-adduct of m/z 174, but the intensity was very low. According to the

close GBs of adenine and c-C3H2 carbene,7 a covalent bond formation should be favored

as experimentally demonstrated in Chapter 3. However, the adjacent carbon of the

primary amine on adenine is not attached to a hydrogen. The covalent product therefore,

would not be able to undergo a loss of 40 Da in the fragmentation pathway as in the cases

of amines. The low yield of CID might serve as a piece of indirect evidence of the

covalent product formation. As for its corresponding nucleoside adenosine, multiple +38

Da adducts as well as the fragments from the carbene products were obtained in Figure

4.1c. Nucleosides have higher GBs than their corresponding nucleobases.8 Simple

complexes should be preferred for adenosine with GB of 957 kJ/mol. In the MS3 CID

spectrum of the reaction product at m/z 306, only a small portion of 38 Da loss (m/z 268)

appeared as the direct loss of C3H2 in Figure 4.1d, while the principle fragment was the

loss of ribose ring (loss of 132 Da). This result indicated that the hydrogen bond in the

proton-bound dimer is stronger than the covalent N-glycosidic bond. The input energy

might be able to trigger further rearrangement of the complex to form covalent product.

A very low amount of m/z 136 also appeared and this was probably due to the further

activation of m/z 174. Similar reaction phenomena were observed for nucleobases of

guanine, cytosine and nucleosides of guanosine, cytidine. All MS2 reaction and MS3 CID

spectra are included in Table 4.2.

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Figure 4.1 MS2 carbene reaction spectra of (a) protonated adenine and (c) protonated

adenosine; and MS3 CID spectra (b) adenine carbene monoadduct and (d) adenosine

carbene monoadduct

Uracil and thymine have significantly lower GBs compared to c-C3H2,9 which makes

them harder for the formation of proton-bound dimers. The major product of the

protonated molecules for these two species was expected to be a proton-transfer product

c-C3H3+, when encountering gas-phase c-C3H2 carbene. Unfortunately, ions of m/z 39

could not be trapped under rf frequency of 950 kHz. Figure 4.2a shows the MS2 reaction

spectrum of protonated uracil with c-C3H2 carbene. No adduct was formed with 38 Da

mass increase, instead only an intensity decrease of the molecular ions was recorded,

probably because of the proton-transfer reaction mentioned above. Although protonated

uracil did not form proton-bound dimer with c-C3H2 due to the low GB, its corresponding

nucleoside uridine showed such a reaction channel in Figure 4.2b since the ribose ring

pushed the electron cloud and thus increased the GB to the same level of c-C3H2.10 The

protonation might locate on the amide amine with two exchangeable hydrogens to induce

a di- carbene adduct. The MS3 CID (Figure 4.2c) of the mono-adduct in uridine reaction

yielded a similar fragmentation pathway as those of adenosine, guanine, and cytidine.

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114

The fact that uracil did not form proton-bound dimer at m/z 151 while uridine-carbene

proton-bound dimer yielded m/z 151 during CID, indicates that the formation of proton-

bound dimer could be manipulated for low GB compounds with the modification of an

easy-to-cleave side chain. For thymine and thymidine, almost identical reactions were

recorded in Table 4.2 as uracil and uridine, except that thymidine has a 2’-deoxyribose

ring instead of a ribose ring. Thus, the neutral loss observed is 116 Da instead of 132 Da

upon activation.

Figure 4.2 MS2 carbene reaction spectra of (a) protonated uracil and (b) protonated

uridine; and MS3 CID spectrum (c) uridine carbene monoadduct

The reactions between nucleobases, nucleosides and pyrolysis-produced carbene

showed that c-C3H2 was able to modify DNA/RNA related gas-phase biomolecular ions.

The reactions of adenine, guanine and cytosine with c-C3H2 tend to form covalent

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products, yet the products lack dissociation pathway to form fragments during MS3 CID.

Uracil and thymine have GBs that are too low to form proton-bound dimers, however

uridine and thymidine are able to form proton-bound dimers and ribose or deoxyribose

ring loss upon CID was observed for the product ions. Adenosine, guanosine, and

cytidine can form proton-bound dimer with c-C3H2. The proton bound energy is higher

than the N-glycosidic bonds and further rearrangements might happen to covert those

dimers into covalent products.

Table 4.2 Ion/carbene reactions of nucleobases, nucleosides and c-C3H2 Reactants MS type MS Spectrum

Protonated

adenine

+

c-C3H2

MS2 Reaction

MS3 CID

Protonated

guanine

+

c-C3H2

MS2 Reaction

MS3 CID

Protonated

cytosine

+

c-C3H2

MS2 Reaction

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116

MS3 CID

Protonated uracil

+

c-C3H2

MS2 Reaction

Protonated

thymine

+

c-C3H2

MS2 Reaction

Protonated

adenosine

+

c-C3H2

MS2 Reaction

MS3 CID

Protonated

guanosine

+

c-C3H2

MS2 Reaction

MS3 CID

Protonated

cytidine

+

c-C3H2

MS2 Reaction

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117

MS3 CID

Protonated uridine

+

c-C3H2

MS2 Reaction

MS3 CID

Protonated

thymidine

+

c-C3H2

MS2 Reaction

4.3.2 Amino acids, peptides, proteins, and lipids

4.3.2.1 Protonated amino acids and singly charged peptides

Protonated amino acids containing both amine and carboxylic acid functional groups

are expected to react with C3H2 via the proton-bound dimer mechanism proposed in the

previous chapter. Compared to the simple amine system studied previously, gas-phase

chemistry should be more complicated with these basic building blocks of macro

biomolecules in terms of fragmentation pathways during CID.

For basic amino acids of Lys, Arg, and His, their GB values are all higher than c-

C3H2, however the MS2 reaction spectra of those three amino acids all showed abundance

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38 Da mass increases and new fragmentation pathway in MS3 CID. This was probably

due to proton relocation in the basic amino acid ions. The cyclopropenylidene would

interact at the proton site which has a similar GB as itself and form a proton-bound dimer.

For instance, the reaction in Figure 2.1a between protonated Lys and c-C3H2 produced

mono- and di- adduct of m/z 185 and 223 as well as some other fragment peaks due to the

excessive internal energy generated by this reaction. MS3 CID of the reaction product of

m/z 185 (Figure 2.1b) yielded all the fragments appeared in the reaction spectrum. A

reaction scheme is presented in Figure 2.1c to illustrate a possible fragmentation pathway

of the proton-bound dimer. The reaction product of m/z 185 could dissociate into the

original reactant protonated Lys of m/z 147 by directly losing c-C3H2 of 38 Da. Thus,

protonated Lys further underwent a NH3 loss, generating m/z 130 cyclic ions upon

activation, which was a common neutral loss for Lys and other amino acids.11 Another

fragmentation pathway was through a covalent product after the carbene reaction. Gas-

phase c-C3H2 could be covalently attached to a nitrogen atom and form a new species.

Although the location of the attachment (side chain or N-terminal) is still unknown, the

covalent product would undergo a NH3 loss similar to the protonated Lys and yield a

cyclic CID product of m/z 168 with an alkyl chain covalently bonded to the amine. This

structure could lose 40 Da C3H4 by the attack of the hydrogen on the adjacent carbon as

the fragmentation pattern of simple amines in the previous chapter, generating a cyclic

iminium of m/z 128. An alternative pathway was for the alkyl chain to grab the hydrogen

on the carboxyl acid to further induce a CO2 (44 Da) loss to form a fragment of m/z 124.

Both (1) and (2) pathways were likely to be electrophilic attacks. The loss of 44 Da did

not appear in low energy fragmentation pathways12 of amino acids and was highly

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possible to be related to the carbene chemistry. Indeed, similar loss was also observed in

His/carbene spectrum (Table 1.1).

Figure 4.3 (a) MS2 carbene reaction spectrum of protonated Lys, (b) MS3 CID spectrum

of Lys/carbene reaction product, and (c) proposed fragmentation pathway of the reaction

product.

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In the case of Arg, CID of protonated Arg (m/z 175) yielded m/z 158 (-H2O), 157 (-

NH3), 140 (-H2O-NH3), 130 (-NH3-CO or -COOH), 116 (-(NH2)2C=NH), and 112 (-H2O-

NH3-CO) (Table 1.1). Therefore, the mono-adduct of m/z 213 in Arg/carbene reaction in

Figure 4.4b yielded: m/z 175 (-C3H2), 169 (-CO2), 152 (-CO2-NH3), 114 (-(NH2)2C=NH -

C3H4), 112 (-C3H2 - H2O-NH3-CO), and 110 (-C3H4 - H2O-NH3-CO) during CID. The

fragments of m/z 175 were due to the direct loss of the carbene from the reaction

monoadduct; m/z 169 and 152 confirmed the CO2 loss pattern by the carbene similar to

Lys and His; and m/z 114 and 110 were possibly the oxidized products of m/z 116 and

112 (two hydrogen removed) in the Arg CID spectrum respectively. The covalent

addition of Arg/carbene reaction might be located at the N-terminal instead of the side

guanidine chain according to the CID fragmentation pathways. However, the most

favorable protonation site was at the Arg residue in singly charged peptide Met-Arg-Phe-

Ala (MRFA) so that the MS3 CID of the complex of singly charged MRFA showed only

dominant fragment at m/z 524 by directly losing C3H2. The alternative N-terminal of Met

residue was unlikely to be protonated.

Figure 4.4 MS2 carbene reaction spectra of (a) protonated Arg and (c) singly charged

MRFA; MS3 CID spectra of (b) Arg/carbene reaction product and (d) MRFA/carbene

reaction product.

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121

The results of protonated basic amino acids and MRFA interacting with

cyclopropenylidene show that although the GBs of the ionic reactants were much higher

than C3H2, covalent products could still be formed based on the MS3 CID fragments, as

long as an alternative protonation site existed close enough to the favored charged

position in the ionic species which had a suitable GB to form a proton-bound dimer for

further rearrangements. Other amino acids were also tested for their modifications with

C3H2 carbene and shown in Table 1.1. A neutral loss of 46 Da (-H2O-CO) was observed

for Phe/C3H2 reaction products;13 a loss of 40 Da (-C3H4) was found for Tyr/C3H2

reaction products; a loss of 17 Da (-NH3) was obtained for Met/C3H2 and Gln/C3H2

reaction products. These results support the formation of a covalent bond.

4.3.2.2 Multiply charged peptides and proteins

Multiply charged peptides could facilitate the C3H2 complex formation through the

proton-bound dimer mechanism due to more charges buried inside one ionic species.

Indeed, triply charged Angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu,

DRVYIHPFHL) of m/z 433 had up to ten adducts when encountering gas-phase C3H2 by

the pyrolysis of allylchloride in Figure 4.5. The number of adducts was related to the

number density of C3H2 carbene generated by pyrolysis, which is as a function of the

pyrolysis temperature. For 750 degree Celsius, the precursor triply charged ions together

with up to four carbene adducts appeared in carbene reaction spectrum (Figure 4.5a),

whereas no precursor ions were present anymore for the pyrolysis temperature of 800 and

900 degree Celsius in Figure 4.5b and c. With the higher temperature for pyrolysis, up to

ten carbene adducts were observed in Figure 4.5c.

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Figure 4.5 MS2 reaction spectra of triply charged Angiotensin I with heated allylchoride

at (a) 750, (b) 800, and (c) 900 degree Celsius.

Multiply charged protein can also react with the gas-phase carbene species. No mass

selection was made to isolate a single ionic population. The purpose of this set of

experiment was to evaluate the carbene reactivity towards different charge states. For

Ubiquitin, seven to twelve multiply charged proteins were observed in MS1 full scan in

Figure 4.6a. The protein-carbene complexes corresponding to the eight to eleven charge

states were formed in Figure 4.6b. The number of carbene adducts has an increasing trend

with the increase of charge states as predicted. The 11+ charge state species yielded

approximately 35 carbene adducts based on the difference of m/z values. The lowest

addition number observed in this experiment was ca. 23 for the 8+ charge state.

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123

Figure 4.6 (a) MS1 spectrum of multiply-charged Ubiquitin, and (b) MS2 spectrum of its

reaction with heated allylchoride.

4.3.2.3 Reactive functional groups

Since carbene is a highly reactive species, it would be interesting to investigate if this

bi-radical species would interact with relatively reactive functional group such as thiyl

group in Cys and disulfide linkages in peptides. Acetyl cysteine with the N-terminal

blocked was selected to test the reactivity of thiyl group and c-C3H2 carbene, which was

designed to minimize the reaction of the protonated amine site. The very low yield of 38

Da mass increase from the protonated species in Figure 4.7a, proved that thiyl group had

limited reactivity when encountering gas-phase c-C3H2 carbene. The mass increase was

most likely due to the complex formation with the protonated amide, similar to the cases

in thymine and uracil.

Disulfide linkage is an important posttranslational modification to construct three

dimensional structure in proteins. A simple disulfide linked di- amino acid cystine was

used in its protonated form to interact with c-C3H2 carbene. Despite the abundant 38 Da

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mass increases in the reaction spectrum (Figure 4.7b), there was no ion observed from

m/z 80-160 (cysteine thiyl radical ion: m/z 121, perthiyl radical ion: m/z 153), which

indicated no disulfide cleavage occurred during the gas-phase carbene reaction. Moreover,

using singly charged oxidized glutathione (single chain sequence: γ-Glu-Cys-Gly, γECG,

disulfide linked), the m/z shift still appeared to be 38 Da, however, no sign of disulfide

cleavage was observed in Figure 4.7c.

Figure 4.7 MS2 carbene reaction spectra of (a) protonated acetyl cysteine, (b) protonated

cystine, and (c) singly charged oxidized glutathione.

Unsaturated phospholipids play an important role in biological functions.14 It was

theoretically calculated and reported that c-C3H2 can react with hydroxyl group for single

bond insertion,1a and neutral unsaturated compounds for double bond addition.1c, 1d

Nevertheless, there has yet to be any experimental evidence for the mentioned

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125

neutral/carbene or ion/carbene reactions. Choline was then used to test the reactivity of c-

C3H2 with hydroxyl group. The advantage of gas-phase choline ion is that it has a fixed

charge to eliminate the proton-bound dimer reaction. In the reaction spectrum of Figure

4.8a, no mass shift was observed for choline ions, inferring insertion reactions did not

occur under the current experimental conditions between choline and c-C3H2.

Furthermore, the sodiated lysophosphatidylcholine (LPC) with an eighteen-carbon acetyl

chain and two double bonds in the 9th and 12th positions (18:2∆9, 12) was generated and

isolated to interact c-C3H2 carbene in Figure 4.8b. The lack of ions at m/z 590 indicated

the gas-phase cyclopropenylidene did not undergo double bond insertion in the LPC

studied.

Figure 4.8 MS2 reaction spectra of (a) hydroxyl group containing choline and (b) double

bond containing LPC (18:2) sodium adduct. No reaction product was observed.

The absence of reactivity of gas-phase c-C3H2 towards neutral thiyl group, disulfide

linkages, hydroxyl group, and double bonds based on the results of Figure 4.7 and Figure

4.8, suggested that the gas-phase chemistry of ion/c-C3H2 was mainly driven by the

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126

proton and the mechanism proposed in the previous chapter. The theoretically calculated

reaction pathway might not occur or be totally shadowed because of the high GB of the

cyclic carbene. All MS2 reaction and MS3 CID spectra are attached in Table 1.1 for all

the biomolecular ions studied in this section.

Table 4.3 Ion/carbene reactions of amino acids, peptides, protein and c-C3H2 Reactant and GB MS type MS Spectrum

Protonated Lysine

951 kJ/mol

MS2 Reaction

MS3 CID

Protonated Histidine

950 kJ/mol

MS2 Reaction

Protonated Arginine

1007 kJ/mol

MS2 Reaction

MS3 CID

MS2 CID

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127

Protonated Leucine

881 kJ/mol

MS2 Reaction

Protonated Methionine

902 kJ/mol

MS2 Reaction

Protonated

Phenylalanine

889 kJ/mol

MS2 Reaction

MS3 CID

Protonated Tyrosine

892 kJ/mol

MS2 Reaction

MS3 CID

Protonated Glutamine

900 kJ/mol

MS2 Reaction

MS3 CID

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128

Protonated Proline

methyl ester

MS2 Reaction

MS3 CID

Proline methyl ester

sodium adduct

MS2 Reaction

MS3 CID

Protonated Cysteine

methyl ester

MS2 Reaction

Protonated Acetyl

Cysteine

MS2 Reaction

Protonated Acetyl

Cysteine methyl ester

MS2 Reaction

Protonated Cystine

MS2 Reaction

with heated

allylchloride

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129

MS3 CID

MS2 Reaction

with heated

propargylbromide

MS2 Reaction

with heated

pentane

MS2 Reaction

with heated d12-

pentane

MS2 Reaction

with heated

dichloromethane

Oxidized glutathione

γ-ECG

|

γ-ECG

Singly charged

MS2 Reaction

Doubly charged

MS2 Reaction

Singly charged

Met-Arg-Phe-Ala

MRFA

MS2 Reaction

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130

MS3 CID

Triply charged

Angiotensin I

Asp-Arg-Val-Tyr-Ile-

His-Pro-Phe-His-Leu

DRVYIHPFHL

MS2 Reaction

Low amount of

C3H2

MS2 Reaction

Medium amount

of C3H2

MS2 Reaction

High amount of

C3H2

Ubiquitin

MS1

MS2 Reaction

Choline

MS2 Reaction

Lysophosphatidylcholine

LPC(18:2) sodium

adduct

MS2 Reaction

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131

4.4 Conclusion

The interaction between biomolecular ions and c-C3H2 have been investigated.

Proton-bound dimers were formed for protonated nucleobases and nucleosides including

adenine, guanine, cytosine, adenosine, guanosine, cytidine, thymidine, and uridine. The

hydrogen bond energy in the proton-bound dimer was determined to be higher than the

N-glycosidic bonds in all nucleosides. The GB values for uracil and thymine are too low

for the complex to form. Protonated basic amino acids were shown to generate covalent

reaction product with c-C3H2 carbene by relocating the proton to a charged site of a

similar GB. Multiply charged peptides and proteins have a wide range of adduct

distribution with different amounts of c-C3H2 generated by various pyrolysis

temperatures. However, the gas-phase reactivity of c-C3H2 carbene was mainly driven by

the charge and high GB of this species. The bi-radical carbene species had limited or no

modification for free thiyl group and disulfide linkages. Furthermore, the calculated

reactivity of double bond addition and single bond insertion did not occur with

biomolecular ions such as choline and LPC. The proton-bound dimer reaction remained

the dominant channel for all the biomolecular ion/c-C3H2 reactions.

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4.5 References

1. (a) Jing, Y.; Liu, H.; Wang, H. L.; Yu, Y.; Tan, X. J.; Chen, Y. G.; Zhang, Y. L.;

Gu, J. S., Quantum Mechanical Study of the Insertion Reaction between

Cyclopropenylidene with R-H (R=F, Oh, Nh2, Ch3). J. Chil. Chem. Soc. 2013, 58 (4),

2218-2221; (b) Tan, X. J.; Li, Z.; Sun, Q.; Li, P.; Wang, W. H.; Wang, M. Y.; Chen, Y.

G., Theoretical study on the reaction mechanisms between propadienylidene and R-H

(R=F, OH, NH2, CH3): an alternative approach to the formation of alkyne. Struct. Chem.

2013, 24 (1), 33-38; (c) Tan, X. J.; Li, Z.; Sun, Q.; Li, P.; Wang, W. H.; Wang, G. R., A

Theoretical Study on the Mechanism of the Addition Reaction between

Cyclopropenylidene and Ethylene. J. Chil. Chem. Soc. 2012, 57 (2), 1174-1177; (d) Wass,

D. F.; Hey, T. W.; Rodriguez-Castro, J.; Russell, C. A.; Shishkov, I. V.; Wingad, R. L.;

Green, M., Cyclopropenylidene carbene Ligands in palladium c-n coupling catalysis.

Organometallics 2007, 26 (19), 4702-4703.

2. (a) Durand, K. L.; Ma, X.; Xia, Y., Intra-molecular reactions between cysteine

sulfinyl radical and a disulfide bond within peptide ions. Int. J. Mass spectrom. (In press);

(b) Ma, X. X.; Love, C. B.; Zhang, X. R.; Xia, Y., Gas-Phase Fragmentation of [M + nH

+ OH](n center dot+) Ions Formed from Peptides Containing Intra-Molecular Disulfide

Bonds. J. Am. Soc. Mass Spectrom. 2011, 22 (5), 922-930; (c) Stinson, C. A.; Xia, Y.,

Radical induced disulfide bond cleavage within peptides via ultraviolet irradiation of an

electrospray plume. Analyst 2013, 138 (10), 2840-2846; (d) Durand, K. L.; Ma, X. X.;

Xia, Y., Intra-molecular reactions as a new approach to investigate bio-radical reactivity:

a case study of cysteine sulfinyl radicals. Analyst 2014, 139 (6), 1327-1330.

3. Haynes, W. M., CRC Handbook of Chemistry and Physics 94th Edition Internet

Version. http://www.hbcpnetbase.com/: 2014.

4. Hunter, E. P. L.; Lias, S. G., Evaluated gas phase basicities and proton affinities

of molecules: An update. J. Phys. Chem. Ref. Data 1998, 27 (3), 413-656.

5. Wu, Q.; Cheng, Q.; Yamaguchi, Y.; Li, Q.; Schaefer, H. F., III, Triplet states of

cyclopropenylidene and its isomers. J. Chem. Phys. 2010, 132 (4).

6. de Meijere, A.; Kozhushkov, S. I., An evolving multifunctional molecular

building block: Bicyclopropylidene. Eur. J. Org. Chem. 2000, (23), 3809-3822.

7. Marek, I.; Simaan, S.; Masarwa, A., Enantiomerically enriched cyclopropene

derivatives: Versatile building blocks in asymmetric synthesis. Angew. Chem., Int. Ed.

2007, 46 (39), 7364-7376.

8. Dookeran, N. N.; Yalcin, T.; Harrison, A. G., Fragmentation reactions of

protonated alpha-amino acids. J. Mass Spectrom. 1996, 31 (5), 500-508.

9. Yao, C.; Syrstad, E. A.; Tureček, F., Electron transfer to protonated beta-alanine

N-methylamide in the gas phase: An experimental and computational study of

dissociation energetics and mechanisms. J. Phys. Chem. A 2007, 111 (20), 4167-4180.

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10. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;

Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji,

H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.;

Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;

Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; ;

Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.;

Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J.

C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.;

Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.;

Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;

Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.;

Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox,

D. J., Gaussian 09, Revision A. 02; Gaussian, Inc., Wallingford, CT 2009.

11. Marion, N.; Nolan, S. P., Well-Defined N-Heterocyclic Carbenes-Palladium(II)

Precatalysts for Cross-Coupling Reactions. Acc. Chem. Res. 2008, 41 (11), 1440-1449.

12. Mebel, A. M.; Jackson, W. M.; Chang, A. H. H.; Lin, S. H., Photodissociation

dynamics of propyne and allene: A view from ab initio calculations of the C3Hn (n=1-4)

species and the isomerization mechanism for C3H2. J. Am. Chem. Soc. 1998, 120 (23),

5751-5763.

13. Vazquez, J.; Harding, M. E.; Gauss, J.; Stanton, J. F., High-Accuracy

Extrapolated ab Initio Thermochemistry of the Propargyl Radical and the Singlet C3H2

Carbenes. J. Phys. Chem. A 2009, 113 (45), 12447-12453.

14. Wass, D. F.; Haddow, M. F.; Hey, T. W.; Orpen, A. G.; Russell, C. A.; Wingad, R.

L.; Green, M., Cyclopropenylidene carbene ligands in palladium C-C coupling catalysis.

Chem. Commun. 2007, (26), 2704-2706.

134

VITA

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134

VITA

Ziqing Lin was born in 1984 in Shanghai, China. In the summer of 2003, he was

admitted to Tsinghua University in Beijing, China, and majored in Chemistry. There, he

obtained a solid knowledge and training in analytical, inorganic, organic, and physical

chemistry. As an undergraduate junior, Ziqing had an undergraduate research program

with Prof. Xinrong Zhang in Key Laboratory for Atomic and Molecular Nanosciences of

the Education Ministry, Department of Chemistry, Tsinghua University. In 2006, he

joined Prof. Zhang’s lab for his senior design and encountered mass spectrometry, where

he found the field fascinating, that analytical chemistry especially mass spectrometry

could be an essential key to monitor and solve biomedical problems. In 2007, Ziqing was

admitted to Department of Chemistry, Tsinghua University for a 3-year Master’s program

after his bachelor degree. He continued to work on applications of ambient mass

spectrometry under the supervision of Prof. Zhang and developed a rapid screening

methodology for clenbuterol, an abused drug and speciation for 8 organic and inorganic

arsenic species. Besides his research as an analyst, Ziqing learned mechanical drawing,

and made compartments to modify the atmospheric pressure interface for mass

spectrometers as well as helping his colleagues design instrumentational devices. He also

drew an amount of cover pictures for scientific journals. Therefore, Ziqing gradually

grew into a combination of scientist, engineer, and artist. In 2010 after obtaining his

Master’s degree in analytical chemistry, Ziqing decided to come to the United States and

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135

pursue his PhD degree in Weldon School of Biomedical Engineering at Purdue

University. He joined Prof. Zheng Ouyang’s group and further worked on

instrumentation and application of mass spectrometry for gas-phase biomolecular ion

reactions, which was a joint program of both science and engineering. After building his

own mass spectrometry system, he discovered for the first time a cyclopropenylidene

reaction with protonated alkyl amines, amino acids and peptides. Ziqing also collaborated

on the development of long-distance ion-transferring tube and other ambient mass

spectrometry applications.

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PUBLICATIONS

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136

PUBLICATIONS

Journals

1. Lin, Z.; Tan, L.; Yang, Y.; Dai, M.; Tureček, F.; Ouyang, Z.; Xia, Y. Gas-phase

reactions of C3H2 with protonated alkyl amines, J. Am. Chem. Soc. In preparation.

2. Lin, Z.; Tan, L.; Garimella, S.; Li, L.; Chen, T.; Xu, W.; Xia, Y.; Ouyang, Z.

Characterization of a DAPI-RIT-DAPI system for gas-phase ion/molecule and ion/ion

reactions, J. Am. Soc. Mass Spectrom. 2014, 25, 48-56.

3. Chen, C.; Lin, Z.; Garimella, S.; Zheng, L.; Shi, R.; Cooks, R. G.; Ouyang, Z.

Development of a mass spectrometry sampling probe for endoscopic analysis, Anal.

Chem. 2014, 85, 11843-11850.

4. Lin, Z.; Zhao, M.; Zhang, S.; Yang, C.; Zhang, X. In situ arsenic speciation on solid

surfaces by desorption electrospray ionization tandem mass spectrometry, Analyst

2010, 135, 1268-1275.

5. Liu, Y. ; Ma, X.; Lin, Z.; He, M.; Han, G.; Yang, C.; Xing, Z.; Zhang, S.; Zhang, X.

Imaging mass spectrometry with a low-temperature plasma probe for the analysis of

works of art, Angew. Chem.-Int. Edit. 2010, 49, 4435-4437

6. Liu, Y.; Lin, Z.; Zhang, S.; Yang, C.; Zhang, X. Rapid screening of active ingredients

in drugs by mass spectrometry with low-temperature plasma probe, Anal. Bioanal.

Chem. 2009, 395, 591-599.

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7. Ma, X.; Zhang, S.; Lin, Z.; Liu, Y.; Xing, Z.; Yang, C.; Zhang, X. Real-time

monitoring of chemical reactions by mass spectrometry utilizing a low-temperature

plasma probe, Analyst 2009, 134, 1863-1867.

8. Lin, Z.; Zhang, S.; Zhao, M.; Yang, C.; Chen, D.; Zhang, X. Rapid screening of

clenbuterol in urine samples by desorption electrospray ionization tandem mass

spectrometry, Rapid Commun. Mass Spectrom. 2008, 22, 1882-1888.

9. Ma, X.; Zhao, M.; Lin, Z.; Zhang, S.; Yang, C.; Zhang, X. Versatile platform

employing desorption electrospray ionization mass spectrometry for high-throughput

analysis, Anal. Chem. 2008, 80, 6131-6136.

Conference Oral and Poster Presentations

1. Lin, Z.; Tan, L.; Yang, Y.; Dai, M.; Tureček, F.; Ouyang, Z.; Xia, Y. Reactions of

Biomolecule Ions with Pyrolysis-formed Carbene, Poster TP353, 62nd ASMS Annual

Conference on Mass Spectrometry and Allied Topics, Baltimore, MD, Jun 17th, 2014.

2. Lin, Z.; Tan, L.; Ouyang, Z.; Xia, Y. Instrumentation and studies for gas-phase

ion/radical reactions, Poster, 26th ASMS Sanibel Conference on Mass Spectrometry,

Clearwater Beach, Fl, US, Jan 30th-Feb 2nd, 2014.

3. Lin, Z.; Tan, L.; Chen, T.; Li, L.; Xia, Y.; Ouyang, Z. Development of a mass

spectrometer for gas phase ion/radical reactions, Oral TOA1450, 61st ASMS Annual

Conference on Mass Spectrometry and Allied Topics, Minneapolis, MN, US, Jun 11th,

2013.

4. Lin, Z.; Garimella, S.; Li, L; Chen, T.; Xu, W.; Xia, Y.; Ouyang, Z. A DAPI-RIT-

DAPI system for in-trap gas phase reactions, Oral TOC850, 60th ASMS Annual

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Conference on Mass Spectrometry and Allied Topics, Vancouver, BC, Canada, May

22nd, 2012.

5. Chen, C.; Lin, Z.; Garimella, S.; Cooks, R. G.; Ouyang, Z. Development of an

endoscopic DESI sampling probe, Poster TP034, 59th ASMS Annual Conference on

Mass Spectrometry and Allied Topics, Denver, CO, Jun 7th, 2011.