Protein Sequencing Research Group

ABRF 2014 annual meeting

» Robert English (co-chair) Shimadzu

» Sara McGrath (co-chair) FDA/CFSAN

» Greg Cavey Southwest Michigan Innovation Center

» Mark Garfield NIH/NIAID

» Pegah Jalili Sigma-Aldrich

» Ejvind Mørtz Alphalyse

» Henriette Remmer (ad-hoc) Univ. of Michigan

» Bill Hendrickson (EB liaison) Univ. of Illinois Chicago

» N-terminal sequencing is in the midst of a technology transition from classical Edman sequencing to mass spectrometry (MS)-based sequencing

» Core laboratories need to have well-defined protocols for terminal sequence analysis by MS

» Practice with types of samples, sample preparation protocols and expected results are critical

» N-terminal sequencing is in the midst of a technology transition from classical Edman sequencing to mass spectrometry (MS)-based sequencing

» Core laboratories need to have well-defined protocols for terminal sequence analysis by MS

» Practice with types of samples, sample preparation protocols and expected results are critical

» This year’s study: use chemical derivitization to enhance N-terminal sequencing of proteins by MS ˃3 known purified proteins, provided separately ˃Successfully derivitize the proteins using a provided protocol ˃Digest and identify the derivitized peptide fragments by MS ˃Correctly interpret spectra and identify the N-terminus

» 22 laboratories requested samples

˃8/22 (36%) international sites

» 13 participants returned data, 12 completed response survey

3

8

1

Participate in 2013 study

Yes

no

Unanswered

1 1

8

1

yes, high yes, low No Unanswered

Experience with N-term derivitization

» Identify the N-terminus of known proteins using chemical derivitization (TMPP or dimethylation)

» Two possible workflows:

» Study requires mass spectrometry (no edman)

(A) In-solution labeling (B) In-gel labeling and digest

Chemical labeling of proteins at N-terminus

SDS-PAGE, excision of bands and in-gel chemical labeling

SDS-PAGE and in-gel or on-membrane cut-off filter tryptic digestion

In-gel tryptic digestion and cleanup

MS analysis including data analysis MS analysis including data analysis

Identification of N-termini Identification of N-termini

TMPP labeling Dimethyl labeling

» 5uL of 100pmol/uL protein

» 5uL of 1mM TMPP

» 10uL 0.1MHepes buffer, pH 8

» 30 min reaction at RT

» 10uL of 10pmol/uL protein

» 90uL 100mM Na acetate, pH 5

» 1uL 4% formaldehyde

» 1uL Na cyanoborohydride

» Incubate 5 mins at RT

>> S Gallien, E Perrodou, C Carapito, C Deshayes, JM Reyrat, A Van Dorsselaer, O Poch, C Schaeffer, O Lecompte. Orthoproteogenomics: Multiple proteomes investigation through orthology and a new MS based protocol. Genome, 2009; 19: 128-135; doi 10.1101/gr.081901.108. >> JL Hsu, SY Huang, NH Chos, SH Chen. Stable-isotope dimethyl labeling for quantitative proteomics. Anal Chem., 2003 Dec 15; 75(24):6843-52.

Protein/Starting

amount (pmol)

Amount of

sample

analyzed by

MS (fmol)

TMPP

Labeling in

solution

TMPP

Labeling

In-Gel

TMPP

Digest in

Solution

TMPP

Digest

In-Gel

Positive

Identification

Hemoglobin

500pmol

α

β

Hemoglobin

100pmol

α

β

Myoglobin

500pmol

Myoglobin

100pmol

BSA

500pmol

BSA

100pmol

Protein name N-term tryptic sequence

Hemoglobin - alpha VLSPADK

Hemoglobin - beta VHLTPEEK

Myoglobin GLSDGEWQQVLNVWGK

Bovine Albumin (BSA) DTHK

» TMPP

˃Addition of fixed-charge improves peptide CID

˃Favors N-term amine labeling over other amino acids

+

+

» Dimethylation

˃Addition of dimethyl by reductive amidation

˃Reacts with N-term and Lysine residues

+ NH2-R N-R

H

NH2-R + NaBH3CN N-R CH2

CH2

Complete in 30 minutes! (almost no unlabeled LeuEnk remaining)

Derivitization reaction: • 21uL Leu-Enk (250 uM final) • 9uL Acetonitrile • 15uL 10mM TMPP in 50% ACN (2.5mM final, 10X molar ratio) • 15uL Hepes (0.1M, pH 8) • Time course: reaction analyzed every 15min for 2 hours

Ion species m/z value

TMPP-YGGFL 1128.45 1+

564.23 2+

YGGFL 556.27

TMPP hydrolysis product

591.19

TMPP - Br 688

MS/MS TMPP hydrolysis product (591 m/z)

TMPP N-term modification C29H34O10P → +572.18 Da

MS/MS TMPP-LeuEnk

Full a- and b- ion series of YGGFL

observed

Derivitization reaction: • 5uL 1000pmol/uL myoglobin in H20 • 10uL Hepes (0.1M, pH 8) • 5uL 10mM TMPP in 50% ACN • Reacted 30mins, inject 3uL on LCT

Ion species m/z value

Myoglobin 16952.46

Myoglobin + TMPP 17525.13

Myoglobin + 2TMPP 18097.90

Myoglobin + TMPP is most abundant species, but unlabeled

and 2x labeled species are present

250 uM myoglobin, 2500 uM TMPP 25uM myoglobin , 250uM TMPP

2.5 uM myoglobin, 25 uM TMPP No labeled myoglobin

Labeling efficiency is dependent on quantity of protein and TMPP, not ratio

M+2H

M+3H

TMPP as Mod

Intact Mass unlabeled Hemoglobin 100pmol in 0.1%FA/H2O

α - 15,126 Da β - 15,867 Da

α + 2 TMPP

b + 2 TMPP

α + 3 TMPP α + 4 TMPP

α + 5 TMPP

b + 3 TMPP

b + 4 TMPP

α + 1 TMPP

50uM hemoglobin reacted with 500uM TMPP

b + 5 TMPP

native

dimethyl

MS/MS verification

Predicted MS/MS fragments for dimethy-labeled Myoglobin

Myoglobin - Labeled peptide

Hemoglobin α and β -Labeled peptides

× BSA - Labeled peptide was not observed

21R (workflow A)

250 150 100 75 50 37 25 20 15

MW Hemo Myo BSA Hemo Myo BSA Myo BSA

(kDa) - - - + + + + + TMPP

21R (workflow A)

MS/MS spectrum of myoglobin N-terminal labeled peptide 1194.6 m/z

Method of

Labeling

Start Amount (pmol) Amount of sample

analyzed by MS (fmol)

Positive Identification

In gel

Digestion

In Solution

Digestion

In gel

Digestion

In Solution

Digestion

In gel Digestion In Solution

Digestion

Hemoglobin

500pmol Protocol

50 200 1800 4000 α Detected α Detected

β Detected β Detected

Hemoglobin100pmol

Protocol

50 200 1800 4000 α Detected α Detected

β Detected β Detected

Myoglobin 500pmol

Protocol

50 200 1500 3200 Detected Detected

Myoglobin 100pmol

Protocol

50 200 1500 3200 Detected Detected

BSA 500pmol

Protocol

50 200 2000 2500 Detected Detected

BSA 100pmol

Protocol

50 200 2000 2500 Detected Detected

Hemoglobin In Gel

Labeling

50 - 1800 - Detected

Myoglobin In Gel

Labeling

50 - 1500 - Detected

BSA In Gel Labeling 50 - 2000 - Detected

uVHLTPEEK

Measured m/z=508.90283+

Theoretical m/z=508.90183+

u= C29H33P1O10

276.19 y2

922.33 b4

200 300 400 500 600 700 800 900 1000 1100 m/z

258.1100

461.7590

356.1453

585.3219 995.4018 328.0609 894.4772 781.4045 1076.1888

405.11 y3

502.34 y4

603.28 y5

716.38 y6

853.44 y7

809.34 b3

672.16 b2

1023.29 b5

560.422+

b6

625.392+

b6

690.052+

b7

HBB (hemoglobin Beta) N-terminus TMPP Modification

Sample ID MS/MS Scan #

HEM_500pmol_InSolutionLabel_InGel_Digestion 3258, 3328,

HEM_500pmol_InSolutionLabel_InSolution_Digestion 3510, 3546, 3579

HEM_100pmol_InSolutionLabel_InGel_Digestion 3064, 3096, 3131

HEM_100pmol_InSolutionLabel_InSolution_Digestion 3985, 4028, 4071

HEM_InGelLabel_InGel_Digestion 3917, 3959, 4000

uDTHK

Measured m/z=536.71822+

Theoretical m/z=536.71742+

u= C29H33P1O10

BSA (albumin) N-terminus TMPP Modification

Sample ID MS/MS Scan #

BSA_500pmol_InSolutionLabel_InGel_Digestion 3244, 3286, 3333

BSA_500pmol_InSolutionLabel_InSolution_Digestion 3761, 3804, 3848

BSA_100pmol_InSolutionLabel_InGel_Digestion 3427, 3471, 3515

BSA_100pmol_InSolutionLabel_InSolution_Digestion 3845, 3889, 3933, 3978

BSA_InGelLabel_InGel_Digestion 3768, 3812, 3857, 3902

385.23 y3

688.24 b2

789.10 b3

926.08 b4

527.682+

M-H2O

200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

m/z

518.61 944.43 367.23 472.64

239.15 623.37 463.57 676.91 590.55 752.14 506.00 908.32 266.24 820.65 717.15 854.17 449.67 349.38 313.34 969.47

» Workflow A ˃in-solution labeling

˃SDS-PAGE cleanup

» Analysis with a Q-Exactive mass spec

» N-termini were identified only for hemoglobin alpha and beta

» The myoglobin terminus was found by mass spectra but was not labeled with TMPP

» The predicted start site for BSA was found as a TMPP labeled peptide, however a large number of other sites were also labeled as well so that the N-terminus identification would be ambiguous

Sequence Prob Mascot Ion scoreModifications Start Stop

(K)vHKEccHGDLLEcADDR(A) 100% 33.34 TMPP-Ac (+572), Carbamidomethyl (+57), Carbamidomethyl (+57), Carbamidomethyl (+57)264 280

(K)vHKEccHGDLLEcADDR(A) 92% 27.74 TMPP-Ac (+572), Carbamidomethyl (+57), Carbamidomethyl (+57), Carbamidomethyl (+57)264 280

(K)yNGVFQEccQAEDK(G) 99% 32.2 TMPP-Ac (+572), Carbamidomethyl (+57), Carbamidomethyl (+57)184 197

(K)yNGVFQEccQAEDK(G) 93% 28.25 TMPP-Ac (+572), Carbamidomethyl (+57), Carbamidomethyl (+57)184 197

(R)lAKEYEATLEEccAK(D) 98% 28.72 TMPP-Ac (+572), Carbamidomethyl (+57), Carbamidomethyl (+57)372 386

(R)lAKEYEATLEEccAK(D) 90% 28.43 TMPP-Ac (+572), Carbamidomethyl (+57), Carbamidomethyl (+57)372 386

(K)vTKccTESLVNR(R) 100% 49.05 TMPP-Ac (+572), Carbamidomethyl (+57), Carbamidomethyl (+57)496 507

(K)lKPDPNTLcDEFK(A) 100% 37.32 TMPP-Ac (+572), Carbamidomethyl (+57)139 151

(K)lKPDPNTLcDEFK(A) 90% 25.62 TMPP-Ac (+572), Carbamidomethyl (+57)139 151

(R)dTHKSEIAHR(F) 100% 21 TMPP-Ac (+572) 25 34

(K)sEIAHR(F) 90% 24.38 TMPP-Ac (+572) 29 34

(K)lKHLVDEPQNLIK(Q) 100% 50.74 TMPP-Ac (+572) 400 412

(K)lKHLVDEPQNLIK(Q) 100% 33.06 TMPP-Ac (+572) 400 412

(K)lKHLVDEPQNLIK(Q) 100% 31.7 TMPP-Ac (+572) 400 412

(K)lKHLVDEPQNLIK(Q) 98% 29.93 TMPP-Ac (+572) 400 412

(K)lKHLVDEPQNLIK(Q) 98% 28.31 TMPP-Ac (+572) 400 412

(R)kVPQVSTPTLVEVSR(S) 100% 57.85 TMPP-Ac (+572) 437 451

(R)kVPQVSTPTLVEVSR(S) 100% 42.88 TMPP-Ac (+572) 437 451

(R)kVPQVSTPTLVEVSR(S) 100% 55.67 TMPP-Ac (+572) 437 451

(R)kVPQVSTPTLVEVSR(S) 100% 46.75 TMPP-Ac (+572) 437 451

(R)kVPQVSTPTLVEVSR(S) 100% 39.52 TMPP-Ac (+572) 437 451

(K)vPQVSTPTLVEVSR(S) 100% 47.26 TMPP-Ac (+572) 438 451

(K)kQTALVELLK(H) 100% 46.24 TMPP-Ac (+572) 548 557

(K)kQTALVELLK(H) 100% 43.94 TMPP-Ac (+572) 548 557

» Lowest detectable protein: 1pmol starting material (BSA) by solution and in-gel labeling

» 50pmol of hemoglobin and myoglobin was detected by 3 labs (4 including LML)

8 10 11

3 2 0

1 0

1

BSA Hemoglobin Myoglobin

Was there sufficient material?

yes no n/a

0 2 2

5 4

7

1 2 2

BSA Hemoglobin Myoglobin

How much did you use?

less than 1 pmol 1-10 pmol

more than 10 pmol

Method Number of correct N-term IDs

In-solution label + In-gel digest 9

In-solution label + In solution digest 6

In-gel label + In-gel digest 6

Protein Number of correct

N-term IDs Number of incorrect

N-term IDs

Hemoglobin alpha 9 2

Hemoglobin beta 9 2

Myoglobin 10 3

BSA 5 7

Protein Starting Material Number of

Participants Number of Positive IDs

Percentage positive ID

Hemoglobin

500pmol 6 6 100

100pmol 6 6 100

50pmol 3 3 100

10pmol 1 1 100

Myoglobin

500pmol 8 7 87.5

400pmol 1 1 100

100pmol 6 5 83

50pmol 2 2 100

10pmol 1 1 100

BSA

500pmol 7 1 14

100pmol 6 2 33

50pmol 2 1 50

10pmol 2 1 50

1pmol 1 1 100

» All participants had at least 1 positive N-terminal ID

» 4 participants identified N-term peptide of all 3 proteins

» In-solution label with in-gel digest was favored protocol

8%

8%

50%

34%

Purification methods used

C-18 spin filter

Used sample as provided (direct analysis)

SDS_PAGE

HPLC

TMPP Labeling Dimethyl Labeling

» Chemistry is straightforward

» Not very sensitive – seems to require a lot of material

» Can get multiple TMPP tags per protein

» Top down MS failed to ID N-term of any of 3 proteins

» Works better for some proteins than others

˃Myoglobin = good

˃Hemoglobin = OK

˃BSA = difficult, several

different opinions of sequence

» More universal labeling efficiency

» Near quantitative/sensitive

» Chemistry requires some hazardous chemicals

Both » Need to recover N-terminal

peptide

» May require enzyme other than trypsin

» Inexpensive

» Adapts to any MS system

2

5

3

1

Estimated time effort

one day or less more than one day more than one week Unanswered

3

8

1

Participate in 2013 study

Yes

no

Unanswered

10

2

Trypsin Chymotrypsin

Digest enzyme used “I feel that the amount of material is not as important as the peptide.

Chymotrypsin yields more n-terminally

labeled peptide for BSA in our experiments.”

» Most participants would like more time for analysis, though most respondents averaged 2-3 days

» Some participants did not like the flexible style of protocol and would like more specific instructions

» Many participants were very interested in this study and in practicing this type of labeling and analysis

» Determine the sensitivity of TMPP/Dimethyl protein labeling with N-terminal peptide identification by mass spectrometry

» Simplify the study to one protein » Allow participants to select the amount for labeling

and mass spectrometry. How low can you go? » Allow participants to perform solution or gel

labeling » Allow participants to optimize conditions as they

wish but provide analysis details

» What would be of most benefit to you?? ˃Plan to send out a survey on these ideas

• Stable base line and reproducible retention times due to isocratic separation

• Sensitivity level: 15-20 cycles of 5 pmol • Would like feedback from community

For more information contact: Robert English (rdenglish@shimadzu.com) Brian Feild (bjfeild@shimadzu.com)

Shimadzu PPSQ-33A: Automated Edman Sequencing

Applied Biosystems: Procise 49x cLC System

For more information contact: Robert English (rdenglish@shimadzu.com) Brian Feild (bjfeild@shimadzu.com)

» PSRG is maintaining a list of Edman providers and repair facilities

» Keep users in contact to share information

» ABRF Marketplace is searchable

» We are always looking for new PSRG members!

» If you have interest in protein sequencing, and skills with either Edman or mass spectrometry, please contact one of our current members

Robert English Shimadzu Scientific

rdenglish@shimadzu.com

Sara McGrath FDA/CFSAN

sara.mcgrath@fda.hhs.gov

» Sponsors of study proteins and reagents:

˃ABRF

˃Biomedical Research Core Facilities, University of Michigan

» Study Design Experiments

˃Francisca Wong, Southwest Michigan Innovation Center

» Anonymizer:

˃Amanda McGinnis, University of Michigan

» ………and study participants!!!!!!