Liu Fan, van Breukelen Bas, Heck Albert J R
From the ‡Biomolecular Mass Spectrometry and Proteomics Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands; §Netherlands Proteomics Center, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
From the ‡Biomolecular Mass Spectrometry and Proteomics Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands; §Netherlands Proteomics Center, Padualaan 8, 3584 CH, Utrecht, The Netherlands
Mol Cell Proteomics. 2014 Oct;13(10):2776-86. doi: 10.1074/mcp.O114.039057. Epub 2014 Jun 30.
Disulfide bond identification is important for a detailed understanding of protein structures, which directly affect their biological functions. Here we describe an integrated workflow for the fast and accurate identification of authentic protein disulfide bridges. This novel workflow incorporates acidic proteolytic digestion using pepsin to eliminate undesirable disulfide reshuffling during sample preparation and a novel search engine, SlinkS, to directly identify disulfide-bridged peptides isolated via electron transfer higher energy dissociation (EThcD). In EThcD fragmentation of disulfide-bridged peptides, electron transfer dissociation preferentially leads to the cleavage of the S-S bonds, generating two intense disulfide-cleaved peptides as primary fragment ions. Subsequently, higher energy collision dissociation primarily targets unreacted and charge-reduced precursor ions, inducing peptide backbone fragmentation. SlinkS is able to provide the accurate monoisotopic precursor masses of the two disulfide-cleaved peptides and the sequence of each linked peptide by matching the remaining EThcD product ions against a linear peptide database. The workflow was validated using a protein mixture containing six proteins rich in natural disulfide bridges. Using this pepsin-based workflow, we were able to efficiently and confidently identify a total of 31 unique Cys-Cys bonds (out of 43 disulfide bridges present), with no disulfide reshuffling products detected. Pepsin digestion not only outperformed trypsin digestion in terms of the number of detected authentic Cys-Cys bonds, but, more important, prevented the formation of artificially reshuffled disulfide bridges due to protein digestion under neutral pH. Our new workflow therefore provides a precise and generic approach for disulfide bridge mapping, which can be used to study protein folding, structure, and stability.
二硫键的鉴定对于深入了解蛋白质结构至关重要，因为蛋白质结构直接影响其生物学功能。在此，我们描述了一种用于快速准确鉴定真实蛋白质二硫键的综合工作流程。这种新颖的工作流程包括使用胃蛋白酶进行酸性蛋白酶消化，以消除样品制备过程中不期望的二硫键重排，以及一种新型搜索引擎SlinkS，用于直接鉴定通过电子转移高能裂解（EThcD）分离的二硫键连接肽段。在二硫键连接肽段的EThcD裂解过程中，电子转移裂解优先导致S-S键的断裂，产生两个强烈的二硫键裂解肽段作为主要碎片离子。随后，高能碰撞裂解主要针对未反应和电荷减少的前体离子，诱导肽主链裂解。SlinkS能够通过将剩余的EThcD产物离子与线性肽数据库进行匹配，提供两个二硫键裂解肽段的准确单同位素前体质量以及每个连接肽段的序列。该工作流程使用含有六种富含天然二硫键的蛋白质的混合物进行了验证。使用这种基于胃蛋白酶的工作流程，我们能够高效且可靠地鉴定出总共31个独特的半胱氨酸-半胱氨酸键（在存在的43个二硫键中），未检测到二硫键重排产物。胃蛋白酶消化不仅在检测到的真实半胱氨酸-半胱氨酸键数量方面优于胰蛋白酶消化，而且更重要的是，防止了由于中性pH条件下蛋白质消化而形成人工重排的二硫键。因此，我们的新工作流程为二硫键图谱绘制提供了一种精确且通用的方法，可用于研究蛋白质折叠、结构和稳定性。
