Using ProSight PTM and Related Tools for Targeted Protein Identification and Characterization with High Mass Accuracy Tandem MS Data

Richard D. LeDuc1, Neil L. Kelleher1

1 University of Illinois at Urbana‐Champaign, Urbana, Illinois
Publication Name:  Current Protocols in Bioinformatics
Unit Number:  Unit 13.6
DOI:  10.1002/0471250953.bi1306s19
Online Posting Date:  September, 2007
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ProSight PTM v2.0, neuroProSight, and the Sequence Gazer allow the identification and characterization of proteins from high mass accuracy tandem mass spectrometric data of intact proteins and large peptides. Input data consists of one or more neutral precursor ion masses and a set of neutral b/y or c/z. fragment ions masses. This data is compared against “shotgun annotated” proteome databases or known protein sequences. With these tools it is possible to not only identify unknown proteins, but to determine the location of post‐translational modifications (PTM) with 100% sequence coverage. Collectively, the tools create a search environment that allows five different search modes, including absolute mass and sequence tag searching, which are conveniently employed via a graphical user interface. Data management and chemical noise reduction tools are also available. These tools provide a complete environment for the identification and characterization of proteins from high resolution tandem mass spectrometry of intact proteins and large peptides. Curr. Protoc. Bioinform. 19:13.6.1‐13.6.28. © 2007 by John Wiley & Sons, Inc.

Keywords: top‐down; intact protein; post‐translational modification (PTM); sequence tag; protein identification; protein characterization; Web

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Performing an Absolute Mass Search
  • Basic Protocol 2: Performing a Sequence Tag Search
  • Basic Protocol 3: Performing a Neuropeptide Search
  • Basic Protocol 4: Using the Sequence Gazer
  • Alternate Protocols
  • Alternate Protocol 1: Performing a GRAM Search
  • Alternate Protocol 2: Performing a Single Protein Mode Search
  • Support Protocols
  • Support Protocol 1: Obtaining and Configuring User Accounts
  • Support Protocol 2: Uploading Data and Maintaining an Account
  • Support Protocol 3: Using Noise Reducer
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

   Horn, D.M., Zubarev, R.A., and McLafferty, F.W. 2000. Automated reduction and interpretation of high resolution electrospray mass spectra of large molecules. J. Am. Soc. Mass Spectrom. 11:320‐332.
   LeDuc, R.D., Taylor, G.K., Kim, Y.B., Januszyk, T.E., Bynum, L.H., Sola, J.V., Garavelli, J.S., and Kelleher, N.L. 2004. Prosight PTM: An integrated environment for protein identification and characterization by top‐down mass spectrometry. Nuc. Acids Res. 32:W340‐W345.
   Mann, M., Meng, C.K., and Fenn, J.B. 1989. Interpreting mass spectra of multiply charged ions. Anal. Chem. 61:1702‐1708.
   Meng, F.Y., Cargile, B.J., Miller, L.M., Forbes, A.J., Johnson, J.R., and Kelleher, N.L. 2001. Informatics and multiplexing of intact protein identification in bacteria and the archaea. Nat. Biotechnol. 19:952‐957.
   Pesavento, J.J., Kim, Y.B., Taylor, G.K., and Kelleher, N.L. 2004. Shotgun annotation of histone modifications: A new approach for streamlined characterization of proteins by top down mass spectrometry. J. Am. Chem. Soc. 126:3386‐3387.
   Reid, G.E., Shang, H., Hogan, J.M., Lee, G.U., and McLuckey, S.A. 2002. Gas‐phase concentration, purification, and identification of whole proteins from complex mixtures. J. Am. Chem. Soc. 124:7353‐7362.
   Southey, B.R., Rodriguez‐Zas, S.L., and Sweedler, J.V. 2006. Prediction of neuropeptide prohormone cleavages with application to rfamides. Peptides 27:1087‐1098.
   Zabrouskov, V., Senko, M.W., Du, Y., Leduc, R.D., and Kelleher, N.L. 2005. New and automated msn approaches for top‐down identification of modified proteins. J. Am. Soc. Mass Spectrom. 16:2027‐2038.
Key References
   Kelleher, N.L. 2004. Top‐down proteomics. Anal. Chem. 76:196A‐203A.
  This is the most current review of the top‐down technique.
   Reid, G.E. and McLuckey, S.A. 2002. ‘Top down’ protein characterization via tandem mass spectrometry. J. Mass Spectrom. 37:663‐675.
  An earlier overview of top‐down proteomics.
Internet Resources
  Home screen for ProSight PTM version 2.0.
  Home screen for neuroProSight.
  Entry point for the standalone Sequence Gazer.
  Entry point into the RESID database. All protein modifications supported by ProSight PTM v2.0 and neuroProSight come from this database.
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