Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Anal Chem. 2013 Jun 18;85(12):5900-8. doi: 10.1021/ac400666p. Epub 2013 May 28.

Discovery of undefined protein cross-linking chemistry: a comprehensive methodology utilizing 18O-labeling and mass spectrometry.

Author information

  • 1Analytical Research and Development, Amgen, One Amgen Center Drive, Thousand Oaks, California 91320, USA.

Abstract

Characterization of protein cross-linking, particularly without prior knowledge of the chemical nature and site of cross-linking, poses a significant challenge, because of their intrinsic structural complexity and the lack of a comprehensive analytical approach. Toward this end, we have developed a generally applicable workflow-XChem-Finder-that involves four stages: (1) detection of cross-linked peptides via (18)O-labeling at C-termini; (2) determination of the putative partial sequences of each cross-linked peptide pair using a fragment ion mass database search against known protein sequences coupled with a de novo sequence tag search; (3) extension to full sequences based on protease specificity, the unique combination of mass, and other constraints; and (4) deduction of cross-linking chemistry and site. The mass difference between the sum of two putative full-length peptides and the cross-linked peptide provides the formulas (elemental composition analysis) for the functional groups involved in each cross-linking. Combined with sequence restraint from MS/MS data, plausible cross-linking chemistry and site were inferred, and ultimately confirmed, by matching with all data. Applying our approach to a stressed IgG2 antibody, 10 cross-linked peptides were discovered and found to be connected via thioethers originating from disulfides at locations that had not been previously recognized. Furthermore, once the cross-link chemistry was revealed, a targeted cross-link search yielded 4 additional cross-linked peptides that all contain the C-terminus of the light chain.

PMID:
23634697
[PubMed - indexed for MEDLINE]
PMCID:
PMC3691076
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Icon for American Chemical Society Icon for PubMed Central
    Loading ...
    Write to the Help Desk