Detecting cross-linked peptides by searching against a database of cross-linked peptide pairs

J Proteome Res. 2010 May 7;9(5):2488-95. doi: 10.1021/pr901163d.

Abstract

Mass spectrometric identification of cross-linked peptides can provide valuable information about the structure of protein complexes. We describe a straightforward database search scheme that identifies and assigns statistical confidence estimates to spectra from cross-linked peptides. The method is well suited to targeted analysis of a single protein complex, without requiring an isotope labeling strategy. Our approach uses a SEQUEST-style search procedure in which the database is comprised of a mixture of single peptides with and without linkers attached and cross-linked products. In contrast to several previous approaches, we generate theoretical spectra that account for all of the expected peaks from a cross-linked product, and we employ an empirical curve-fitting procedure to estimate statistical confidence measures. We show that our fully automated procedure successfully reidentifies spectra from a previous study, and we provide evidence that our statistical confidence estimates are accurate.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Cytochrome P-450 CYP2E1 / chemistry
  • Cytochrome P-450 CYP2E1 / metabolism
  • Cytochromes b5 / chemistry
  • Cytochromes b5 / metabolism
  • Databases, Protein*
  • Escherichia coli
  • Humans
  • Linear Models
  • Mass Spectrometry / methods*
  • Molecular Sequence Data
  • Peptides / chemistry*
  • Peptides / metabolism
  • Protein Interaction Mapping / methods*
  • Proteomics / methods*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism

Substances

  • Peptides
  • Recombinant Proteins
  • Cytochromes b5
  • Cytochrome P-450 CYP2E1