i i ( ) i ifi i f i i i i#260 Protein Sequencing Research Group (PSRG) 2015 Study: N terminal Identification of a Standard Protein at Low Picomole Levels by Dimethyl Labeling and Bottom up Mass Spectrometry#260 Protein Sequencing Research Group (PSRG) 2015 Study: N-terminal Identification of a Standard Protein at Low Picomole Levels by Dimethyl Labeling and Bottom-up Mass Spectrometry#260 Protein Sequencing Research Group (PSRG) 2015 Study: N-terminal Identification of a Standard Protein at Low Picomole Levels by Dimethyl Labeling and Bottom-up Mass Spectrometry#260 Protein Sequencing Research Group (PSRG) 2015 Study: N terminal Identification of a Standard Protein at Low Picomole Levels by Dimethyl Labeling and Bottom up Mass Spectrometry q g p ( ) y y y g p p yS C M G th1 H di E dj t B 2 G S C 3 R b t D E li h4 X i L 5 H i tt A R 6 B tt S Phi 7Sara C McGrath1 Hediye Erdjument-Bromage2 Gregory S Cavey3 Robert D English4 Xuemei Luo5 Henriette A Remmer6 Brett S Phinney7Sara C. McGrath , Hediye Erdjument-Bromage , Gregory S. Cavey , Robert D. English , Xuemei Luo , Henriette A. Remmer , Brett S. PhinneyS C G , y j g , G g y S C y , g , , , S y1FDA C t f F d S f t d A li d N t iti 2M i l Sl K tt i C C t 3L h MI L b S th t Mi hi I ti C t 4Shi d S i tifi I t t 5U i it f T M di l B h 6U i it f Mi hi 7UC D i G C t1FDA Center for Food Safety and Applied Nutrition 2Memorial Sloan-Kettering Cancer Center 3Launch MI Lab Southwest Michigan Innovation Center 4Shimadzu Scientific Instruments 5University of Texas Medical Branch 6University of Michigan 7UC Davis Genome CenterFDA Center for Food Safety and Applied Nutrition, Memorial Sloan-Kettering Cancer Center, Launch MI Lab, Southwest Michigan Innovation Center, Shimadzu Scientific Instruments, University of Texas Medical Branch, University of Michigan, UC Davis Genome Centery pp , g , , g , , y , y g ,

B k d d Obj ti St d D iBackground and Objective Study DesignBackground and Objective Study DesignBackground and Objective Study Design Dimethyl labeling • Nt dimethyl peptides are best sequenced from b ionsDimethyl labeling • Nt-dimethyl peptides are best sequenced from b ionsDimethyl labeling y p p qN t i l i i i th id t f t h l t iti f l i l Ed Additi f di th l b d ti Internal dimethylations can confound results• N-terminal sequencing is in the midst of a technology transition from classical Edman O• Addition of dimethyl by reductive • Internal dimethylations can confound results• N-terminal sequencing is in the midst of a technology transition from classical Edman O• Addition of dimethyl by reductive Internal dimethylations can confound resultsq g gy

i t t t ( S) b d i ║W kfl (A) W kfl (B)y y

b3 ion is not concl si e dimeth l can be at either N termin s or R3sequencing to mass spectrometry (MS) based sequencing ║Workflow (A) Workflow (B) amidation • b3 ion is not conclusive – dimethyl can be at either N-terminus or R3sequencing to mass spectrometry (MS)-based sequencing CH║Workflow (A) Workflow (B) amidation b3 o s o co c us e d e y ca be a e e e us o 3q g p y ( ) q g CH3H HIn solution labeling In gel labeling

amidation• Must verify that b ions indicate correct placement of dimethyl group• Core laboratories need well defined protocols for terminal sequence analysis by MS

3H HIn-solution labeling In-gel labeling i h d i id• Must verify that b ions indicate correct placement of dimethyl group• Core laboratories need well-defined protocols for terminal sequence analysis by MS NH peptide N peptideIn solution labeling In gel labeling

• Reacts with N term and Lysine residuesgCore laboratories need well defined protocols for terminal sequence analysis by MS NH2-peptide N-peptide• Reacts with N-term and Lysine residuesPractice with types of samples sample preparation protocols and expected results are

2 p p p pCHSDS-PAGE separation of proteins*

y• Practice with types of samples, sample preparation protocols and expected results are NaBH3CN CH3Chemical labeling of proteins at N-terminus SDS-PAGE separation of proteinsPractice with types of samples, sample preparation protocols and expected results are NaBH3CN 3Chemical labeling of proteins at N-terminus *This study used only a single purified proteiniti l f f l l b b b3

This study used only a single purified proteini di icritical for successful analyses b1 b2 b3Reaction conditionscritical for successful analyses 1 2 3Reaction conditionsy

Cleanup with SDS-PAGE or membrane cut-off filter In-gel chemical labelingCleanup with SDS-PAGE or membrane cut-off filter In-gel chemical labeling 10 L f 10 l/ L t i• 10uL of 10pmol/uL protein• This year’s study: use dimethylation of protein to enhance identification of N terminal • 10uL of 10pmol/uL protein• This year s study: use dimethylation of protein to enhance identification of N-terminal p / p

Myoglobin: P68082This year s study: use dimethylation of protein to enhance identification of N terminal Digestion in-gel or on cut-off filter In-gel tryptic digestion and cleanup 90 L 100 M N t t H 5

Myoglobin: P68082peptide sequence Digestion in-gel or on cut-off filter In-gel tryptic digestion and cleanup • 90uL 100mM Na acetate pH 5y gpeptide sequence • 90uL 100mM Na acetate, pH 5peptide sequence p

GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHP1 L 4% f ld h d GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHP• 1 known purified protein (myoglobin) MS analysis including data analysis MS analysis including data analysis • 1uL 4% formaldehyde GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHP• 1 known purified protein (myoglobin) MS analysis including data analysis MS analysis including data analysis 1uL 4% formaldehydeETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGIL

p p ( y g )ETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILSuccessfully derivatize the proteins using a provided protocol 1uL Na cyanoborohydride ETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGIL• Successfully derivatize the proteins using a provided protocol • 1uL Na cyanoborohydride KKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHS

Successfully derivatize the proteins using a provided protocolLysineIdentification of N-termini Identification of N-termini 1uL Na cyanoborohydride KKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSDi t d id tif th d i iti d tid f t b MSLysineIdentification of N-termini Identification of N-termini KKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHS• Digest and identify the derivitized peptide fragments by MS • Incubate 5 mins at RT• Digest and identify the derivitized peptide fragments by MS • Incubate 5 mins at RT KHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG

g y p p g yH C CHIncubate 5 mins at RT KHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG• Determine amount of protein needed for practical use H3C CH3G G QG G QG• Determine amount of protein needed for practical use 3 3p p

i i Other examples:Method Sensitivity Chemistry and Sample Preparation LC MS and Data Analysis: Other examples:Method Sensitivity Chemistry and Sample Preparation LC-MS and Data Analysis: Other examples:Method Sensitivity Chemistry and Sample Preparation LC MS and Data Analysis: py y p p yM t ti i t f l ith 5 l t ti t i l P t l ti l b li dditi f 4% i h d id i ti l t h l ti di t S h i f d t t fit Di th l t tid ith i t l l i E id f tid [M H]+ d MS/MS t• Most participants were successful with 5 pmol starting material • Post-solution labeling addition of 4% ammonium hydroxide is essential to quench solution digest • Search engines may force data to fit Dimethyl to peptides with internal lysines • Evidence of peptide [M+H]+ and MS/MS spectra• Most participants were successful with 5 pmol starting material • Post-solution labeling, addition of 4% ammonium hydroxide is essential to quench solution digest • Search engines may force data to fit Dimethyl to peptides with internal lysines • Evidence of peptide [M+H] and MS/MS spectrap p p g g y q g g y y p p y p p [ ] p

• Several could detect 0 3 pmol of starting protein • Failure to quench may result in uncontrolled dimethylation of peptides during in solution digest • Matched y and b ions cannot distinguish between Nt and internal K dimethylation • Detection of peptide at 0 3 pmol using MALDI/TOF• Several could detect 0.3 pmol of starting protein • Failure to quench may result in uncontrolled dimethylation of peptides during in-solution digest • Matched y and b ions cannot distinguish between Nt and internal K dimethylation • Detection of peptide at 0.3 pmol using MALDI/TOFSe e a cou d de ec 0.3 p o o s a g p o e a u e o que c ay esu u co o ed d e y a o o pep des du g so u o d ges Matched y and b ions cannot distinguish between Nt and internal K dimethylation e ec o o pep de a 0.3 p o us g / ODi th l t d l i i hi h• Dimethylated lysines receive higher scoresGLSDGEWQQVLNVWGK m/z 908 4539 (2+) • Dimethylated lysines receive higher scores b b b b b bTable 1 Dimethyl In Solution Labeling bGLSDGEWQQVLNVWGK m/z 908.4539 (2+) y y g

5 pmolb6 b7 b8 b10 b11 b13Table 1. Dimethyl In-Solution Labeling b12

• Must use Nt dimethyl as well as K dimethyl among the variable search parameters 5 pmol6 7 8 10 11 13y g 12

• Must use Nt-dimethyl as well as K dimethyl among the variable search parameters pAmount of Protein in Reaction Must use Nt dimethyl as well as K dimethyl among the variable search parametersAmount of Protein in Reaction

Reagent 5 pmol 1 pmol 0 3 pmolReagent 5 pmol 1 pmol 0.3 pmol

i f i l/Lowest Quantity of Protein 1 pmol/uL protein in 100 mM Na acetate pH 5 5 uL 1 uL 0 3 uLLowest Quantity of Protein 1 pmol/uL protein in 100 mM Na acetate, pH 5 5 uL 1 uL 0.3 uLyD t t d N L i di th l ti i ifi d i di th l d i bl d100 M N H 5 4 4 7Detected No Lysine dimethylation is specified: Lysine dimethyl used as a variable mod:100 mM Na acetate, pH 5 - 4 4.7Detected

1 pmolNo Lysine dimethylation is specified: Lysine dimethyl used as a variable mod:100 mM Na acetate, pH 5 4 4.7

1 pmolsearch engines fit MS/MS to we will obtain the following hits for4% F ld h d 1 L 1 L 1 Lpsearch engines fit MS/MS to we will obtain the following hits for 4% Formaldehyde 1 uL 1 uL 1 uL

more 5 pmol 1 pmol 0 3 pmol lessg /

ifi Nt di th l tig

i t l L i di th l ti4% Formaldehyde 1 uL 1 uL 1 uL

more 5 pmol 1 pmol 0.3 pmol less non-specific Nt-dimethylation: internal Lysine dimethylation:260 M N b h d id 1 L 1 L 1 L non specific Nt dimethylation: internal Lysine dimethylation:260 mM Na cyanoborohydride 1 uL 1 uL 1 uLy yReaction Conditions vortex 5 min RT vortex 5 min RT vortex 5 min RT3 NDIAAQYKELGFQG NDIAAQYKELGFQGReaction Conditions vortex 5 min RT vortex 5 min RT vortex 5 min RT3 NDIAAQYKELGFQG NDIAAQYKELGFQG

0 3 lNDIAAQYKELGFQG Q Q

4% ammonium hydroxide (quench reaction) 0.3 pmolASEDLKKHGTVVLTALGGILKK ASEDLKKHGTVVLTALGGILKK4% ammonium hydroxide (quench reaction) 1 uL vortex 1 uL vortex 1 uL vortex pASEDLKKHGTVVLTALGGILKK ASEDLKKHGTVVLTALGGILKK y (q )* ti l b t i d f i l ti di t 1 uL, vortex 1 uL, vortex 1 uL, vortex ASEDLKKHGTVVLTALGGILKK *optional, but required for in-solution digest , , ,

ALELFRNDIAAQYKELGFQG ALELFRNDIAAQYKELGFQGoptional, but required for in solution digest

2 2 2 ALELFRNDIAAQYKELGFQG ALELFRNDIAAQYKELGFQG Final Volume 8 uL 8 uL 8 uL2 2 2 DiMethyl-GLSDGEWQQVLNVWGK m/z 922 4694 (2+)ALELFRNDIAAQYKELGFQG Q QFinal Volume 8 uL 8 uL 8 uL2 2 2 DiMethyl-GLSDGEWQQVLNVWGK m/z 922.4694 (2+) YKELGFQG YKELGFQGYKELGFQG YKELGFQG YKELGFQG Q

1 1 11 • 6 participants identified Nt dimethylated myoglobin as the miscleaved peptide spanning K161 1 11 • 6 participants identified Nt-dimethylated myoglobin as the miscleaved peptide spanning K161 1 11 p p y y g p p p gMS/MS spectra of m/z 1843 7

S t f M l bi di th l t d tidMany also identified the Ct fragment with internal lysine dimethylationMS/MS spectra of m/z 1843.7

Spectrum of Myoglobin dimethylated peptide:− Many also identified the Ct fragment with internal lysine dimethylation Spectrum of Myoglobin dimethylated peptide: Many also identified the Ct fragment with internal lysine dimethylationIPIKYLEFISDAIIHVAHSKAll d W kfl A i l ti d i ti ti IPIKYLEFISDAIIHVAHSK − All used Workflow A in-solution derivatization0 0 Where is the dimethyl group located?

All used Workflow A, in-solution derivatization0 0 Where is the dimethyl group located? • Detection of peptide at MS1 level0 0 y g pb b b t d t t d (b l t ff)

• Detection of peptide at MS1 level• b1, b2, b3 not detected (below cutoff)

p pW kfl A (i W kfl B (i l

b1, b2, b3 not detected (below cutoff)b t i 28 Dnative dimethyl 2x dimethyl • No MSMS were acquired for the 1pmol standardWorkflow A (in- Workflow B (in-gel • b4 contains +28 DaPeptide native dimethyl 2x dimethyl • No MSMS were acquired for the 1pmol standardWorkflow A (in Workflow B (in gel b4 contains +28 Da

4Peptide [M H]

y[M H]

y[M H] id f i l l b l d id [ ]2solution labeling) labeling) confirms dimethyl at Nt I1 OR at internal K4

p [M+H]+ [M+H]+ [M+H]+ • Evidence for n-terminal labeled peptides [M+H]2+solution labeling) labeling) confirms dimethyl at Nt I OR at internal K[ ] [ ] [ ] Evidence for n terminal labeled peptides [M+H]GLSDGEWQQVLNVWGK 1815 90 1843 93 1871 96 Th h ld f S MSMS 30 000C l iGLSDGEWQQVLNVWGK 1815.90 1843.93 1871.96 • Threshold for Survey MSMS = 30 000 countsConclusion: Threshold for Survey MSMS = 30,000 countsConclusion:GLSDGEWQQVLNVWGKVEADIAGHGQEVLIR 3403 74 3431 77 3459 80 MS/MS spectrum does not support the exact positionGLSDGEWQQVLNVWGKVEADIAGHGQEVLIR 3403.74 3431.77 3459.80 MS/MS spectrum does not support the exact position

ProteinMW of the Dimethyl group5 5 1 1 0 3 0 3

ProteinMW of the Dimethyl groupNDIAAKYKELGFQG (c term fragment) 1553 79 1581 83 1609 86 5 5 1 1 0.3 0.3 (pmol)LaddergNDIAAKYKELGFQG (c-term fragment) 1553.79 1581.83 1609.86 (pmol)Ladder

h l [ ]2 /Dimethyl-GLSDGEWQQVLNVWGK [M+2H]2+ at m/z 922 47Dimethyl GLSDGEWQQVLNVWGK [M+2H] at m/z 922.47 922 97

• Excise protein bands: For 1 pmol and 300 fmol amounts 922.97z=2922.47 100 922.47• Excise protein bands: For 1 pmol and 300 fmol amounts, 100 z=2922.47

z=2 100 922.47Excise protein bands: For 1 pmol and 300 fmol amounts, z 2

905 l 1 lband may not be visible 90 905 pmol 1 pmolband may not be visible80

p py80 80

Excise gel area migrating at same place as 5 0 70 922.97– Excise gel area migrating at same place as 5 70

ce

70

ce

9 9Excise gel area migrating at same place as 5 60an

c

60anc

picomole myoglobin 60nda

923.47 60

nda

picomole myoglobin 50bun

z=2 50Abu 923.47p y g 50

e A 50

e A 9 3

M difi d il t i b d t i li 40tive

40tive

– Modified silver stain can be used to visualize 40

elat 923.97

40

ela

927 92Modified silver stain can be used to visualize 30R

e z=2 929 99 30Re 927.92

lower protein quantities30 929.99

z=?927.98924.3030

927.42926.27lower protein quantities 20z=?z=?z=? 20 920.89 923 97 924 85lower protein quantities 20

928.32921.16 924.81 927.48926 66 928 97

20 923.97 924.85 928.46 929.10922.41 926 89

Aft i l l b li i l i ith 4% NH OH 10 z=?z=?924.81

z=?927.48

z=?926.66

z=?928.97

?920.41

?922.31 925.85 10

926.89

• After in-gel labeling rinse gel pieces with 4% NH4OH z ? z ?z=? z=?z=? z=? z=?10After in gel labeling, rinse gel pieces with 4% NH4OH 0 0g g g 4

920 921 922 923 924 925 926 927 928 929 930 920 921 922 923 924 925 926 927 928 929 930

Aids in quenching of dimethylation reactants m/z m/z– Aids in quenching of dimethylation reactants 15%T SDS PAGE followed by Ag stain/q g y 15%T SDS-PAGE followed by Ag stainy g

Whi h kfl did ?Participant Res lts Lessons Learned AcknowledgementsDid ti t i th Which workflow did you use?Participant Results Lessons Learned AcknowledgementsDid you particpate in the Which workflow did you use?Participant Results Lessons Learned AcknowledgementsDid you particpate in the p gS f t d t i d t

y p p2014 t d ? • Sponsors of study proteins and reagents:2014 study?• 25 laboratories requested samples Sponsors of study proteins and reagents:2014 study?• 25 laboratories requested samples Wh t i t t ti did ? • ABRF

yq p What instrumentation did you use? Both A Sample prep is cr cial • ABRF• 13 participants returned data 4 reported incomplete resultsWhat instrumentation did you use? Both A • Sample prep is crucial • Manual inspection of data is required Biomedical Research Core Facilities University of Michigan• 13 participants returned data, 4 reported incomplete results and B Sample prep is crucial • Manual inspection of data is required • Biomedical Research Core Facilities, University of Michiganp p , p p and B p p p

Mi i i l f t i lManual inspection of data is required Biomedical Research Core Facilities, University of Michigan

i d Gi i i i f i hiAll participants reported this as a challenging study 15% Minimize loss of materialp q

V if th t b i t l ti f • Anonymizer: Amanda McGinnis University of Michigan• All participants reported this as a challenging study 1S t 15% – Minimize loss of material – Verify that b-ions can support location of • Anonymizer: Amanda McGinnis, University of MichiganY

All participants reported this as a challenging study 1Synapt 15% – Verify that b-ions can support location of • Study participants!!!!!!Yes 1Synapt

Desalting/removal of excess reagenty pp

d i iti ti • Study participants!!!!!!Yes – Desalting/removal of excess reagent derivitization y p p18% W kfl

Desalting/removal of excess reagent derivitizationD h i ith N t l b li ? 18% 1Axima QIT TOF Workflow

R fDo you have experience with N-term labeling? 1Axima QIT-TOF Workflow Workflow Dimethylation of internal lysines can complicate ReferencesDo you have experience with N term labeling? AWorkflow • Tight control of reaction conditions is – Dimethylation of internal lysines can complicate ReferencesA

B • Tight control of reaction conditions is Dimethylation of internal lysines can complicate AB T i l T f 54%B Tight control of reaction conditions is

data interpretation Orthoproteogenomics: Multiple proteomes investigation through orthology and a7 2AB Triple Tof 54%B gdata interpretation • Orthoproteogenomics: Multiple proteomes investigation through orthology and a 7 2AB Triple Tof 31% necessary data interpretation p g p p g g gy

new MS based protocol S Gallien et al Genome 19 128 135 (2009)7 31% necessary new MS based protocol, S Gallien, et al., Genome, 19, 128-135 (2009)necessary

2AB Sciex 4800/5800 TOF pH is critical Dimeth l labeling is a sef l tool for • Stable-isotope dimethyl labeling for quantitative proteomics JL Hsu et alN 2AB Sciex 4800/5800 TOF – pH is critical • Dimethyl labeling is a useful tool for Stable isotope dimethyl labeling for quantitative proteomics. JL Hsu, et al., A l ti l Ch i t 75 24 6843 52 (2003)No / pH is critical Dimethyl labeling is a useful tool for Analytical Chemistry, 75, 24, 6843-52. (2003)No

Adj t t f H ft ti ff ty g

iAnalytical Chemistry, 75, 24, 6843 52. (2003)

82% – Adjustment of pH after reaction affects Proteomics Labs • In gel digestion for mass spectrometric characterization of proteins and3 82% 1Bruker Ultraflex Adjustment of pH after reaction affects Proteomics Labs • In-gel digestion for mass spectrometric characterization of proteins and 3 1Bruker Ultraflex j pffi i f t i di t

Proteomics Labsproteomes A Shevchenko et al Nature Protocols 1 6 p 2856 2860 (2006)2 efficiency of trypsin digest I d d t f id tit f Nt A i A id proteomes. A Shevchenko, et al., Nature Protocols, 1, 6, p 2856-2860 (2006) 1 2 efficiency of trypsin digest – Independent of identity of Nt-Amino Acid can

i l l i h d f l i iś i ki l1 2

7O bity yp g Independent of identity of Nt Amino Acid, can

• Universal sample preparation method for proteome analysis. JR Wiśniewski, et al.,7Orbitrapp y

b i ll li d Universal sample preparation method for proteome analysis. JR Wiśniewski, et al., N t M th d 6 359 362 (2009)

7Orbitrap be universally applied Nature Methods 6, 359-362 (2009)No Yes a little Yes a lot n/a be universally applied , ( )No Yes, a little Yes, a lot n/a y pp