Discovery
of Undefined Protein Cross-Linking Chemistry:
A Comprehensive Methodology Utilizing <sup>18</sup>O‑Labeling
and Mass Spectrometry
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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 workflowXChem-Finderthat
involves four stages: (1) detection of cross-linked peptides via <sup>18</sup>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