021611 abrf2011 sprgtalk ari final...# unique peptides uc id yes 3,571 2 084 0 1000 2000 2,084 1,623...
TRANSCRIPT
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
Research Group (sPRG)
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
Proteomics Standards
A BR F Rationale
Research Group (sPRG)
• 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?
Proteomics Standards
A BR F Background
Research Group (sPRG)
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
Proteomics Standards
A BR F Background
Research Group (sPRG)
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)
Proteomics Standards
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
Proteomics Standards
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#
%
Proteomics Standards
A BR F sPRG 2011 Study Objectives
Research Group (sPRG)
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.
Proteomics Standards
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
Research Group (sPRG)
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
Proteomics Standards
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) …
Proteomics Standards
A BR F “Bonus” peptides. Synthesis byproducts.
Research Group (sPRG)
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)
Proteomics Standards
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
Proteomics Standards
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%
Proteomics Standards
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
Research Group (sPRG)
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:
Research Group (sPRG)
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:
Research Group (sPRG)
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
Proteomics Standards
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
Proteomics Standards
A BR F Sulfotyrosine‐containing peptides
Research Group (sPRG)
• 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
Proteomics Standards
A BR F Sulfated Peptides: Neutral Loss
Research Group (sPRG)
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
Research Group (sPRG)
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)
Proteomics Standards
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
Proteomics Standards
A BR F Nitro‐Tyr, Acetyl‐Lys, and Methyl‐Lys/Arg
Research Group (sPRG)
• 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;
Proteomics Standards
A BR F Differentially modified peptide isoforms
NL: 2.44E7Base Peak
Research Group (sPRG)
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)
Proteomics Standards
A BR F Differentially modified peptide isoforms
Research Group (sPRG)
MS/MS Non‐Modified
MS/MS Y‐Sulfated
MS/MS Y‐Nitrated
Proteomics Standards
A BR F Methylated Peptides
Research Group (sPRG)
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)
Proteomics Standards
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
Proteomics Standards
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/
Proteomics Standards
A BR F Standard Stability Study
Research Group (sPRG)
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.
Proteomics Standards
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!!!
Proteomics Standards
A BR F Acknowledgements
Research Group (sPRG)
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
Proteomics Standards
A BR F Questions for the audience
Research Group (sPRG)
• 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?
Proteomics Standards
A BR F
Research Group (sPRG)
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]