De Novo Peptide Sequencing via Manual Interpretation of MS/MS Spectra

Terri Addona1, Karl Clauser1

1 Millennium Pharmaceuticals, Cambridge
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 16.11
DOI:  10.1002/0471140864.ps1611s27
Online Posting Date:  May, 2002
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Abstract

Databases of complete genome sequences are rapidly appearing, and a recent trend in interpretation of peptide MS/MS spectra is database matching. A caveat of database matching is that it can only be successful if the database being searched contains the identical amino acid sequence, or one with high homology, to the peptide being investigated. In instances where high‐quality MS/MS spectra are obtained for which there is no corresponding database match (e.g., when the peptide of interest is derived from a novel protein from an organism whose genome is not known, has post‐translationally modified amino acids, or has multiple amino acid substitutions as compared to the homologous sequence from a different species), de novo (or manual) interpretation of the MS/MS spectrum, as described here, is required. Methods are also provided for verification of the results of the de novo analysis through chemical modification and limited manual Edman degradation of the peptide.

     
 
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Table of Contents

  • Basic Protocol 1: Manual Interpretation of MS/MS Spectra
  • Support Protocol 1: Confirmation of the De Novo Sequence Through Methyl Esterification
  • Support Protocol 2: Confirmation of the De Novo Sequence Through Acetylation
  • Support Protocol 3: Manual Edman Degradation
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Manual Interpretation of MS/MS Spectra

  Materials
  • Peptides generated from a tryptic digest of a reduced and alkylated single protein or protein mixture
  • Liquid chromatography (LC) apparatus coupled in‐line to a microelectrospray ionization source on a tandem mass spectrometer (quadrupole ion trap, triple quadrupole, or hybrid quadrupole/time‐of‐flight) with <200 nl/ml flow rates and 75‐µm‐i.d. capillary reversed‐phase columns
  • Additional equipment and reagents for confirming sequence information via chemical modification (see protocol 2 and protocol 32) and manual Edman degradation (see protocol 4)

Support Protocol 1: Confirmation of the De Novo Sequence Through Methyl Esterification

  Materials
  • Sample
  • Acetyl chloride
  • Anhydrous methanol, ice cold
  • Additional reagents and equipment for LC/MS/MS (see protocol 1)

Support Protocol 2: Confirmation of the De Novo Sequence Through Acetylation

  • Sample
  • Acetic anhydride
  • 200 mM ammonium bicarbonate, pH 8.5
  • Additional reagents and equipment for LC/MS/MS (see protocol 1)

Support Protocol 3: Manual Edman Degradation

  Materials
  • Sample
  • Phenylisothiocyanate
  • Pyridine
  • Trifluoroacetic acid, concentrated
  • Butyl acetate
  • Additional reagents and equipment for LC/MS/MS (see protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
   Clauser, K.R., Baker, P.R., and Burlingame, A.L. 1996. Peptide Fragment‐Ion Tags from MALDI/PSD for Error Tolerant Searching of Genomic Databases. In Proceedings of the 44th ASMS Conference Mass Spectrom. p. 365. Allied Topics, Portland, Oreg.
   Eng, J.K., McCormick, A.L., and Yates, J.R. 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.
   Falick, A.M., Hines, W.M., Medzihradszky, K.F., Baldwin, M.A., and Gibson, B.W. 1993. Low‐mass ions produced from peptides by high‐energy collision‐induced dissociation in tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 4: 882‐893.
   Fenyo, D., Qin, J., and Chait, B.T. 1998. Protein identification using mass spectrometric information. Electrophoresis 19: 998‐1005.
   Mann, M. and Wilm, M. 1994. Error tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66: 4390‐4399.
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