Protein Sequencing Research Group ABRF 2015 annual meeting

Transcription

1 Protein Sequencing Research Group ABRF 2015 annual meeting

2 » 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

3 » 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 dimethylation of protein to enhance identification of N-terminal peptide sequence 1 known purified protein (myoglobin) Successfully derivitize the proteins using a provided protocol Digest and identify the derivitized peptide fragments by MS Determine amount of protein needed for practical use

4 » 25 laboratories requested samples 10/25 (40%) international sites» 13 participants returned data, 4 reported incomplete results» All participants reported this as a challenging study Do you have experience with N-term labeling? Did you particpate in the 2014 study? 7 18% Yes % No No Yes, a little Yes, a lot n/a

5 » Identify the N-terminus of known proteins using chemical derivitization (dimethylation)» Determine lowest amount of protein identified Workflow (A) In-solution labeling Chemical labeling of proteins at N-terminus Cleanup with SDS-PAGE or membrane cutoff filter Digestion in-gel or on cut-off filter MS analysis including data analysis Identification of N-termini Workflow (B) In-gel labeling SDS-PAGE separation of proteins* In-gel chemical labeling In-gel tryptic digestion and cleanup MS analysis including data analysis Identification of N-termini *This study used only a single purified protein

6 Which Workflow did you perform? What instrumentation did you use? Synapt 1 Both A and B 15% Axima QIT-TOF 1 Workflow B 31% Workflow A 54% AB Triple Tof 2 AB Sciex 4800/5800 TOF 2 Bruker Ultraflex 1 Orbitrap

7 Reaction conditions» 5uL of 1pmol/uL protein (5pmol)» 100mM Na acetate, ph 5» 4% formaldehyde» Na cyanoborohydride» Incubate at RT Myoglobin: P68082 NH 2 -peptide Dimethyl labeling» Addition of dimethyl by reductive amidation» Reacts with N-term and Lysine residues H O H NaBH 3 CN CH 3 CH 3 N-peptide GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKT EAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYL EFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG

8 » Nt-dimethyl peptides are best sequenced from b ions b 1 b 2 b 3 H 3 C H O H H O H H O H H O N C C N C C N C C N C C OH H 3 C R 1 R 2 R 3 R 4 y 3 y 2 y 1 y ions do not contain N-terminal information

9 » Nt-dimethyl peptides are best sequenced from b ions» Internal dimethylations can confound results b 1 b 2 b 3 H 3 C H O H H O H H O H H O N C C N C C N C C N C C OH H 3 C R 1 R 2 KR 3 R 4 H 3 C CH 3

10 » Nt-dimethyl peptides are best sequenced from b ions» Internal dimethylations can confound results b 1 b 2 b 3 H 3 C H O H H O H H O H H O N C C N C C N C C N C C OH H 3 C R 1 R 2 KR 3 R 4 H 3 C CH 3 b 3 ion is not conclusive dimethyl can be at either N-terminus or R 3 Must verify that b ions indicate correct placement of dimethyl group

11 » SDS-PAGE purified» In-gel derivatized» In-gel digested SDS-PAGE Myoglobin ~17 kda 5 pmol 85 ng 3 pmol 51 ng 1 pmol 17 ng 0.3 pmol 5 ng Myoglobin Amount Loaded (pmol)

12 Base Peak Underivatized Peptide Dimethyl Nt Peptide Dimethyl Nt, 1xDeam Peptide Dimethyl Nt, 2xDeam Peptide

13 Extracted Ions for GLSDGEWQQVLNVWGK:

14 Extracted Ions for GLSDGEWQQVLNVWGK: Estimation of Dimethylated Myoglobin

15

16 GLSDGEWQQVLNVWGK m/z (2+) DiMethyl-GLSDGEWQQVLNVWGK m/z (2+)

17 » Most participants were successful with 5 pmol starting material» Several could detect 0.3 pmol of starting protein 3 Lowest Quantity of Protein Detected more 5 pmol 1 pmol 0.3 pmol less Workflow A (in-solution labeling) Workflow B (in-gel labeling)

18

19 In this sample, only the N-terminal peptide was labeled on N-terminus

20 Spectrum of Labeled N-terminal Peptide Most b ions are represented

21 » Detection of peptide at MS1 level No MSMS were acquired for the 1pmol standard Evidence for n-terminal labeled peptides [M+H] 2+» Threshold for Survey MSMS = 30,000 counts WF-A: In-Solution Labeling In-gel Digest Dimethyl-GLSDGEWQQVLNVWGK [M+2H] 2+ at m/z Relative Abundance z=? z=? z= z=? z= z= z= z=? z=? z=? z=? z=? z=? z=? m/z pmol 90 1 pmol Relative Abundance m/z

22 WF-B: In-gel Labeling In-gel Digest» Evidence of peptide [M+H] + and MS/MS spectra» Detection of peptide at 0.3 pmol using MALDI/TOF pmol pmol 0.3 pmol m/z m/z MS detection of intact peptides MS/MS spectra of m/z

23 WF-B: In-gel Labeling In-gel Digest» Evidence of peptide [M+H] + and MS/MS spectra» Detection of peptide at 0.3 pmol using MALDI/TOF b 6 b 7 b 8 b 10 b 11 b 13 5 pmol 1 pmol 0.3 pmol MS/MS spectra of m/z

24 In-Solution Labeling In-Solution Digest» Post-solution labeling, addition of 4% ammonium hydroxide is essential to quench solution digest Table 1. Dimethyl In-Solution Labeling Amount of Protein in Reaction Reagent 5 pmol 1 pmol 0.3 pmol 1 pmol/ul protein in 100 mm Na acetate, ph 5 5 ul 1 ul 0.3 ul 100 mm Na acetate, ph 5-4 ul 4.7 ul 4% Formaldehyde 1 ul 1 ul 1 ul 260 mm Na cyanoborohydride 1 ul 1 ul 1 ul Reaction Conditions vortex 5 min RT vortex 5 min RT vortex 5 min RT 4% ammonium hydroxide (quench reaction) *optional, but required for in-solution digest 1 ul, vortex 1 ul, vortex 1 ul, vortex Final Volume 8 ul 8 ul 8 ul

25 In-Solution Labeling In-Solution Digest» Post-solution labeling, addition of 4% ammonium hydroxide is essential to quench solution digest Table 1. Dimethyl In-Solution Labeling Amount of Protein in Reaction Reagent 5 pmol 1 pmol 0.3 pmol 1 pmol/ul protein in 100 mm Na acetate, ph 5 5 ul 1 ul 0.3 ul 100 mm» Na acetate, ph 5-4 ul 4.7 ul 4% Formaldehyde 1 ul 1 ul 1 ul 260 mm Na cyanoborohydride 1 ul 1 ul 1 ul Reaction Conditions vortex 5 min RT vortex 5 min RT vortex 5 min RT 4% ammonium hydroxide (quench reaction) *optional, but required for in-solution digest 1 ul, vortex 1 ul, vortex 1 ul, vortex Final Volume 8 ul 8 ul 8 ul» Failure to quench may result in uncontrolled dimethylation of peptides during in-solution digest

26 In-Solution Labeling In-gel Digest» Ensure neutral ph: 1M TrisHCl, ph 8.3, is added to each tube until bromophenol blue color is achieved. In-gel Labeling In-gel Digest» After in-gel labeling, rinse gel pieces with 4% NH 4 OH Aids in quenching of dimethylation reactants

27 Q: Cannot easily visualize bands for 1 and 0.3 pmol A: Recommend Silver Stain for 0.3 pmol protein» Some labs excised the invisible gel band comigrating with the visible myoglobin in appearing at 1 and 5 pmol MW Ladder Protein amount (pmol) 15%T SDS-PAGE followed by Ag stain Lab 21R

28 Q: Why did we lose myoglobin after in-solution labeling? A: Many participants reported major loss of protein» Some possibilities: Losses during de-salting of the intact protein Losses may occur if the reaction solution is not fully neutralized before SDS-PAGE

29 » 6 participants identified Nt-dimethylated myoglobin as the miscleaved peptide spanning K16» Many also identified the Ct fragment with internal lysine dimethylation peptide native [M+H]+ dimethyl [M+H]+ 2x dimethyl [M+H]+ GLSDGEWQQVLNVWGK GLSDGEWQQVLNVWGKVEADIAGHGQEVLIR NDIAAKYKELGFQG (c-term fragment) » All used Workflow A, in-solution derivatization

30 Q: In the tryptic peptide GLSDGEWQQVLNVWGK, why was the C-terminal Lysine not methylated? A: N-terminal of the intact protein is the primary site for Dimethylation reaction» Secondary reactions at the epsilon side chain of Lysine also occur (minor) Dimethylated Lysines are miscleaved during in-gel digestion Typically reported as internal, dimethylated Lysines rather than C terminal Lysines» e.g.: in ASEDLKKHGTVVLTALGGILK, internal Lysines are often dimethylated.

31 » Workflow B approach performed on several projects from Memorial Sloan Kettering Cancer Center laboratories» PIs volunteered gel-bound and PVDF-bound proteins LC-MS with dimethylation Comparative Edman degradation experiments» In all instances, the majority of Lysine dimethylated peptides are found to reside in a miscleaved peptide.» Chang et al., ABRF 2015 Poster # 145: N-terminal amino acid sequence determination of proteins by N-terminal dimethyl labeling; pitfalls and advantages when compared with Edman degradation sequence analysis

32 » LC-MS with vented column/trap loading helps remove excess reagent» For in-solution labeling, manual desalting or FASP protocol improves sensitivity» For database searching, include a net mass addition of (C 2 H 4 ) to the N-terminus and lysine amino acids as a variable modification

33 » Search engines may force the data to fit N- terminal Dimethyl to peptides with internal lysines Matched y and b ions cannot distinguish between Nt dimethyl and internal Lysine dimethylation No Lysine dimethylation is specified: search engines fit MS/MS to non-specific Nt-dimethylation: NDIAAQYKELGFQG ASEDLKKHGTVVLTALGGILKK ALELFRNDIAAQYKELGFQG YKELGFQG Lysine dimethyl used as a variable mod: we will obtain the following hits for internal Lysine dimethylation: NDIAAQYKELGFQG ASEDLKKHGTVVLTALGGILKK ALELFRNDIAAQYKELGFQG YKELGFQG

34 » Search engines may force the data to fit N- terminal Dimethyl to peptides with internal lysines Matched y and b ions cannot distinguish between Nt dimethyl and internal Lysine dimethylation No Lysine dimethylation is specified: search engines fit MS/MS to non-specific Nt-dimethylation: Lysine dimethyl used as a variable mod: we will obtain the following hits for internal Lysine dimethylation: NDIAAQYKELGFQG ASEDLKKHGTVVLTALGGILKK ALELFRNDIAAQYKELGFQG YKELGFQG NDIAAQYKELGFQG ASEDLKKHGTVVLTALGGILKK ALELFRNDIAAQYKELGFQG YKELGFQG Dimethylated lysines receive higher scores Must use Nt-dimethyl as well as Lysine dimethyl among the variable search parameters

35 Observation of dimethylation at the N-terminus of I 99 Spectrum of Myoglobin dimethylated peptide: IPIKYLEFISDAIIHVAHSK Where is the dimethyl group located?

36 Observation of dimethylation at the N-terminus of I 99 Spectrum of Myoglobin dimethylated peptide: IPIKYLEFISDAIIHVAHSK Where is the dimethyl group located? b 1, b 2, b 3 not detected (below cutoff) y 17, y 18, y 19 not detected (above cutoff) b 4 contains +28 Da confirms dimethyl at Nt I 1 OR at internal K 4

37 Observation of dimethylation at the N-terminus of I 99 Spectrum of Myoglobin dimethylated peptide: IPIKYLEFISDAIIHVAHSK Where is the dimethyl group located? b 1, b 2, b 3 not detected (below cutoff) y 17, y 18, y 19 not detected (above cutoff) b 4 contains +28 Da confirms dimethyl at Nt I 1 OR at internal K 4 Conclusion: MS/MS spectrum does not support the exact positioning of Dimethyl group

38 » After doing both 2014 and 2015 studies, I would choose TMPP labeling method over dimethylation because the TMPP reagent is less likely to hit epsilon lysine.» Worst offender was probably the size of the N-terminal peptide at 3460 Da. The C-terminal peptide was half that size and was easily seen and fragmented. Perhaps I should have investigated using a different cleavage enzyme.» An in-gel digest was performed and the 3460 Da mass was difficult to extract from the gel.» Digestion was really poor even with good ph control. The sodium acetate was a real pain.» With all attempts made the recoveries were very poor and hardly any peptide could be identified.» Protocol and explanations should be more detailed.» You should deliver the FASTA file to process more objectively.» This was fun!

39 » Sample prep is crucial Minimize loss of material Desalting/removal of excess reagent» Tight control of reaction conditions is required ph is critical Adjustment of ph after reaction may affect efficiency of trypsin digest» Manual inspection of data Verify that b-ions can support location of derivitization Dimethylation of internal lysines can complicate data interpretation» Dimethyl labeling may be a useful tool for Proteomics Labs Independent of identity of Nt-Amino Acid, can be universally applied

40 » Updated protocol to be added to the PSRG/ABRF webpage» See our other related posters: Chang et al., ABRF 2015 Poster # 145: N-terminal amino acid sequence determination of proteins by N-terminal dimethyl labeling; pitfalls and advantages when compared with Edman degradation sequence analysis ABRF PSRG Poster # 260: N-terminal identification of a standard protein at low picomole levels by dimethyl labeling and bottom-up mass spectrometry

41 » 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 Sara McGrath FDA/CFSAN

42 » Sponsors of study proteins and reagents: ABRF Biomedical Research Core Facilities University of Michigan» Anonymizer: Amanda McGinnis, University of Michigan» and study participants!!!!!!

43 » Sara McGrath (co-chair) FDA/CFSAN» Robert English (co-chair) Shimadzu» Greg Cavey Southwest Michigan Innovation Center» Hediye Erdjument-Bromage MSKCC/Rockefeller Research Laboratories» Mark Garfield NIH/NIAID» Xuemei Luo Univ. of Texas Medical Branch» Henriette Remmer Univ. of Michigan» Brett Phinney (EB liaison) UC Davis

44 » Orthoproteogenomics: Multiple proteomes investigation through orthology and a new MS based protocol, S Gallien, E Perrodou, C Carapito, C Deshayes, JM Reyrat, A Van Dorsselaer, O Poch, C Schaeffer, O Lecompte, Genome, 19, (2009)» Stable-isotope dimethyl labeling for quantitative proteomics. JL Hsu, SY Huang, NH Chos, SH Chen Analytical Chemistry, 75, 24, (2003)» In-gel digestion for mass spectrometric characterization of proteins and proteomes. Andrej Shevchenko, Henrik Tomas, Jan Havlis, Jesper V Olsen & Matthias Mann, Nature Protocols, 1, 6, p (2006)» Universal sample preparation method for proteome analysis. Jacek R Wiśniewski, Alexandre Zougman, Nagarjuna Nagaraj & Matthias Mann, Nature Methods 6, (2009)