Characterization of protein cross-links via mass spectrometry and an open-modification search strategy

Anal Chem. 2008 Nov 15;80(22):8799-806. doi: 10.1021/ac801646f. Epub 2008 Oct 24.


Protein-protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein-protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying cross-linked peptides from single LC/MS/MS data sets, without using specialized cross-linkers or custom-written software. To achieve this, each peptide in the pair of cross-linked peptides was considered to be "post-translationally" modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of cross-linked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich data sets for cross-linked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native cross-links in proteins. The pipeline was validated by characterizing the known interactions in the chemically cross-linked CYP2E1-b5 complex. Utility of this method in identifying native cross-links was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / metabolism
  • Catalytic Domain
  • Cross-Linking Reagents / pharmacology*
  • Cytochrome P-450 CYP2E1 / chemistry
  • Cytochrome P-450 CYP2E1 / metabolism
  • Cytochromes b5 / chemistry
  • Cytochromes b5 / metabolism
  • Disulfides / metabolism
  • Humans
  • Mass Spectrometry
  • Molecular Sequence Data
  • Peptides / chemistry
  • Peptides / metabolism
  • Protein Binding / drug effects
  • Proteins / metabolism*
  • Reproducibility of Results


  • Bacterial Proteins
  • Cross-Linking Reagents
  • Disulfides
  • Peptides
  • Proteins
  • Cytochromes b5
  • Cytochrome P-450 CYP2E1