Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
Mol Syst Biol. 2019 Sep;15(9):e8994. doi: 10.15252/msb.20198994.
We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.
我们提出了一个简洁的工作流程，通过引入顺序消化和交联识别软件 xiSEARCH 来增强交联肽的质谱检测。顺序消化通过选择性缩短长胰蛋白酶肽来增强肽检测。我们展示了我们用于交联多蛋白复合物和细胞裂解物的简单 12 馏分方案，进行定量分析和高密度交联，而不需要特定交联剂的特征。这种整体方法揭示了动态的蛋白质-蛋白质相互作用位点，这些位点是可及的，具有基本的功能相关性，因此非常适合小分子抑制剂的开发。
