Biomolecule analysis with electrospray mass spectrometry

Dr. Harry BrumerWood Biotechnology LaboratoryKTH Department of Biotechnologyharry@biotech.kth.se

Molecular Biotechnology Prof Mathias Uhlén Prof Joakim Lundeberg Prof Per-Åke Nygren Prof Stefan Ståhl

Biochemistry Prof Karl Hult Prof Pål Nyrén

Dept. of Biotechnology~200 personer

ca 80 pers

ca 20 pers

Wood Biotechnology Prof Tuula Teeri

ca 25 pers

Bioprocess technology Prof Sven-Olof Enfors Prof Lena Häggström ca

30 pers

Environmental Microbiology Assoc. Prof Gunnel Dalhammar

ca 10 pers

Structural BiochemistryProf Torleif Härd

ca 15 pers

Theoretical ChemistryProf Hans Ågren

Prof Faris Gelmukhanov

ca 15 pers

Adm

ca 5 pers

KTH Department of Biotechnology

http://www.biotech.kth.se/woodbiotechnology/

MS in the Wood Biotechnology Lab

Equipment purchase funded by the Wallenberg Consortium North for Functional Genomics

Micromass Q-TOF2 orthogonal acceleration quadrupole/time-of-flight MS

Orthogonal (Z-spray) electrospray ionisation interface

Nanoflow LC system (200 nL/min)

MS Components

Ionisation source

ion optics, including one or more mass analysers

detector

analyte ionisation mass selection

detection

EI, CI, FAB, MALDI, ESI

mag. sector, quadrupole, TOF, ion trap, FT-ICR

faraday cup, dynode, scintillation, MCP

Hybrid quadrupole-TOF

Hybrid quadrupole-TOF

Hybrid quadrupole-TOF

Electrospray ionisation (ESI)

Biological molecules are often non-volatile and/or thermally labile:

proteins/peptidesnucleic acids (DNA, RNA)carbohydrates

EI and CI not applicable w/o derivitisationFAB mass range limited

OK for small carbohydrates, peptides

MALDI good for biologicalsnot dynamic, typically gives [M+H]+ ions

ESIsuitable for on-line methods (CE, LC)gives multiply charged ions

Electrospray ionisation (ESI)

ESI: Ion production

“Z-spray” ESI source

ESI yields multiply-charged ions

Normal quadrupoles can be used for high-mass biomolecules

Hybrid quadrupole-TOF

Time-of-flight

analyzers (TOF)

K.E. = 1/2 mv2

Orthogonal acceleration TOF (oaTOF)

used in ESI-TOF applications

Hybrid quadrupole-TOF

Quadrupole mass analyzer

Q1 can be tuned to allow only ions of a single m/z to pass

Hybrid quadrupole-TOF

The collision cell

1 pmol/uL in 1:1 MeOH/water, 0.2 % formic 05-Mar-2002 11:09:19

600 800 1000 1200 1400m/z0

100

%

GLUFIB5 47 (2.014) Cm (24:56) TOF MS ES+ 7.62e3785.848

786.339

786.842

Glu-fibrinogen peptide: TOF MS

783 784 785 786 787 788 789 790 791m/z0

100

%

GLUFIB5 47 (2.014) Cm (24:56) TOF MS ES+ 7.62e3785.848

786.339

786.842

0.5 m/z separation of isotopic peaks: [M+2H]2+

Peptide sequencing by MS/MS

Amino acids differ in their side chains

Predominant fragmentation

Weakest bonds

Glu-fib ESI-TOF MS/MS1 pmol/uL, 10ul/min 23-Oct-2001 18:54:19

200 400 600 800 1000 1200 1400 1600 1800m/z0

100

%

GLUFIB_MSMS_23OCT2001 121 (5.362) Cm (87:125) TOF MSMS 785.80ES+ 2.55e3333.188

187.073

684.339

480.252

497.199

813.396

1056.469

1285.511

1286.520

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000M/z0

100

%

intermediate soln.GLUFIB_NANO_MSMS_19NOV MaxEnt 3 95 [Ev-188973,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) 1: TOF MSMS 785.85ES+

R A S F F G E E N D N V G E yMax684.36

y6

480.27y4

333.20y3

187.09b2

175.13y1

169.0772.10V

246.17y2

382.19

497.21

627.35y5

612.24

813.41y7

740.30

1570.72(M+H) +1285.58

y11

1056.52y9942.47

y8

924.431039.46

1171.53y10

1057.44

1268.551535.71

1517.661384.67y12

1515.68

1571.86

1588.94

Raw data

MaxEnt 3 deisotoped, deconvoluted data

Glu-Gly-Val-Asn-Asp-Asn-Glu-Glu-Gly-Phe-Ph-Ser-Ala-Arg

31-Oct-2001 15:00:03

500 600 700 800 900 1000 1100 1200 1300 1400m/z0

100

%

BOV_INSULIN 148 (6.527) Cm (148:176) TOF MS ES+ 4.05e4956.3642

956.8810

1147.4683

ESI-TOF MS of bovine insulin, 5736 Da

955 956 957 958m/z0

100

%

BOV_INSULIN 148 (6.527) Cm (148:176) TOF MS ES+ 4.05e4956.3642

956.2003

956.0491

956.5406

956.7046

956.8810

Isotopic peaks sep’d. by 0.16 m/z -> (M+6H)6+

horse heart myoglobin 24-Oct-2001 17:05:29

600 800 1000 1200 1400 1600 1800m/z0

100

%

HHM 1 (0.094) Cm (1:32) TOF MS ES+ 7.62e3616.1976

942.7773617.1996 1060.4655

1131.0928

1304.9095

Horse heart myoglobin (HHM)

ESI-TOF MS

1056 1058 1060 1062 1064 1066 1068m/z0

100

%

HHM 1 (0.094) Cm (1:32) TOF MS ES+ 3.26e31060.4655

1061.7930 1063.0416

1065.5413

No charge state information available - insufficient resolution

HHM - deconvoluted masshorse heart myoglobin 24-Oct-2001 17:05:29

10000 12000 14000 16000 18000mass0

100

%

HHM 10 (0.869) M1 [Ev-176947,It11] (Gs,0.400,751:1961,1.00,L33,R33); Cm (4:21)6.89e416951.176

From system of equations: [M+n(H+)]/n

MaxEnt 1

Charge state distributions

600 800 1000 1200 1400 1600 1800m/z0

100

%

HHM 1 (0.094) Cm (1:32) TOF MS ES+ 7.62e3616.1976

942.7773617.1996 1060.4655

1131.0928

1304.9095

(M+21H)21+

(M+9H)9+

in ACN/water + 1% formic 19-Nov-2001 15:29:30

600 800 1000 1200 1400 1600 1800m/z0

100

%

YADH_NANO_19NOV 15 (0.673) Cm (13:44) TOF MS ES+ 1.52e31149.3616

1050.9436

855.6663

799.9018

1268.1848

1362.0247

1414.3512

1470.913936749.4 Da

ca. 20+ to 50+

16952 Da

ca. 9+ to 21+

Monitoring deglycosylation of XET

Intact protein mass analysis of a glycoprotein

Glycosylation removed enzymatically

Kinetics followed by ESI-MS

Extent of deglycosylation can be correlated w/ changes in protein function

LC-MS & LC-MS/MS

LC-MS/MS for glycopeptide discovery

Reconstructed chromatogram showing only sugar signature ions (m/z 163, 204, & 366)

TOF MS “survey” total ion chromatogram

MS/MS spectra acquired in real time

Detailed MS/MS analysis

Localise site of glycan attachment in the protein

Comparison of MS/MS sequence information

with other XETs

Allows us to define the site of glycosylation for the entire protein family

Henriksson, et al. (2003) Biochemical Journal, 375, 61-73.

Oligosaccharide MS/MS analysis: Borriss, R., et al. (2003) Carbohydrate Research, 338, 1455-1467.

ArabidopsisTCH4 SAGTVTTLYLKSPGTTWDEIDFEFLGNS SGEPYTLHTNVYTQGKGDKEQQFKLWFDPTANFH 140XET1 SAGTVTAFYLSSQNSEHDEIDFEFLGNR TGQPYILQTNVFTGGKGDREQRIYLWFDPTKEFH 150Kiwi SAGTVTAFYLSSQNSEHDEIDFEFLGNR TGQPYILQTNVFTGGKGDREQRIYLWFDPTKDYH 142Tomato SAGVVTAFYLSSNNAEHDEIDFEFLGNR TGQPYILQTNVFTGGKGNREQRIYLWFDPTKGYH 150Tobacco SAGVVTAFYLSSNNAEHDEIDFEFLGNR TGQPYILQTNVFTGGKGDREQRIYLWFDPTKGYH 149Soybean SAGTVTAFYLSSQNAEHDEIDFEFLGNR TGQPYILQTNVFTGGKGDREQRIYLWFDPTKEYH 147MS/MS seq. YLSSTNNEHDELDFEFLGDR TGQPVLLQTNVFTGGK ***.**::**.* **********. :*:** *:***:* ***::**:: ****** :*