Protein Identification Using a Quadrupole Ion Trap Mass Spectrometer and SEQUEST Database Matching

Roger E. Moore1, Mary K. Young1, Terry D. Lee1

1 Beckman Research Institute of the City of Hope, Duarte
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 16.10
DOI:  10.1002/0471140864.ps1610s22
Online Posting Date:  May, 2001
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Abstract

A procedure is described for rapid, sensitive protein identification utilizing liquid chromatography‐‐ tandem mass spectrometry. The analysis is performed on mixtures of peptides obtained by enzyme digestion. The SEQUEST computer program is used to match the sequence information in the spectra to a database of known protein sequences.

     
 
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Table of Contents

  • Basic Protocol 1: Collection of Peptide MS/MS Spectra and Analysis by SEQUEST
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Collection of Peptide MS/MS Spectra and Analysis by SEQUEST

  Materials
  • Protein sample digested into peptides with an appropriate enzyme (unit 11.1 )
  • Capillary HPLC system (unit 16.9)
  • Quadrupole Ion Trap Mass Spectrometer (QIT‐MS) with up‐to‐date software (Thermoquest LCQ with Excaliber version 1.1)
  • Fast computer for database searching (Microsoft Windows NT 4.0 with Service Pack 5 or higher)
  • SEQUEST and SEQUEST Browser software (Version 26 or higher; Eng et al., )
  • Protein or nucleotide sequence database in FASTA format (also see unit 2.1)
  • Additional reagents and equipment for desalting by gel filtration chromatography (unit 8.3)
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Figures

Videos

Literature Cited

Literature Cited
   Ducret, A., Van Oostveen, I., Eng, J.K., Yates, J.R. III. , and Aebersold, R. 1998. High throughput protein characterization by automated reversed‐phase chromatography/electrospray tandem mass spectrometry. Protein Sci. 7: 706‐719.
   Eng, J.K., Mccormack, A.L., and Yates, J.R. III. 1994. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5: 976‐989.
   Henzel, W.J., Billeci, T.M., Stults, J.T., Wong, S.C., Grimley, C., and Watanabe, C. 1993. Identifying proteins from two‐dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc. Natl. Acad. Sci. USA. 90: 5011‐5015.
   James, P., Quadroni, M., Carafoli, E., and Gonnet, G. 1993. Protein identification by mass profile fingerprinting. Biochem. Biophys. Res. Commun. 195: 58‐64.
   Link, A.J., Eng, J., Schieltz, D.M., Carmack, E., Mize, G.J., Morris, D.R., Garvik, B.M., and Yates, J.R. III. 1999. Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17: 676‐682.
   Mann, M. and Wilm, M. 1994. Error‐tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66: 4390‐4399.
   Moore, R.E., Young, M.K., and Lee, T.D. 2000. Method for screening peptide fragment ion mass spectra prior to database searching. J. Am. Soc. Mass Spectrom 11: 422‐426.
   Pappin, D.J.C., Hojrup, P., and Bleasby, A.J. 1993. Rapid identification of proteins by peptide‐mass fingerprinting. Curr. Biol. 3: 327‐332.
   Yates, J.R. III, Morgan, S.F., Gatlin, C.L., Griffin, P.R., and Eng, J.K. 1998. Method to compare collision‐induced dissociation spectra of peptides: Potential for library searching and subtractive analysis. Anal. Chem. 70: 3557‐3565.
Internet Resources
   ftp://ncbi.nlm.nih.gov/blast/db/
  Web site for downloading a number of databases available from the National Center for Biotechnology Information.
   http://www.cityofhope.org/microseq/download.html
  Web site for downloading the winnow.pl program to screen spectra prior to a SEQUEST search.
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