Lecture 5

Condensed phase ionisation techniques: spray methods

At the end of this lecture you should be able to:

• describe the ion formation models in ESI-MS

• calculate molecular weights and charge states from low- and high-resolution ESI-MS spectra

Ionisation Techniques: Overview

Gas-Phase Methods• Electron Impact (EI)• Chemical Ionization (CI)

Desorption Methods• Secondary Ion MS (SIMS) and Liquid SIMS• Fast Atom Bombardment (FAB)• Laser Desorption/Ionization (LDI)• Matrix-Assisted Laser Desorption/Ionization (MALDI)

Spray Methods• Atmospheric Pressure Chemical Ionization (APCI)• Electrospray (ESI)

Condensed phase ionisation techniques (2): spray methods

Overview

• Thermospray

• APCI: Atmospheric pressure chemical ionisation

• APPI: Atmospheric pressure photoionisation

• Electrospray ionisation

Atmospheric pressure chemical ionisation (APCI)

• For non-polar and thermally stable compounds < 1500 Da

• Useful to combine with liquid chromatography

www.chm.bris.ac.uk/ms/theory/apci-ionisation.html

Flow

Spray needle/Capillary Spray

Cone

Atmospheric pressure

Skimmers

Vacuum

To mass spectrometer

Nebuliser gas Corona discharge

Ions

Electrospray Ionisation (ESI)• Nobel Prize to Fenn in 2002• Also atmospheric pressure ionisation• Very versatile• Also works for (very) large (bio)molecules, including

proteins, nucleic acids, carbohydrates• Softest ionisation technique of all

3-4 keV2-10 l/min

http://www.chm.bris.ac.uk/ms/theory/esi-ionisation.html

Flow

Spray needle/Capillary Spray

Cone

Atmospheric pressure

Skimmers

Vacuum

To mass spectrometer

Electrospray Ionisation (ESI)

• Flowrates: 2 to 10 l/min: Best interface for LC/MS

• Can be combined with almost any mass analyser Common: TOF, Ion Trap, Quadrupole, FT-ICR

• Uses: Mass detection, structure elucidation, protein folding, H/D exchange, protein sequencing….

Sample characteristics

• Common solvents: mixtures of water with acetonitrile or methanol

• Usually with added acid (acetic, formic), < 1%• Can’t tolerate (non-volatile) salt or buffers • Can do positive or negative electrospray:

selected by capillary voltage

What it looks like

Ion evaporation model

Ionisation models

Charged residue model

www.chm.bris.ac.uk/ms/theory/esi-ionisation.html

Analyte molecule

Multiply charged droplet

Spray needle tip

Rayleigh limit is reached

Multiply charged droplet

1. Spray generates multiply charged droplets

2. Solvent evaporation leads to increasing charge density

3. When charge density too high: Coulombic explosion

Charged amino acids

N

O

O

O-

AspartateN

OO

O-

GlutamateAcidic

3.65 4.24

N

O

N

NHHistidine

6.00

N

ON

NN

+

ArginineN

OH3N+

Lysine

Basic

10.8

12.5

Usually negatively charged at pH 7

Positively/uncharged at pH 7

Always positively charged

Charges on surface of proteins

Red: Negatively chargedBlue: Positively chargedWhite: No charge; hydrophobic

Examples• ESI of large molecules usually produces multiply charged

ions• e.g. proteins:

• Each peak corresponds to the same protein, but with different number of protons attached: Observed ions are

[M+nH]n+

750 1000 1250 1500 1750 2000 2250 2500 2750

13+

12+11+

10+9+

8+7+

6+

Charge state series

m/z

942.8

893.3

848.7

808.3

771.6

738.1

707.4

998.1

1060.5

How to determine the molecular mass of a protein from an ESI-MS spectrum

• Observed ions have composition [M+nH]n+

• Let m1,m2,…, mn : m/z values of the different peaks

• mn =

• Its neighbouring peak to the left:

• mn+1 =

• Solving both equations for n and M:

• n =

• M = n(mn – H)

• e.g. m2 = 998.1 and m1 = 1060.5• n = 16, and M = 16952 Da

m/z

1n1)H](n[M

n must be an integer

nnH][M

1nn

1n

mmHm

Deconvolution of ESI mass spectra

Charge statesDeconvolutedspectrum

M = n(mn – H)

3000 30000 Mass (Da)

Quick reminder: average and monoisotopic mass

The distance between isotopic peaks reveals charge state

protein_modelingLCTmix of 6 proteins

m/z915 916 917 918

%

0

100

prot_mix_0724a 350 (5.837) Sm (SG, 2x6.00); Cm (343:374) TOF MS ES+ 1.86e3915.7363

915.4818

915.2274

915.9765

916.2311

916.4857

916.7402

protein_modelingLCTmix of 6 proteins

m/z1084 1085 1086 1087 1088 1089 1090

%

0

100

prot_mix_0724a 655 (10.923) Sm (SG, 2x6.00); Cm (645:675) TOF MS ES+ 4541086.5515

1086.0433

1087.0444

1087.5529

1088.0460

protein_modelingLCTmix of 6 proteins

m/z500 501 502 503 504 505 506 507 508 509 510 511 512

%

0

100

prot_mix_0724a 651 (10.856) Sm (SG, 2x6.00); Cm (648:651) TOF MS ES+ 783505.3506

506.3584

507.3566

915.2247

915.4818 915.7363

915.9765

916.2311

916.4857

505.3506

506.3584

507.3566

1086.0433

1086.5515

1086.0444

1087.5529

1088.0460

+1

1.00

0.51 +2

0.25

+4

Jonathan A. KartyJonathan A. Karty

1695.7 1696.2 1696.7 m/z

0.0

0.5

1.0

1.5

a.i.

Charge States and distance between isotopic peaks

0.1 +10

Example beyond molecular mass: Protein folding

• Calbindin: Calcium binding induces protein folding: increase in charge states with higher m/z (=lower charge, more folded)

No Ca2+

excess Ca2+

deconvolutedraw data

Recent developments: ambient mass spectrometry:

DESI and DART• DESI: Desorption electrospray ionisation• DART: Direct analysis in real time• Applicable to solids, liquids, and gases• No prior sample treatment !

Ionsiation techniques: SummaryIonisation Vola-

tileThermal Size Amount Examples

EI Yes Stable Small 1-2 mg organics

CI Yes Stable Small 1-2 mg organics

FAB No Stable Medium 0.5-1 mg Polar/ionic organics, organometallics, peptides, biomolecules

FD No Labile Medium 1-2 mg Non-polar organics, organometallics

MALDI No Labile Large 250 fmol-500 pmol

Peptides, proteins, polymers

ESI No labile Large 1-300 pmol/l

Polar/ionic organics, peptides, proteins, biomolecules, organometallics, polymers

Table adapted from http://www.scs.uiuc.edu/~msweb/SLM530.pdf

Summary: Application ranges of various techniques

masspec.scripps.edu/MSHistory/whatisms.php

Self-assessment questions• Q1 Describe the two ion formation models in ESI• Q2 A positive-ion ESI spectrum shows the following adjacent signals at m/z 4348.8,

4546.5, 4762.9, 5001.0 5264.2. Calculate the molecular mass of the molecule. • Q3 The following m/z (979,1040,1109,1189,1280,1387,1512,1664) were obtained by

electrospray ionisation of a protein from an aqueous solution. – Calculate the molecular mass of the protein within 10 Da. – Describe how the mass spectrum would have looked if the same protein had

been ionised by MALDI.• Q4 What distance between isotopic peaks would you expect for a +12 charge state ?• Q5 The following peaks arose from the different isotopes contributing to the ESI

mass spectrum resulting from the protonation of a species of relative molecular mass m to reach a charge z.:m/z=848.40, 848.45, 848.50, 848.55, 848.60, 848.65, 848.70. – What is the value z and what is the mass of the species giving rise to the peak at

m/z 848.55

Lecture 6

Tandem MS

Peptide/protein identification by MS

At the end of this session you should be able to

• explain how structural information can be obtained by Tandem MS and MALDI-TOF/PSD

• explain how mass spectrometry data can be used to identify known and unknown proteins

Tandem MS(also termed MS2 and MSn)

• Used for:– Identify and quantify compounds in complex

mixtures– Structure elucidation of unknown compounds

• Applied in:– Proteomics– Metabolomics– Biomarker discovery– De novo protein sequencing

Tandem MS

• Multistage technique:

mass selection of precursor ion

Ion source MS-1

Activation and fragmentation

MS-2

Mass analysisof product ions

Normalspectrum

MS/MS spectrum

Tandem MSFragmentation techniques

• Collision-induced dissociation (CID): – most common– Possible with ESI coupled to triple-quad, Ion trap, FT-ICR,

and MALDI-TOF

• Electron Capture Dissociation (ECD): – only for multiply charged biopolymers– Primarily with FT-ICR

• Electron-Transfer Dissociation (ETD)• Absorption of electromagnetic radiation• Not Tandem MS, but useful fragmentation technique: Post-

source decay (PSD) combined with MALDI reflectron TOF

Reflectron-TOF and Post-Source decay for MALDI-TOF

D e t e c t o r

r e f l e c t r o n

i n d u c e s p o s t

s o u r c e d e c a y

• (Some) parent ions fragment in field-free drift region• Parent and product ions arrive at reflectron

simultaneously (same velocity)• Product ions leave Reflectron earlier (smaller Ekin)

Field-free region

Example: MALDI-PSD TOF spectrum of a neuropeptide

Necla Birgül, Christoph Weise, Hans-Jürgen Kreienkamp and Dietmar Richter , The EMBO Journal (1999) 18, 5892–5900

Parent Ion

Simplified schematic for protein identification from biological samples (“Proteomics”)

Cell cultureTissueBiofluid

Complexprotein mixture

Peptide-massfingerprints

Peptides

Identified proteinsPeptide

sequences

Individual or small sets of proteins

ExtractionSeparation

Cleavage

MALDI

Peptide mass mapping

Sequencing(LC-MS/MS)

Database search

Database search

Peptide mass mapping/ fingerprinting

• Makes use of specific cleavage agents– Chemical cleavage: e.g. CNBr– Digestion with endoproteases (proteolytic

enzymes): Trypsin, pepsin, chymotrypsin etc.

– See exercises

Peptide mass mapping/fingerprinting

ProteinSequence

(in database)

TheoreticalMass Spectrum

TheoreticalDigest

QNICPRVNRIVTPCVAYGLGRAPIAPCCRALNDLRFVNTRNLRRAACRCLVGVVNRNPGLRRNPRFQNIPRDCRNTFVRPFWWRPRIQCGRIN

NTFVRPFWWRPRIVTPCVAYGLGRCLVGVVNRAPIAPCCRFQNIP...

m/z600 900 1200 1500 1800A

bundance

Protein Peptides Mass Spectrum

m/z600 900 1200 1500 1800

Abundance

Digest

Peptide sequences

Compare

De novo protein discovery

• Mass fingerprinting only practicable if protein is already in a database

• If previously undiscovered protein: Need to sequence

• Can be done by sequencing peptides

Peptide sequencing:fragmentation rules

• Three types of bonds along backbone• amino alkyl bond• alkyl-carbonyl bond• amide bond

+NH3―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2―

R4R1 R2 R3

N-terminus C-terminus

y3

b1

NH2―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2H

R4R1 R2R3

z3

c1

x3

a1

Peptide sequencing:fragmentation rules

• Each bond can be broken by fragmentation Six possible product ions

• But: peptide bond most likely to break in low energy CID (and MALDI/PSD):

Mostly b and y fragments

Peptide fragments generated by low energy CID or PSD

N-terminus C-terminus

y3

b1

y2

b2

y1

b3

NH2―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2H

R4R1 R2 R3

m/z values of b ions: residue masses + 1 (+H+)m/z values of y ions: residue masses +17 (OH-) + 2 (2H+) = 19

y2: +NH3―CH―CO―NH―CH―CO2H

R4R3

b3: NH2―CH―CO―NH―CH―CO―NH―CH―C=O

R1 R2 R3

+

For example:

Example: Peptide analysed by MALDI TOF reflectron and PSD

Proposed sequence: HTH[LIN]FA[LIN]R

HisThrHisPheAlaLeu/

Ile/AsnArg

Leu/Ile/Asn

Leu/Ile: 113.08Asn: 114.04

y1 y2

y3

y4

y5

y6

y7 y8

Self-assessment questions

• Q1 How are MALDI and ESI used for the identification of proteins ?

• Q2 Describe how Peptide Fingerprinting works

• Q3 A MALDI-PSD spectrum of a peptide shows the following y-peaks:174.8 / 288.3 / 359.0 / 506.1 / 619.6 / 756.0 / 857.6 / 995.2Find out the masses for amino acids (e.g. at Wikipedia). Calculate the differences between peaks in this spectrum and suggest possible sequences for this peptide

Exercises• Exercise 1: Protein cleavage/digestion

1. Go to http://www.expasy.ch/tools/peptidecutter/ 2. In the box, enter ALBU_HUMAN (this is the swissprot name of human

serum albumin) - you can also choose a different protein if you like. Sequences and swissprot codes can for example be found in the swissprot database (at www.expasy.ch).

3. Scroll down, and tick the box “only the following selection of enzymes and chemicals”,and then select one chemical or enzyme, which you want to use for cleaving the albumin protein, from the list

4. Scroll back up, and click “Perform”

5. Inspect the output. How many times is albumin cleaved by your chosen cleavage agent ? Find out what the specificity of your cleavage agent is.

• Exercise 2: Calculation of molecular masses of proteins and peptides– 1. Copy a peptide fragment from the output of

Exercise 1 (or make one up yourself), go to http://www.expasy.ch/tools/protparam.html

– and paste your sequence into the appropriate box (the large one).

– 2. Click “Compute Parameters”.– 3. Inspect the output. What is the molecular formula

of the peptide ? How many positively and negatively charged side-chains does your peptide have ? What charge would it have at pH 7 ?

• Exercise 3. Average and monoisotopic masses– 1. Copy the molecular formula (or the one-letter code

sequence) of the peptide from exercise 2, and go to http://education.expasy.org/student_projects/isotopident/htdocs/

– 2. Paste the formula or sequence in the appropriate box. Make sure that you have selected the correct “Type of composition” (e.g. “chemical formula”) in the respective pulldown menu.

– 3. Click “Submit query”.– 4. Inspect the output. How many isotopic peaks are there?

Which is the most abundant peak ? What are the monoisotopic and the average masses ?