Department of Chemistry , University of Texas , Austin , Texas 78712 , United States.
Anal Chem. 2018 Jul 17;90(14):8523-8530. doi: 10.1021/acs.analchem.8b01556. Epub 2018 Jun 28.
Deciphering disulfide bond patterns in proteins remains a significant challenge. In the present study, interlinked disulfide bonds connecting peptide chains are homolytically cleaved with 193 nm ultraviolet photodissociation (UVPD). Analysis of insulin showcased the ability of UVPD to cleave multiple disulfide bonds and provide sequence coverage of the peptide chains in the same MS/MS event. For proteins containing more complex disulfide bonding patterns, an approach combining partial reduction and alkylation mitigated disulfide scrambling and allowed assignment of the array of disulfide bonds. The 4 disulfide bonds of lysozyme and the 19 disulfide bonds of serotransferrin were characterized through LC/UVPD-MS analysis of nonreduced and partially reduced protein digests.
解析蛋白质中的二硫键模式仍然是一项重大挑战。在本研究中，通过 193nm 紫外光解（UVPD）使连接肽链的交联二硫键均裂。胰岛素的分析展示了 UVPD 切断多个二硫键并在相同的 MS/MS 事件中提供肽链序列覆盖的能力。对于含有更复杂二硫键模式的蛋白质，结合部分还原和烷基化的方法减轻了二硫键重排，并允许分配数组二硫键。通过非还原和部分还原蛋白质消化物的 LC/UVPD-MS 分析，鉴定了溶菌酶的 4 个二硫键和转铁蛋白的 19 个二硫键。
