Discovery of Undefined Protein Cross-Linking Chemistry: A Comprehensive Methodology Utilizing 18O-Labeling and Mass Spectrometry

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• 作者：Min Liu ; Zhongqi Zhang ; Tianzhu Zang ; Chris Spahr ; Janet Cheetham ; Da Ren ; Zhaohui Sunny Zhou
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：June 18, 2013
• 年：2013
• 卷：85
• 期：12
• 页码：5900-5908
• 全文大小：544K
• 年卷期：v.85,no.12(June 18, 2013)
• ISSN：1520-6882

文摘

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鈥擷Chem-Finder鈥攖hat involves four stages: (1) detection of cross-linked peptides via ¹⁸O-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.