Protein Sequencing (PSRG)

Mission

The mission of the Protein Sequencing Research Group (PSRG) is to design studies to assess techniques for determining the primary structure of protein termini that will allow participating laboratories to gauge their expectations and competence in performing these analyses. 

Questions or interest in joining an ABRF research group? Contact us

PSRG Guidelines:
The PSRG has the responsibility to regularly design and distribute samples to the ABRF membership following a reasonable time line decided mutually by the PSRG and the Executive Board of ABRF (EB). This includes sample design (EB approval required), sample preparation, test evaluation by PSRG members, sample distribution, data receipt and evaluation, oral and/or poster presentation at the ABRF or other international meeting (e.g., Protein Society or ASBMB), and finally, publication in a peer reviewed journal. The study should be designed for N – and/or C-terminal sequence analysis of proteins containing the 20 common amino acids plus (optionally) other post-translationally modified amino acids that are commonly found in proteins. Any technology that yields information about the termini of proteins may be used. The PSRG should be comprised of 6-8 members plus one EB Liaison whose purpose is to assist the research group and to facilitate communication with the EB. The PSRG should meet about once a month via a conference call to discuss the current sample and ensure continued progress. Once a year, one of the members will be chosen as chairperson in a fashion determined by the PSRG. The chair has the ultimate responsibility for ensuring the success of the RG and its studies, from initial design to the final publication. This includes communication with the EB, management office, JBT or other journal, chairs of the other protein research groups, ABRF membership, and the yearly ABRF meeting organizers. Every year, 2-3 members rotate off the RG and are replaced by new members who are nominated by the PSRG for approval by the EB. The PSRG should use this as a guideline but is not limited to it, either in order presented or type of sample. To help ensure that the PSRG meets the continuing needs of ABRF members, each sample submission form would contain a few survey questions to better gauge the kinds of samples that would be most useful to our members and to solicit suggestions for future studies.

Current Membership

David Wood, Saint Louis University, Chair
Hediye Bromage, New York University School of Medicine 
Allis Chien, Stanford University
Greg Cavey, Innovative Mass Spectrometry, LLC
Robert English, Shimadzu Scientific Instruments
Brian Feild, Shimadzu Scientific Instruments
Xuemei Luo, UTMB - Galveston
Sara McGrath, U.S. Food and Drug Administration
Magnus Palmblad, Leiden University Medical Center, (EB liaison)

Studies
1)

PSRG 2015-2016 Study:
    - View Study Announcement (34K)
    - View Sample Insert (504K)
    - View Oral Presentation (2.1M)

2) PSRG 2014-2015 Study: N-terminal sequencing of standard proteins by dimethyl labeling and bottom-up mass spectrometry
    - View Study Announcement (34K)
    - View Sample Insert (359K)
3) PSRG 2013-2014 Study: N-terminal sequencing of standard proteins by N-terminal labeling and mass spectrometry
    - View Study Announcement (30K)
    - View Sample Insert (237K)
4) PSRG 2012-2013 Study : Sample preparation and terminal sequencing of a protein mixture
    - View Document 2012-2013 Study Announcement 
    - View Document 2012 Participant Results Survey 
5) PSRG 2011 Study: Comparing sensitivity limits for N-terminal Sequencing Technologies
    - View PSRG 2011 Study Announcement 
    - View PSRG 2011 Study Sample Insert (36K)
6) PSRG 2010 Study: Comparing Edman and MS Sequencing of a Monoclonal Antibody (study closed)
    - View Study Announcement (94K)
    - View Cover Letter/Instructions (20K)
    - View Google Doc's data reporting form and survey 
7) ESRG 2009 Study: Comparing Edman and Mass Spectrometry Techniques for N-terminal Sequencing
    - ESRG 2009 Study Announcement 
    - Cover Letter/Sample Instructions (15K)
    - Vendor ABRF Study Participation Guidelines (18K)
    - Data Sheet - Excel file for both Edman and MS Data 
8) ESRG 2008 Study "Investigation into homopolymeric amino acid N-terminal sequence tags and their effects on automated Edman degradation"
    - ESRG 2008 Study Announcement 
    - Cover Letter/Sample Instructions 
    - Data Sheet (41K)
    - Vendor ABRF Study Participation Guidelines (18K)
9) ESRG 2007 Study "Procedures used to Determine the N-Terminal Sequence of a Blocked Protein"
    - ESRG 2007 Study Announcement 
    - Cover Letter/Sample Instructions 
    - Deblocking Cycle 
    - Data Sheet (25K)
10) ESRG 2006 Study "Edman Sequencing as a Method for Polypeptide Quantitation"
    - ESRG 2006 Study Announcement 
11) ESRG 2005 Study "Identification of Modified Amino Acids by Edman Sequencing"
    - Instructions for Filling Out the Datasheet 
    - Sample Description Including Modified Amino Acids 
    - Data Sheet (19K)
12) ESRG 2004 Study "Modified Amino Acids in Edman Sequencing"
    - Sample Description Including Modified Amino Acids 
13)

ESRG 2004 Study Aid: Pages 10-12 from "Identification of Modified PTH-Amino Acids in Protein Sequence Analysis" 1st ed. compiled by Mark Crankshaw and Greg Grant
    - Chromatogram: page 10 
    - Legend to chromatogram: page 11 
    - Legend continued: page 12 

 

Activities

1) ESRG Newsletter Vol.1 No.1, - Report on the ESRG at ABRF 2008, Salt Lake City, Utah and a Tribute to Pehr Edman

Electronic Posters

  PSRG 2015 - ABRF Poster and Oral Presentations: N-terminal Identification of a Standard Protein at Low Picomole Levels by Dimethyl Labeling and Bottom-up Mass Spectrometry
  PSRG 2014 - ABRF Poster and Oral Presentations: N-terminal Sequencing of Standard Proteins by N-terminal Labeling and Mass Spectrometry
  PSRG 2013 - ABRF Poster and Oral Presentations: Results of the PSRG 2013 Study Year 2: Terminal Sequencing of Standard Proteins in a Mixture
  PSRG 2012 - ABRF Poster and Oral Presentations: Results of the PSRG 2012 Study: Terminal Sequencing of Standard Proteins in a Mixture, Year 1 of the 2 Year Study
  PSRG 2011 - ABRF Presntations: Results of the PSRG 2011 Study: SensitivityAssessment of Edman and Mass Spectrometric Terminal Sequencing of an Undisclosed Protein
    - PSRG 2011 Poster (4,387K)
    - PSRG 2011 Oral Presentation (9,213K)
  PSRG 2010 Poster: Edman and Mass Spectrometric Terminal Sequencing of a Monoclonal Antibody
  PSRG 2010 Oral Presentation: N-Terminal Sequencing of an Antibody
  ESRG 2009 Poster: Comparison of Edman and Mass Spectrometry Techniques for N-terminal Sequencing
  ESRG 2009 Oral Presentation. "N-Terminal Sequencing Techniques"
  ESRG 2008 Oral Presentation. "Effects of a Homopolymeric Amino Acid Tag on Edman Degradation"
  ESRG 2008 Poster. "Investigation into Homopolymeric Amino Acid N-terminal Sequence Tags and their Effects on Automated Edman Degradation"
  ESRG 2007 Poster: "Procedures used to Determine the N-Terminal Sequence of a Blocked Protein"
  ESRG 2007 Oral Presentation: "Procedures used to Determine the N-Terminal Sequence of a Blocked Protein"
  ESRG 2006 poster: "Edman Sequencing as a Method for Polypeptide Quantitation"
  ESRG 2006: Oral presentation: Edman Sequencing as a Method for Polypeptide Quantitation
  ESRG 2005 "State of Edman Sequencing" Survey, used for both the poster and oral presentation at the ABRF meeting, Savannah, GA, February 5-8, 2005
  ESRG 2005 poster "Identification of Modified Amino Acids by Edman Sequencing"
  ESRG 2005: Oral presentation: Modified amino acids by Edman sequencing
  "ABRF ESRG 2004 Study: Modified Amino Acids in Edman Sequencing"; ESRG study poster presented at the 2004 ABRF meeting February 28-March 2 in Portland, Oregon.
  "Modified Amino Acids in Edman Sequencing"; ESRG study results presented at the 2004 ABRF meeting February 28-March 2 in Portland, Oregon.
  "ABRF-ESRG 2003: Analysis of a PVDF-Bound Known Protein with a Homogeneous Amino-Terminus" ; ESRG study poster presented at the 2003 ABRF meeting February 10-13 in Denver, Colorado.
  Slides from tutorial, "Making the Most of Your Edman Sequence Data; A Primer on Data Calling, Analysis, Interpretation and Reporting" in pdf format, presented at the 2003 ABRF meeting February 10-13 in Denver, Colorado.
  Slides from the 2003 ESRG Study oral presentation given at the 2003 ABRF meeting, Feb 10-13 in Denver, Colorado.
  "ABRF-2002 ESRG, a Difficult Sequence: Analysis of a PVDF-Bound Known Protein With a Heterogeneous Amino-Terminus"; poster presented at the 2002 ABRF meeting March 10-12 in Austin, Texas.
  Slides from ESRG 2002 Study oral presentation given at 2002 ABRF meeting March 10-12 in Austin, Texas
  The first half of the tutorial session "N-terminal Edman sequencing sample preparation" slides in pdf format.
  Second half of the tutorial session "N-terminal Edman sequencing sample preparation" slides in pdf format.
  Here is the method briefs handout from the tutorial session. Please use these methods and references only as guides for you to develop similar methods more fully in your own labs, no liability to ABRF for their use.

Membership History

Member NameOrganizationDetails
Daniel Brune Arizona State Univ  Chair: 02/05 - 02/06
Member: 02/06 - 04/07
Scott Buckel Amgen, Inc.  Member: 01/01 - 03/03
Dr. Allis Chien Stanford University EB Liaison: 03/17 - 09/18
Richard G. Cook Baylor Col of Med  Member: 01/01 - 03/04
J. M. Crawford Yale Univ. Keck Biotech Res. Lab.  Member: 01/01 - 03/04
Dr Nancy D Denslow Univ of Florida-Gainesville  Chair: 03/04 - 02/05
Member: 03/02 - 03/04
EB liaison: 02/05 - 02/09
David R. Dupont Applied Biosystems  Member: 01/01 - 03/02
Dr Robert English Univ. of Texas Medical Branch  Chair_(co-chair): 04/12 - 04/13
Member: 05/11 - 04/12
Dr Hediye Erdjument-Bromage Sloan Kettering Inst  Member: 04/14 - 04/15
Joseph Fernandez Rockefeller Univ  Chair: 01/01 - 03/02
Member: 03/02 - 03/03
Mr. Mark K. Garfield NIAID/NIH  Member: 05/12 - 04/15
Brian Hampton Universtiy of Maryland School of Medicine  Member: 02/05 - 04/07
Member: 02/08 - 02/09
Ad hoc: 02/09 - 04/10
Co-Chair: 04/07 - 02/08
Dr William G Hendrickson Univ. of Illinois, Chicago  EB Liason: 03/13 - 04/14
Peter Hunziker University of Zurich  Chair: 02/08 - 02/09
MemberBecome Ad Hoc: 02/09 - 04/10
Member: 03/06 - 02/08
Ad hoc: 04/10 - 04/11
Pegah R Jalili Sigma-Aldrich  Member: 06/11 - 04/14
Viswanatham Katta Genentech  Member: 06/10 - 03/13
Ryuji Kobayashi UT MD Anderson Cancer Center  Member: 03/03 - 03/06
William S. Lane Harvard University  EB Liason: 10/03 - 02/05
Joseph W. Leone Pfizer  Chair: 02/06 - 04/07
Member: 03/04 - 02/06
Member chair-emeritus: 04/07 - 02/08
Joseph F. Leykam Michigan State University  Member: 04/07 - 08/07
Dr. Klaus D. Linse Biosynthesis Inc.  Member: 02/05 - 02/08
Benjamin J Madden Mayo Clinic  Chair: 02/03 - 03/04
Member: 03/04 - 02/05
Member: 01/01 - 02/03
Kwasi G Mawuenyega Washington University School of Medicine  Member: 05/09 - 04/12
Dr. Sara C. McGrath US FDA  Chair_(co-chair): 04/13 - 04/15
Member: 05/12 - 03/13
Dr. Ejvind Mortz Alphalyse  : 03/13 - 04/14
John M Neveu Harvard University  Chair: 04/02 - 01/03
Member: 01/01 - 03/02
Member: 02/03 - 02/05
Dr. Ronald L. Niece Research Resources & Technologies  Member: 03/88 - 03/91
Dr. Brett S Phinney Proteomics Core UC Davis Genome Center  EB Liason: 04/14 - 04/15
Dr Jan Pohl Emory Univ Sch of Med  Member: 03/04 - 04/07
Dr. Henriette A. Remmer University of Michigan  Member: 06/10 - 04/11
Ad hoc: 03/13 - 04/15
co-chair: 04/11 - 04/12
Co-chair: 04/12 - 03/13
Wendy Sandoval Genentech, Inc.  Member: 04/10 - 04/11
Member changing to cochair: 04/07 - 02/09
Co-chair: 02/09 - 04/10
Jack Simpson US Pharmacopeia  EB liaison: 02/09 - 03/13
John S. Smith Univ of Texas Med Branch  Member: 04/10 - 04/11
Member changing to cochair: 09/07 - 02/09
Co-chair: 02/09 - 04/10
Laurey Steinke University of Nebraska Medical Center  EB liason: 01/01 - 09/03
Dr. Detlev Suckau Bruker Daltonik GmbH  MemberBruker Daltonics: 05/10 - 03/13
Richard S Thoma Monsanto  Member: 02/08 - 02/09
Member: 02/05 - 04/07
Ad hoc: 02/09 - 04/10
Co-Chair: 04/07 - 02/08
James Walters Sigma-Aldrich  Chair: 04/10 - 04/11
Member: 04/09 - 04/10
Ad hoc: 04/12 - 03/13
co-chair: 04/11 - 04/12
Dr. Frances Weis-Garcia Memorial Sloan Kettering Cancer Center EB Liaison: 02/16 - 03/17
Ken R. Williams Yale Univ, Keck Lab  Member: 03/88 - 03/90
Dr. Bosong Xiang Monsanto Company  Member: 04/09 - 04/12

Publications

  1. A Synthetic Peptide for Evaluating Protein Sequencing Capabilities: Design of ABRF-93SEQ and Results. In (J.W. Crabb, ed.) Techniques in Protein Chemistry V, Academic Press, San Diego, pp133-141 (1994)
    Rush, J., Andrews, P. C., Crimmins, D. L., Gambee, J. E., Grant, G. A., Mische, S. M. and Speicher, D. W.
  2. ABRF ESRG 2005 Study: identification of seven modified amino acids by Edman sequencing. J Biomol Tech, 2006. 17(5): p. 308-326.
    Brune, D., N.D. Denslow, R. Kobayashi, W.S. Lane, J.W. Leone, B.J. Madden, J.M. Neveu, and J. Pohl
  3. ABRF-2002 ESRG, A Difficult Sequence: Analysis of a PVDF-Bound KNown Protein With a Heterogeneous Amino-Terminus Journal of Biomolecular Techniques 13:246-257 (2002).
    Buckel, S.D., Cook,R.G., Crawford, J.M., Dupont, D.R., Madden,B.J., Neveu,J.M., Steinke, L., Fernandez, J
  4. ABRF-2003 ESRG: Analysis of a PVDF-Bound KNown Protein With a Heterogeneous Amino-Terminus Journal of Biomolecular Techniques 14:278-288 (2003).
    Buckel, S.D., Cook,R.G., Crawford,M.J., Denslow, N., Madden,B.J., Steinke, L., Fernandez, J., Neveu,J.M
  5. ABRF-97SEQ: Sequencing Results of a Low-Level Sample. J Biomol Tech, 10:26-32 (1999)
    Stone, K., Fernandez, J., Admon, A., Henzel, W., Lane, W., Rohde, M., and Steinke, L.
  6. ABRF-98SEQ: Evaluation of peptide sequencing at high sensitivity J. Biomol. Tech., Jun 2000; 11: 92 - 99.
    WJ Henzel, A Admon, SA Carr, G Davis, K De Jongh, W Lane, M Rohde, and L Steinke
  7. Amino Acid Analysis and Sequencing - What Is State-of-the-Art?
    Niece, R.L., L.H. Ericsson, A.V. Fowler, A.J. Smith, D.W. Speicher, J.W. Crabb, and K.R. Williams.

    "Methods in Protein Sequence Analysis" (H. Jörnvall, J.-O. Höög, and A.-M. Gustavsson, Eds.), Birkhäuser Verlag, Basel, 133-141, 1991

  8. Assignment of Cysteine and Tryptophan Residues during Protein Sequencing: Results of ABRF-94SEQ. In (J. W. Crabb, ed.) Techniques in Protein Chemistry VI, Academic Press, San Diego, pp 209-217 (1995)
    Gambee, J. E., Andrews, P. C., Grant, G. A., Merrill, B., Mische, S. M. and Rush, J.
  9. Design and Analysis of ABRF-95SEQ, a Recombinant Protein with Sequence Heterogeneity. In (D. Marshak, ed.) Techniques in Protein Chemistry VII, Academic Press, San Diego, pp 347-358 (1996)
    DeJongh, K. S., Fernandez, J., Gambee, J. E., Grant, G. A., Merrill, B., Stone, K. L. and Rush, J.
  10. Design, Characterization and Results of ABRF-89SEQ: A Test Sample for Evaluating Protein Sequencer Performance in Protein Microchemistry Core Facilities. In (T. E. Hugli, ed.) Current Research in Protein Chemistry, Academic Press, pp 159-166 (1989)
    Speicher, D. W., Grant, G. A., Niece, R. L., Blacher, R. W., Fowler A. V. and Williams, K. R.
  11. Edman Sequencing Research Group 2004 Study: Modified Amino Acids in Edman Sequencing, J. Biomol. Tech., Sep 2005; 16: 272 - 284.
    D. Brune, J.M. Crawford, R.G. Cook, N.D. Denslow, R. Kobayashi, B.J. Madden, J. M. Neveu, and L. Steinke
  12. Evaluation of ABRF-96SEQ: A Sequence Assignment Exercise. In (D. R. Marshak, ed.) Techniques in Protein Chemistry VIII, Academic Press, San Diego, pp 69-78 (1997)
    Fernandez, J., Admon, A., DeJongh, K., Grant, G., Henzel, W., Lane, W. S., Stone, K. L. and Merrill, B.
  13. Evaluation of Protein Sequencing Core Facilities: Design, Characterization, and Results form a Test Sample (ABRF-91SEQ). In (R. H. Angeletti, ed.) Techniques in Protein Chemistry III, Academic Press, San Diego, pp 35-35 (1992)
    Crimmins, D. L., Grant, G. A., Mende-Muller, L. M., Niece, R. L., Slaughter, C., Speicher, D. W. and Yuksel, K. U.
  14. Instrumentation Used in Protein and Nucleic Acid Resource Facilities: A Survey of Users
    Beach, C.M. G.M. Hathaway, T.K. Hayes, A.J. Smith, and R.L. Niece.

    ABRF News, Vol. 2.1 Supplement: 1-7, 1991

  15. Protein Sequencing from Polyvinylidene Difluoride Membranes: Design and Characterization of a Test Sample (ABRF-90SEQ) and Evaluation of Results. In (J. J. Villafranca, ed.) Techniques in Protein Chemistry II, Academic Press, San Diego, pp 151-162 (1991)
    Yuksel, K. U., Grant, G. A., Mende-Muller, L. M., Niece, R. L., Williams, K. R. and Speicher, D. W.
  16. Protein Sequencing of Post-translationally Modified Peptides and Proteins: Design, Characterization and Results of ABRF-92SEQ. In (R. H. Angeletti, ed.) Techniques in Protein Chemistry IV, Academic Press, San Diego, pp 453-461 (1993)
    Mische, S. M., Yuksel, K. U., Mende-Muller, L. M., Matsudaira, P., Crimmins, D. L. and Andrews, P. C.
  17. Synthetic Peptide for Evaluating Protein Sequencer and Amino Acid Analyzer Performance in Core Facilities: Design and Results
    Niece, R.L., K.R. Williams, C.L. Wadsworth, J. Elliott, K.L. Stone, W.J. McMurray, A. Fowler, D. Atherton, R. Kutny, and A.J. Smith. A.

    in "Techniques in Protein Chemistry" (Hugli, T., Ed.), Academic Press, San Diego, 89-110, 1989

     


     

    2000 ABRF Peptide Sequencing by Mass Spectrometry Quiz

     Introduction

    Prior to its demise, the ABRF Mass Spectrometry Committee prepared four "tryptic" peptides whose sequences did not match anything in the public nucleotide or protein sequence databases. For two of these peptides, data was acquired using an ion trap mass spectrometer, and for the other two a quadrupole / time-of-flight hybrid mass spectrometer (Qtof) was used. Using these data, a MS/MS peptide sequencing quiz was prepared for the ABRF membership, where the aim was four-fold. First, this was a sort of late final exam for those who took the 1999 ABRF course taught by Prof. Don Hunt. Second, the results might indicate the need for similar additional ABRF courses. Third, the anonymous results allowed individuals to assess their own level of proficiency at this task. Fourth, it allowed a comparison of the relative merits of two types of mass spectrometers most commonly used for peptide sequencing.

    This study is closed, and the results and answers can be found here. I suppose one could still take the quiz, but you'll have to grade yourself.

    The Quiz

    Check out the sequencing tutorial, if you need instructions on sequencing tryptic peptides using low energy CID data.

    Ion trap data

    Data for two peptides was acquired, where the first one is the easiest. To aid in the verification of your proposed sequences, MSspectra were acquired for both peptides. The following are all standard gif files:

    Qtof data

    The next two problems were obtained from a quadrupole / time-of-flight hybrid mass spectrometer (Qtof), where the third peptide is easier than the fourth. The fourth peptide was also subjected to trypsin in the presence of a 1:1 mixture of 16O and 18O water, which labeled the C-terminus with a mixture of the two stable isotopes. The precursor resolution was set wide enough to pass both the 16O and 18O labeled peptide, such that C-terminal fragment ions in the MS/MS spectrum exhibited two mass unit doublets. There are two spectra shown, where the raw data is on top, and the spectrum beneath it is the same data subjected to a y-ion filter.

     

    Questions or interest in joining an ABRF research group? Contact us