Discovery of undefined protein cross-linking chemistry: a comprehensive methodology utilizing 18O-labeling and mass spectrometry.
Discovery of undefined protein cross-linking chemistry: a comprehensive methodology utilizing 18O-labeling and mass spectrometry.
复制标题
DOI：
10.1021/ac400666p
复制
发表时间：
2013-06-18
影响因子：
7.4
通讯作者：
Zhou, Zhaohui Sunny
中科院分区：
文献类型：
--
作者：
Liu, Min;Zhang, Zhongqi;Zang, Tianzhu;Spahr, Chris;Cheetham, Janet;Ren, Da;Zhou, Zhaohui Sunny
蛋白质交联的表征，特别是在没有交联的化学性质和位点的先验知识的情况下，由于其固有的结构复杂性和缺乏全面的分析方法而构成了重大挑战。为此，我们已经开发了一个普遍适用的工作流程XChem的，包括四个阶段。(1)通过C-末端的18 O-标记检测交联肽。(2)使用碎片离子质量数据库搜索已知蛋白质序列，结合从头序列标签搜索，确定每个交联肽对的推定部分序列。(3)基于蛋白酶特异性、质量的独特组合和其他限制条件延伸至全序列。(4)交联化学和交联部位的推断。两个推定的全长肽的总和与交联肽之间的质量差提供了每个交联中涉及的官能团的公式（元素组成分析）。结合来自MS/MS数据的序列限制，推断出合理的交联化学和位点，并最终通过与所有数据匹配来确认。将我们的方法应用于应激的IgG 2抗体，发现了十个交联肽，并且发现它们通过源自先前未识别的位置处的二硫化物的硫醚连接。此外，一旦揭示了交联化学，靶向交联搜索产生了4个额外的交联肽，它们都含有轻链的C-末端。
Characterization of protein crosslinking, particularly without prior knowledge of the chemical nature and site of crosslinking, poses a significant challenge due to their intrinsic structural complexity and the lack of a comprehensive analytical approach. Towards this end, we have developed a generally applicable workflow—XChem-Finder that involves four stages. (1) Detection of crosslinked peptides via 18O-labeling at C-termini. (2) Determination of the putative partial sequences of each crosslinked 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. (4) Deduction of crosslinking chemistry and site. The mass difference between the sum of two putative full-length peptides and the crosslinked 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 crosslinking chemistry and site were inferred, and ultimately, confirmed by matching with all data. Applying our approach to a stressed IgG2 antibody, ten cross-linked peptides were discovered and found to be connected via thioether originating from disulfides at locations that had not been previously recognized. Furthermore, once the crosslink chemistry was revealed, a targeted crosslink search yielded four additional crosslinked peptides that all contain the C-terminus of the light chain.
登录
查看更多内容
影响因子：
7.4
作者：
Koehler, Christian J.;Arntzen, Magnus O.;Thiede, Bernd
通讯作者：
Thiede, Bernd
影响因子：
15
作者：
Cohen, Steven L.;Price, Colleen;Vlasak, Josef
通讯作者：
Vlasak, Josef
影响因子：
3.4
作者：
Datola, Antonio;Richert, Sophie;Jone, Carl
通讯作者：
Jone, Carl
影响因子：
7.4
作者：
Alfaro, Joshua F.;Gillies, Laura A.;Zhou, Zhaohui Sunny
通讯作者：
Zhou, Zhaohui Sunny
影响因子：
7.4
作者：
Liu, Min;Cheetham, Janet;Zhou, Zhaohui Sunny
通讯作者：
Zhou, Zhaohui Sunny