Disulfide Linkage Characterization of Disulfide Bond-Containing Proteins and Peptides by Reducing Electrochemistry and Mass Spectrometry
Disulfide Linkage Characterization of Disulfide Bond-Containing Proteins and Peptides by Reducing Electrochemistry and Mass Spectrometry
复制标题
DOI：
10.1021/acs.analchem.5b03148
复制
发表时间：
2016-02-02
影响因子：
7.4
通讯作者：
Nielsen, Peter Kresten
中科院分区：
文献类型：
--
作者：
Cramer, Christian N.;Haselmann, Kim F.;Nielsen, Peter Kresten
解开二硫键模式是蛋白质表征的关键部分，无论是在基础研究中以前未表征的蛋白质还是重组药物蛋白。在生物制药工业中，阐明半胱氨酸连接是必要的，以避免在最终产品中的二硫无序和不正确折叠形式。质谱（MS）是一种高度利用的分析工具，由于快速和准确的表征。然而，二硫键是蛋白质结构中额外的共价键，这对串联质谱（MS/MS）的蛋白质测序提出了挑战。电化学（EC）还原二硫键最近被证明提供了高效的还原效率，显着提高了在线耦合与质谱表征的序列覆盖率。在这项研究中，评估了EC二硫还原结合质谱表征在二硫定位中的潜在应用。我们采用了两种方法，基于(1)输注EC-MS中关于还原程度的高灵活性和即时信息，在完整蛋白水平上生成部分还原物种；(2)在LC-EC-MS非还原蛋白消化平台中，亲本二硫化物连接片段和游离还原肽之间保留联系。在这里，我们报告成功地使用EC作为部分还原方法在绘制完整的人胰岛素（HI）和溶菌酶的二硫键。此外，我们建立了一个LC-EC-MS平台，通过在线EC还原非还原蛋白水解消化，有利于对复杂和高度二硫化物结合的蛋白质（如人血清白蛋白（HSA））进行二硫化物表征。
Unravelling, of disulfide linkage patterns is a crucial part of protein characterization, whether it is for a previously uncharacterized protein in basic research or a recombinant pharmaceutical protein. In the biopharmaceutical industry, elucidation of the cysteine connectivities is a necessity to avoid disulfide scrambled and incorrectly folded forms in the final product. Mass spectrometry (MS) is a highly utilized analytical tool for this due to fast and accurate characterization. However, disulfide bonds being an additional covalent bond in the protein structure represent a challenge in protein sequencing by tandem MS (MS/MS). Electrochemical (EC) reduction of disulfide bonds has recently been demonstrated to provide efficient reduction efficiencies, significantly enhancing sequence coverages in online coupling with MS characterization. In this study, the potential use of EC disulfide reduction in combination with MS characterization for disulfide mapping was assessed. We employed two approaches based on (1) the high flexibility and instant information about the degree of reduction in infusion EC-MS to generate partially reduced species on the intact protein level and (2) the preserved link between parent disulfide-linked fragments and free reduced peptides in an LC-EC-MS platform of nonreduced proteolytic protein digestions. Here we report the successful use of EC as a partial reduction approach in mapping of disulfide bonds of intact human insulin (HI) and lysozyme. In addition, we established a LC-EC-MS platform advantageous in disulfide characterization of complex and highly disulfide-bonded proteins such as human serum albumin (HSA) by online EC reduction of nonreduced proteolytic digestions.