In vivo chemical cross-linking combined with LCMSMS of digested extracts (in vivo CX-MS) can reveal stable and dynamic protein-protein interactions at a proteome wide-scale and at the peptide level. Here we explore the use of the search engine pLink 2 for analysis of a previously obtained LCMSMS dataset from exponentially growing Bacillus subtilis treated in culture with the cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). Cross-linked peptide pairs were identified by pLink 2 at an overall FDR of < 5%. To also obtain a FDR < 5% for inter-protein cross-linked peptide pairs additional thresholds values were applied for matched fragment intensity and for the numbers of unambiguous y and b ions to be assigned for both composite peptides. Also the mass- and charge-dependent retention times of target peptides purified by diagonal strong cation exchange chromatography were used as a criterion to distinguish true from false positives. After this filtering, pLink 2 identified more than 80% of previously reported protein-protein interactions. In addition the use of pLink 2 revealed interesting new inter-protein cross-linked peptide pairs, among others showing interactions between the global transcriptional repressor AbrB and elongation factor Tu and between the essential protein YlaN of unknown function and the ferric uptake repressor Fur.

中文翻译：

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通过体内交联检测低蛋白-蛋白质相互作用的FDR的复合过滤器

体内化学交联与消化提取物的LCMSMS结合（体内CX-MS）可以在蛋白质组学广泛范围和肽水平上揭示稳定和动态的蛋白质-蛋白质相互作用。在这里，我们探索了搜索引擎pLink 2的用途，该方法用于分析以前用交联剂双（琥珀酰亚胺基）-3-叠氮基甲基戊二酸酯（BAMG）处理的指数增长的枯草芽孢杆菌获得的LCMSMS数据集。通过pLink 2以总FDR <5％鉴定出了交联的肽对。为了使蛋白质间交联的肽对的FDR小于5％，对匹配的片段强度以及为两种复合肽指定的y和b离子的明确数量应用了其他阈值。通过对角强阳离子交换色谱纯化的目标肽的质量和电荷依赖性保留时间也用作区分真假阳性的标准。经过此过滤后，pLink 2鉴定出了先前报道的蛋白质与蛋白质相互作用的80％以上。此外，pLink 2的使用揭示了有趣的新的蛋白间交联肽对，其中还显示了全局转录阻遏物AbrB和延伸因子Tu之间以及功能未知的必需蛋白YlaN与铁摄取阻遏物Fur之间的相互作用。