021611 ABRF2011 sPRGtalk ARI Final...# unique Peptides UC ID Yes 3,571 2 084 0 1000 2000 2,084 1,623 14941 87133 22730 86010 13800 84940v 20899i 53706 92536i 870486i 45682 870484i

A BA BR F

Proteomics Standards Research Group (sPRG)www.abrf.org/sprg

ABRF‐sPRG2011 study: development and characterization of aand characterization of a

comprehensive standard for analysis of l i l difi ipost‐translational modifications

Proteomics Standards

A BR F sPRG

Alexander R. Ivanov (Chair) Harvard UniversityChristopher Colangelo Y l U i it

Research Group (sPRG)

Christopher Colangelo Yale University

Craig Dufresne Thermo Fisher Scientific

James Farmar University of Virginia

David Friedman Vanderbilt UniversityChris Kinsinger NIH NCI

Kathryn S Lilley University of Cambridgey y y f g

Karl Mechtler Research Institute of Molecular Biology

Brett Phinney University of California, DavisKristie Rose Vanderbilt UniversityKristie Rose Vanderbilt UniversityScott A. Shaffer University of Massachusetts

Susan T. Weintraub University of Texas HSC

Corporate CollaborationProteomics Standards

A BR F

Sigma‐Aldrich – intact proteins


Sigma Aldrich intact proteins Kristin Rolwes, Shantanu Roychowdhury, George Lipscomb

Thermo Fisher Scientific – synthetic peptidesRainer Gebhart, Georgi Videnov, Joel Louette, Manuela Schaffrath

Michrom Bioresources– STTR grant application for futureMichrom Bioresources STTR grant application for future collaborationsKerry Nugent


A BR F Rationale


• Characterization of PTMs and post‐translationally regulated ll l i f h j f icellular processes is one of the major reasons for proteomics

to stay

Post‐translational modifications of proteins:• Major role in regulation of cellular processes.• Analysis of PTM sites is a major challengeAnalysis of PTM sites is a major challenge.• Every PTM poses characteristic analytical difficulties.• New techniques and approaches are emerging.M ffi i t PTM h t i ti• More efficient PTM characterization may open new

landscapes for the sudy of biology in health and disease.• What works best? •How to get better?•How to expend the toolkit?


A BR F Background


PubMed # of Publications ("PTM" or "post‐translational modification" or "post‐translational protein modification“)

5000

6000

modification or post translational protein modification )

3000

4000

2000

0

1000

1980 1985 1990 1995 2000 2005 2010


A BR F Background


PRG 2003 Study 2 synthetic phosphopeptides and 2 digested proteins

sPRG 2007 Study‐‐ phosphopeptide standard ver 1.0(mixture of seven protein digests)

sPRG 2010 Study – phosphopeptide standard ver 2.0(23 synthetic phosphopeptides (mono‐, di‐, tri‐, tetra‐)

including 2 peptides from PRG 2003 in the background of equimolar digest of six proteins

iPRG 2010 Study – Informatic Evaluation of PhosphopeptideIdentification and Phosphosite Localization

sPRG 2011 Study – PTM standard (phosphorylation, acetylation, sulfation, nitration, methylation)


A BR F

2010 2003

Background: sPRG2010 take home messagesResearch Group (sPRG)

2010 2003

Protein Sequence # of times ID % ID # of times

ID % ID

PDIA1_BOVIN SVpSDYEGK (PDIA1) 17 40% 8 15%PDIA1 BOVIN THILLFLPKpSVSDYEGK (PDIA1) 26 62% 8 15%PDIA1_BOVIN THILLFLPKpSVSDYEGK (PDIA1) 26 62% 8 15%

Phosphopeptide identification rate for 2003 and 2010 sPRG studies.95% 93% 95%

90%90%100% Blue - 1 Phosphosite

R d 2 Ph h it81% 83% 81%

60%

48%

90%90%

60%55%

74%74%

60%62% 62%

50%

60%

70%

80%

90% Red - 2 PhosphositesGreen - 3 Phosphosites

48%

24%

36%

24%21%

38%40%

10%

20%

30%

40%

50%

0%

10%

Success of Detection by Number of Phosphosites per Peptide


A BR F Background: iPRG2010

S f iPRG2010 Id ifi i R lResearch Group (sPRG)

6000

7000

8000# spectra Id Yes# spectra Loc Yes# unique Peptides UC ID Yes

Summary of iPRG2010 Identification Results

3000

4000

5000

6000 # unique Peptides UC ID Yes

3,571

2 084

0

1000

2000

3000 2,084

1,623

1494

187

133

2273

086

010

1380

084

940v

2089

9i53

706

9253

6i87

0486

i45

682

8704

84i

8524

613

867

2044

1v40

816i

2010

950

308i

2985

0v56

365

6639

891

943i

4758

771

263

6521

163

103

9721

9i20

814

6196

3v18

621

7463

715

769

7711

466

514

7711

5

100%

40%

60%

80%

% distin

ct pep

tides

3P 2P 1P

# phosphosites

Courtesy of iPRG

0%

20%

2 3 4 5 6 7 8 9 10 11 12

SCX fr#

%


A BR F sPRG 2011 Study Objectives


Goals:To develop a readily available standard for:To develop a readily available standard for:•assessment of a laboratory's ability to detect an array of PTMs in a complex proteomic sample, •development of new approaches for characterization of post‐translationally modified proteins,•generate guidelines for effectual analysis of the selected g g ymodifications.

Study Design: g• To establish collaborative partnership with commercial companies to enable the study and to secure prospective commercialization of the standardcommercialization of the standard.•To allocate two years for the study instead of the typical 1‐year long RG study time frame.


A BR F sPRG 2011 Study Design

Research Group (sPRG) Thermo

Synthetic peptides

Sigma

Intact ProteinsSynthetic peptides~30 O‐phosphopeptides (S,T, and Y):~15 multi‐phospho (di, tri, & tetra)including positional isomers

Intact ProteinsALBU PDIA1PRDX1 UBIQ NQO2 SYHCincluding positional isomers

5 sulfotyrosine5 nitrotyrosine5 acetylated Lys

NQO2 SYHC

5 acetylated Lys5 monomethylated Arg5 monomethylated Lys5 dimethylated Lys

SPE cleanup

sPRG5 dimethylated Lys5 trimethylated Lys3 asymmetric dimethylated Arg3 symmetric dimethylated Arg

Trypticdigestion

y y gsPRG2011standardQC QC

Proteomics Standards R h G ( PRG)

A BR F sPRG 2009 Sample Development


Synthesize Modified Peptides

Characterize by MALDI MS and LC‐ESI MS

Purify Synthetic Peptides

Subaliquot & Freeze All Synthetic P tid

DefinePeptide / Protein Ratios p p Peptides

Selection of

Mix Synthetic Peptides and Subaliquot, Dry

Selection of Peptide Candidates

Characterize by MALDI MS

Create a Spectral Digests of

Proteins& Mail Samples

Selection of Protein C did

by MALDI MS and LC‐ESI MS

Spectral Library

Digest

Candidates

Isolate Proteins

Purify Proteins

Subaliquot & Freeze All Proteins

Digest Proteins; Mix TrypticPeptides

Characterize by MALDI MS and LC‐ESI MS


A BR F

“Bonus” proteins

Preliminary ObservationsResearch Group (sPRG) Bonus proteins

Protein Standards Used: Proteins Detected:Histidyl‐tRNA synthetase (human)

1. Histidyl‐tRNA synthetase(human)2 P i d i 1 (h )

Histidyl tRNA synthetase (human)Peroxiredoxin 1 (human) Protein disulfide isomerase (bovine)Q i d t 2 (h )2. Peroxiredoxin 1 (human)

3. Protein disulfide isomerase(bovine)

Quinone reductase 2 (human)Serum albumin (Human)Ubiquitin (human)

4. Quinone reductase 2 (human)5. Serum albumin (Human)

Apolipoprotein A‐I (bovine)CalmodulinTropomyosin alpha chain (human)5. Serum albumin (Human)

6. Ubiquitin (human)Tropomyosin alpha chain (human)Cytochrome b5 (bovine)Glyceraldehyde 3‐phosphate dehydrogenase (E coli)dehydrogenase (E. coli)Senescence marker protein‐30 (bovine) …


A BR F “Bonus” peptides. Synthesis byproducts.


40.60465.20911 53.17

639.6458749.83325.23627BIC

442.71463

38.08964.38153 52.70

279.1658349.88

325.23520

RT: 38.08

BIC???

???47.72

252.9800938.80

284.2148136.76450.71310

46.17263.91248

RT: 40.60MA: 129378BP: 465.19696

XIC +2

AA: 3382229BP: 482.69373

RT: 37.41AA: 14623BP: 482.69357

RT: 37.58MA: 58829426

XIC, +2

34 36 38 40 42 44 46 48 50 52 54

40.06465.20813

XIC, +2

34 36 38 40 42 44 46 48 50 52 54

MA: 58829426BP: 442.71466

XIC, +2 Neutral loss or non‐modified

884.47899.48

869.04 MALDI TOF MALDI TOF???

% In

tens

ity

869.05

% In

tens

ity

913.46

915.47

858.62 ???

800 1040

887.481012.59

800 1040

SVSDnYEGK ; MW 928.37738; m/z 465.1965 (z=2) SVSDsYEGK , MW 963.3491; m/z 482.6824 (z=2)


A BR F Instrument Platforms Used by the

sPRG to Validate the Sample andResearch Group (sPRG) sPRG to Validate the Sample and Individual Sample Constituents

NanoLC ESI MS: MALDI MS:

nLC – LTQ Orbitrap (Thermo)nLC – LTQ Orbitrap Velos (Thermo)

Axima TOF/TOF (Shimadzu)AB4700 TOF/TOF (AppliedBio)

nLC – LTQ (Thermo)QQQ TSQ Vantage (Thermo)

AB4800 TOF/TOF (AppliedBio)

Fragmentation ModesCID, HCD, ETD CID, PSD


A BR F Results of the Initial Sample

Validation by the sPRGResearch Group (sPRG)

100%

Average detection success rate, %

Validation by the sPRG

60%

70%

80%

90%

30%

40%

50%

60%

0%

10%

20%


A BR F Preliminary Observations. Multiply

Phosphorylated Peptides.Research Group (sPRG)

Phosphorylated Peptides.• Difficult to detect without any enrichment • Often elute in broad peaksOften elute in broad peaks• Poor ionization efficiency• Predominant neutral loss in MS2. Poor CID fragmentation efficiency• Some detected as ion species corresponding to partially dephosphorylated forms possibly due to in‐source decay

• Additional use of alternative to CID fragmentation modes and multistageAdditional use of alternative to CID fragmentation modes and multistage activation CID fragmentation was helpful

• Additives to the sample mixture (EDTA, phosphate, citrate, etc) helped i th MS i l f tidimprove the MS signal for some peptides

• Optimization of LC separation conditions and MS data acquisition settings helped improve detection sensitivity for closely eluting isobaric positional isomers and sulfopeptides

• RT prediction and site localizition algorithms might be helpful

Alternative Fragmentation Modes: CID z = 3Proteomics Standards

A BR F

CID, z = 3Pros:

‐ Nearly 100% efficient for non‐neutral loss phosphorylations, and >50% efficient for neutral loss phosphorylations


50% efficient for neutral loss phosphorylations‐ Same collision voltage is used for all peptides

Cons:‐Many phosphorylations show loss of 98, sometimes making

y11²?‐NH3, [M+3H]³?‐P644 02

20

Extracted from: C:\Xcalibur\data\ABRF_peptide_mix\111510_ABRF_peptides_03.raw #6490 RT: 42.33 ITMS, CID, z=+3, Mono m/z=676.34625 Da, MH+=2027.02420 Da, Match Tol.=0.8 Da

assignment of the site difficult

y15²?895.22

644.02

15

s] (1

0^3)

y13²?

b2?, y4²?‐H2O239.15

b3?

y15²?‐P846.30

5

10

Inte

nsity

[cou

nts

y 3782.13y4?

496.27

b3?352.29

500 1000 1500 2000

m/z

0

Alternative Fragmentation Modes:ETD z = 3Proteomics Standards

A BR F

ETD , z = 3Pros:

‐ Does not cleave phosphorylationCons:


Cons:‐ Efficiency ~30% maximum for observed phosphorylations (% of ions that are assignable as b and y‐type ions / total estimated ions)

Alternative Fragmentation Modes: HCD z = 3Proteomics Standards

A BR F

HCD, z = 3Pros:

‐ Can be used to produce transitions for triple quadrupole workCons:


Cons:‐ Hard to get the optimal energy for each different peptide, either leaving behind undissociated precursor or producing second generation product ions which are no usable in a database search

b2?8000

Extracted from: C:\Xcalibur\data\ABRF_peptide_mix\111510_ABRF_peptides_04.raw #4674 RT: 41.70 FTMS, HCD, z=+3, Mono m/z=676.34619 Da, MH+=2027.02402 Da, Match Tol.=0.05 Da

g p

239.11411

5000

6000

7000

ount

s]

y15²?894.96179

b4?465.28238

y10?1189.51489

y1?‐H2O129.10233

y8?‐P866.38965

y10?‐P1091.53723

b6?725.43500

y10²?595.26099

b3?352.19800

2000

3000

4000

Inte

nsity

[co

y8?964.36365

465.28238 1189.51489

500 1000 1500 2000

m/z

0

1000


A BR F Multiply Phosphorylated Peptides:

Peak Broadening, Co‐elution ofResearch Group (sPRG)

BIC44.91

451.14520 49 BIC

Peak Broadening, Co elution of Positional Isoforms, Low Signal

BIC 451.14520 49307.

RT: 45.31AA: 436473BP: 614.29517

RT: 42.27MA: 2528064BP: 550.71387

43.05550 71417

BIC

XIC, +2

40 42 44 46 48 32 34 36 38 40 42 44 46 48Time (min)

550.71417

XIC, +2

EpSpTLHLVLR ; MW 1226.5461; m/z 614.2809 (z=2) DIpSLpSDpYK ; MW 1179.3539; m/z 590.6848 Time (min)

RT 42 58

BICXIC +1

Elution with RT: 42.58MA: 8077884BP: 550.71448

42.89550.71423

XIC, +2DISLpSDpYK DIpSLpSDpYK

XIC, +1 10%TFE

4 36 38 40 42 44 46 48Time (min)

DISLpSDpYK; MW 1099.3876; m/z 550.7016

DISLpSDpYK p p p

XIC, +2


A BR F Sulfotyrosine‐containing peptides


• Predominant neutral loss peaks• Poor fragmentation efficiency g y• Intense Na and K adducts• Elution times and m/z values for sulfo‐ and phospho‐isopeptides are very close but possible to differentiate with higher mass accuracy, optimized separation conditions and RT predictionprediction

• Some sulfopeptides reveal several isobaric LC‐MS1 peaks with close RTs


A BR F Sulfated Peptides: Neutral Loss


1107.96

MALDI TOF/TOF

1.0E+4100 1066

.54

y

[M+H] ‐80 [M+H] ‐80

MS1MS1

823 0 1324 0

n t e

n s it

% In

tens

ity

MS1MS1

1802.2100 1067.0

823.0 1324.0

a t iv e I

[M+H] ‐80

638.3

R e l

1121.95

1105.951236.081050.88 1139.96

MS2

TVIDsYNGER; m/z 1146.47

69.0 1126.0

mass/chargeTIAQDsYGVLK ; m/z 1187.56

00 1040 1280

mass/charge

Sulfated Peptides: Neutral Loss and Poor FragmentationProteomics Standards

A BR F

Poor Fragmentation

8

10

12 326.81604


MS1

510.7183

[M+2H+] ‐80, z=2

2

4

6

8

940.48566

266.95218 529.699221020.44458

652 95874 830 82104 1298 15613 1544 18982

MS1 [M 2H ] 80, z 2

8

10

12

bund

ance

0652.95874 830.82104 1298.15613 1544.18982

470.76807

MS2 on the sulfopeptide

0

2

4

6

Rel

ativ

e A

b

712.30310

429.11224310.28967 625.41229 792.52570 1000.38275

712.34772201.09686 461.85806

sulfopeptide(m/z 510.71, z=2)

6

8

10

12

625.34052

310.16345

MS2 on the non‐modified peptide

/

200 400 600 800 1000 1200 1400 1600m/z

0

2

4

794.24518919.19049

(m/z 470.74, z=2)


A BR F Sulfated and Phosphorylated

Peptides

50

100

0 RT: 39.42AA: 159126640BP: 482.69855

RT: 38.09AA: 46135BP 482 69742

PhosphopeptideSulfopeptideXIC 2

Research Group (sPRG)Peptides

50

100

0 BP: 482.69742

39.42964.39020

XIC, +2

XIC, +10

20 25 30 35 40 45 50 50

XIC, +2

0

5

elat

ive

Abu

ndan

ce

RT: 41.24AA: 331340BP: 482.69827

20 25 30 35 40 45 50 5Time (min)

60

80

100

Abu

ndan

ce

964.38032 Very similar m/z values;

l

60

80

1000

20

40

Rel

ativ

e A 965.38333

966.38741964.88098 965.15249

964.39054

MS1 Close retention times;

Predominant neutral

964.0 964.5 965.0 965.5 966.0 966.5 967.m/z

0

20

40

60

965.39357

966.39610964.89209965.89491963.87108

MS1 loss of sulfate


A BR F Nitro‐Tyr, Acetyl‐Lys, and Methyl‐Lys/Arg


• Nitro‐TyrIntense neutral loss ions and Na and K adducts (not as pronounced as in sulfotyrosine peptides)

• Acetyl‐Lys Some result in low intensity signal

• Methyl‐Lys and Argo Close elution or co‐elution of mono‐, di‐, and tri‐o Close elution or co elution of mono , di , and trimethylated peptides (shallower gradients are helpful)

o Co‐elution of symDIMETH‐R and asymDIMETH‐R peptides on C18on C18

o Some symDIMETH‐R and asymDIMETH‐R peptides were indistinguishable in CID and ETD spectra

o Some DIMETH‐R peptides demonstrate predominant neutral loss in MS2 spectra;


A BR F Differentially modified peptide isoforms

NL: 2.44E7Base Peak


NL: 5.74E6m/z= 431.7328-431.7414 ALAPEYAK431.7380

NL: 3.94E5m/z= 471.7108-471.7202ALAPEsYAK

471.7167m/z 471.7108 471.7202

NL: 1.89E7m/z= 454 2251-454 2341ALAPEnYAK

454.2305

20 22 24 26 28 30 32 34 36 38Time (min)

m/z 454.2251 454.2341 ALAPEnYAK

Time (min)


A BR F Differentially modified peptide isoforms


MS/MS Non‐Modified

MS/MS Y‐Sulfated

MS/MS Y‐Nitrated


A BR F Methylated Peptides


NL: 1.94E7Base PeakBase Peak

NL: 6.65E6m/z= 508.7853-508.7955 L(me)KAEGSEIR508.7913

NL: 5.62E6m/z= 515.7928-515.8032

L(dime)KAEGSEIR515.7990

NL: 2.27E6m/z= 522.8008-522.8112

522.8066 L(trime)KAEGSEIR

19 20 21 22 23 24 25 26 27 28 29 30 31 32

NL: 1.48E7m/z= 522.7826-522.7930

L(ac)KAEGSEIR522.7888

19 20 21 22 23 24 25 26 27 28 29 30 31 32Time (min)


A BR F R‐Dimethylated Peptides: Co‐elution and

Similarity of Fragmentation PatternsResearch Group (sPRG)

ETD: AsymDIMETH‐R CID: AsymDIMETH‐R

Similarity of Fragmentation Patterns

ETD: SymDIMETH‐R CID: SymDIMETH‐R


A BR F A Spectral Library for the sPRG 2011

S d d i B i C dResearch Group (sPRG) Standard is Being Created

http://peptide.nist.gov/http://peptide.nist.gov/


A BR F Standard Stability Study


For a standard to be of general utility to the mass spectrometry community it must be stable upon transportation and storage sPRG will test the following:storage. sPRG will test the following:

Stability of freeze dried standardwith time/ temperature of storagewith time/ temperature of storage

Stability of standard upon reconstitution in recommended reagentsreagents

an array of different common reconstitution reagents

ill b t t d d t bilit ith ti / twill be tested and stability with time/ storage

Issue: a method to disentangle the stability of the standard from instrument performance will be established This is necessary toinstrument performance will be established. This is necessary to ensure fluctuations in instrumentation performance do not confound measurements made within longitudinal studies.


A BR F

PRG 2011 St d A tResearch Group (sPRG) sPRG 2011 Study Announcement and Call for Study Participants

P ti i t i th PRG2011 St d d h fParticipate in the sPRG2011 Study and have fun with characterizing more PTMs and refining your

l ti l h !!!analytical approaches!!!

[email protected]


A BR F Acknowledgements


Research Institute of Molecular Biology:

Sigma‐Aldrich:Kristin RolwesBiology:

Andras SchmidtOtto Hudecz

Kristin Rolwes Shantanu RoychowdhuryGeorge Lipscomb

Harvard School of Public Health:Emily Freeman

Thermo Fisher ScientificRainer Gebhart

Alexander Zolotarev

NIST:

Georgi VidenovJoel LouetteManuela SchaffrathNIST:

Paul Rudnick

ABRF

Manuela Schaffrath

Michrom Bioresources:K N tABRF Kerry Nugent


A BR F Questions for the audience


• Should we distribute the sample releasing all information about i d i ?mixed constituents ?

OR• Should we rather run the study in two stages: 1. send the sample without providing any specific information about peptide sequences and proteins and collect data (similar to PRG studies);PRG studies);2. provide all information and request reanalysis of acquired data and sample leftovers?

• Would it be helpful to provide the standard in two formulations: with and without addition of background digests?


A BR F


Please Visit Our Website www.abrf.org , Click on Research Groups then sPRGClick on Research Groups then sPRG

Questions Ideas or Interested in Joining Us?Questions, Ideas or Interested in Joining Us?

Contact Alexander Ivanov (sPRG Chair)Contact Alexander Ivanov (sPRG Chair)[email protected]

Participate in the sPRG2011 Study and have fun with PTMs (PTMomics or Modificomics☺)!!!with PTMs (PTMomics or Modificomics☺)[email protected]

Documents

021611 ABRF2011 sPRGtalk ARI Final...# unique Peptides UC ID Yes 3,571 2 084 0 1000 2000 2,084 1,623 14941 87133 22730 86010 13800 84940v 20899i 53706 92536i 870486i 45682 870484i