Webinar, Agilent, May 8, 2008

Application of iontrap ETD/MS/MS in protein studies

Ole Nørregaard Jensen, PhDProtein Research Group

Department of Biochemistry and Molecular BiologyDepartment of Biochemistry and Molecular BiologyUniversity of Southern Denmark

www.protein.sdu.dk

Post-translational modifications of proteins have many regulatory roles in the cell

ON Jensen (2006) Nature Rev. Mol. Cell. Biol.

Modification-specific proteomics:p pInterpreting the biological roles of PTMs

Id tif PTM’ d t iIdentify PTM’ed proteins

Determine PTM sites of proteinsDetermine PTM sites of proteins

Determine functional PTMs by site-specific tit tiquantitation

Determine interdependence and cooperativityDetermine interdependence and cooperativity between multiple PTMs

M d l/ i l l l lModel/simulate complex molecular systems

PTM-specific ion signal

N

C MS/MS

N

Δm( PTM, position )Stable PTM

PTM-specific neutral loss

MS/MS/MS

N

CPTM MS/MS

Δm(PTM = )

Labile PTM(aa)PTM

- Amino acid residue modified with stable PTM

- Amino acid residue modified with labile PTMC - C-terminal amino acid residueN - N-terminal amino acid residue PTM

Challenges in functional phosphoproteomics

• Technology– Sensitivity (do we find all relevant phospho-sites?)– Specificity (false positives?)– Quantitation (differential phosphorylation events)

• Biology– Phosphorylation motifs– Phosphorylation site occupancy vs. biological activity– Spatial and temporal phosphorylation profiles– Interplay between phosphorylation and other post-translational y y

modifications

MS data acquisitionMS (Δm)

Modification-specific phoshoprotein analysis by mass spectrometry

Optimized sample preparationfor phosphopeptides

MS/MS sequencingMS/MS neutral loss

MS/MS diagnostic ionsMultistage MS

MALDI MS/MSESI MS/MS

MRM

Mass spectra

LysisFractionationP

Computational data analysis and data miningProtease treatment

Phosphopeptide enrichmentand data mining

Tissue Phosphorylationsite assignments

TissueBio-fluidsCells

ValidationBiological function

Recovery/enrichment of phosphopeptides y p p p pprior to mass spectrometry

• Fe(III)-IMAC (+/- O-Methylesterification)

• TiO2, ZrO2,…TiO2, ZrO2,…• SCX and SAX • Isoelectric focusingIsoelectric focusing• HILIC• Antibodies (α-pY Ab)• Antibodies (α-pY Ab)

ON Jensen (2006) Nature Rev. Mol. Cell. Biol.( )Thingholm et al (2006) Nat. Prot., Thingholm et al (2008) Mol. Cell. Proteomics

Phosphopeptide sequencing by ion-electron reactions (ECD)p p p q g y ( )Stensballe et al. (2000) Rapid Commun. Mass Spectrom. 14, 1793-1800

ETD

• Electron Transfer Dissociation: Fluoranthene anions transfer an electron to multiply protonated peptides inducing fragmentation along p p p g g gpathways that are analogous to those observed in electron capture dissociation (ECD)

[M+nH]n+ + e- => [M+nH](n-1)+·

Syka et al, PNAS USA 2004; 101: 9528-33

Agilent TechnologiesAgilent TechnologiesHPLC-Chip/MS ETD System

BSA - 50 fmol BSA tryptic digest

- CID and ETD is sequentially performed on selected ions within msecs

Gradient

80

90

3

8x10Intens.

Gradient %B

30

40

50

60

70

%B

1

2

0

10

20

0 1.5 3 4.5 6 7.5 9

Time (min)0 1 2 3 4 5 6 7 8 Time [min]

0

1

Ch t h ditiMS conditionsD i fl 4 L/ iChromatography conditions

A = 0.1% formic acid in waterB = 90% acetonitrile + 0.1% formic acid in water

Flow rate = 300 nL/minGradient

Drying gas flow 4 L/minDry gas 300 °C Vcap Typically 1800-1900 V Skim1 30 VCapillary exit 100 VTrap drive 85Gradient

Time (min) % B0 37 807.01 30

Trap drive 85Averages 1Accumulation time 150 msSmart target 500,000MS scan range 300-2200Precursor ions 5

Stoptime 9 minutes Fragmentation amplitude 1.3VMS/MS scan range 100-2000, charge state ≥2Active exclusion On, 2 spectra, 1 minMS/MS ICC target 500,000

Ion-Trap with ETD moduleIon Trap with ETD module

Agilent XCT Ultra/ETD

MS/MS peptide fragmentation

x2 x1

y2 y1

O O O

z2 z1

H2N CH

R1

C NH CH

R2

C NH CH

R3

C OH

a1 a2

b1

c

b2

c

• ETD produces c and z fragment ions

c1 c2

p g• CID produces b and y fragment ions

Stability of PTMs:Th f h h hi tidiThe case of phosphohistidine

Anal. Chem. (2007) 79 (19) 7450-7456

MS/MS sequencing of phosphohistidine peptidesS/ S seque c g o p osp o st d e pept des

ETD/ECD enable sequencing of pHis peptides

Kleinnijenhuis et al (2007) Anal. Chem. 79 (19) 7450-7456

More efficient fragmentation of 3+ ions than 2+ ionsETD MS/MS of phosphopeptides

Peptide sequencing by ETD/CID MS/MS

– ETD MS/MS (3+, 4+, ..)– Extensive fragmentation (c, z´)– ETD maintains phosphorylation-sites intact– ETD seems more efficient than CID for

phosphopeptide sequencing by ion trap MS– CID and ETD generate distinct datasets

Chi et al (2007) PNAS 104 (7) 2193 2198Chi et al (2007) PNAS 104 (7) 2193-2198Molina et al (2007) PNAS 104 (7) 2199-2204

Coon et al, ASMS 2007

How to increase the charge state f t ti tid f ETD MS/MS?of tryptic peptides for ETD MS/MS?

– Tryptic peptides often generate 2+ ions

Generate larger peptides to begin with– Generate larger peptides to begin with….• Endoproteinase Lys-C, Glu-C, …• Limited proteolysis by trypsin

– Chemical derivatization to increase peptide charge state

– Add a ’supercharge’ reagent to sample/solvent

Supercharging of ions by m-NBA

47% ater/50% methanol/3% acetic acid

Cytochrome c (10−5 M)

47% water/50% methanol/3% acetic acid

43% glycerol/54% water/3% acetic acid

46.5% water/49.5% methanol/1% m-NBA/3% acetic acid.

A.T. Iavarone, E.R. Williams (2002) Int. J. Mass Spectrom. 219 63–72

Frank Kjeldsen

Supercharging of peptides by m-NBALC-ETD/CID-MS/MS analysis of BSA tryptic digest

Frank Kjeldsen

Improved ETD/MS/MSp(3+ vs. 2+ ions)

m-NBA is compatible with LC-MS/MSs co pat b e t C S/ S

Agilent XCT Ultra/ETD w. ChipCube interface

Supercharging of peptidesSupercharging of peptides

• 0.1% m-NBA increases the average charge state % g gof tryptic peptides (2.2+ to 2.6+)

0 1% NBA i tibl ith LC MS• 0.1% m-NBA is compatible with LC-MS– m-NBA has low ionization efficiency– Minor effect on retention time– Minor effect on retention time

• Supercharging of peptides leads to– Increase in DDA MS/MS analysis of 3+ peptides– more efficient ETD

improved scores for peptide/protein identification– improved scores for peptide/protein identificationKjeldsen et al (2007) Anal Chem. 79 (24) 9243-9252.

Hydrogen Exchange Mass Spectrometry (HX-MS)

is a biophysical method that probes the t i t t l d i b l iprotein structural dynamics by applying

mass spectrometry to measure the deuterium incorporation into the backbone amide groups of proteinsamide groups of proteins.

CID MS/MS of deuterium-labeled peptides causes ‘h d bli ’‘hydrogen scrambling’

LE-CID, HE-CID,SORI-CID, PSD

ECD, ETD,MALDI-ISD

Easy procedure to obtain a polarised labeled peptide

H2N-His−His−His−His−His−His−Ile−Ile−Lys−Ile−Ile−Lys-COOH

Fast intrinsic exchange rate Slow intrinsic exchange rate

Labeling procedure:Labeling procedure:

1. complete deuteration by dissolution in D2O

D D D DDD D D DDH2N COOH

D D D DDD D D DD

2. Dilution into cold acidic H2O

H H H HH D D D DDH2N COOH

2

3. Direct infusion via a cooled syringe into an electrospray ion sourceThomas Jørgensen et al

ETD-generated c and z ions of polarised labeled peptides serve a probes for the level of scrambling

c6 z7

z

serve a probes for the level of scrambling

non-labeled peptide

H H H H H H H H H H HH2N COOH

z7

c66

z7

low-scrambling

H H H H H H D D D D DH2N COOH

c6

high scrambling

H2NH D H D H D H D H D H

COOH

z7

high-scrambling

840 842 844 846 848 850 852 854 856m/z

c6

ETD retains the deuterium-labeling pattern g pupon peptide fragmentation

H2N

COOH

H2N COOH

The activation energy for the mobilization of protons is significantly lower than the one for peptide fragmentationsignificantly lower than the one for peptide fragmentation

M Zehl et al (submitted)

ETD MS/MSETD MS/MS

• ETD MS/MS is of great utility in our laboratoryETD MS/MS is of great utility in our laboratory– Peptide sequencing– Mapping of post-translational modificationspp g p– Investigations by HX-MS of protein conformation,

protein-protein and protein-ligand interactions

• ChipCube is a robust interface for LC-MSF t ti– Fast separation

– Easy to use and very robust ( > 200 runs per chip)– Availability of various chromatographic resins– Availability of various chromatographic resins

Acknowledgements

Anne Kleinnijenhuis

Protein Research Group Department of Biochemistry & Molecular Biology

www.protein.sdu.dkAnne KleinnijenhuisFrank KjeldsenAnders GiessingChristian R. Ingrell

Martin ZehlKasper Rand

Thomas Jørgensen Martin R LarsenMartin R. LarsenFinn KirpekarPeter HøjrupPeter Roepstorff

Instrument manufacturersEuropean Union

Danish Nat’l Research FoundationDanish Research Councils

Lundbeck Foundation