Quantitative cross-linking/mass spectrometry (QCLMS) is an emerging approach to study conformational changes of proteins and multi-subunit complexes. Distinguishing protein conformations requires reproducibly identifying and quantifying cross-linked peptides. Here we analyzed the variation between multiple cross-linking reactions using bis[sulfosuccinimidyl] suberate (BS³)-cross-linked human serum albumin (HSA) and evaluated how reproducible cross-linked peptides can be identified and quantified by LC-MS analysis. To make QCLMS accessible to a broader research community, we developed a workflow that integrates the established software tools MaxQuant for spectra preprocessing, Xi for cross-linked peptide identification, and finally Skyline for quantification (MS1 filtering). Out of the 221 unique residue pairs identified in our sample, 124 were subsequently quantified across 10 analyses with coefficient of variation (CV) values of 14% (injection replica) and 32% (reaction replica). Thus our results demonstrate that the reproducibility of QCLMS is in line with the reproducibility of general quantitative proteomics and we establish a robust workflow for MS1-based quantitation of cross-linked peptides.

中文翻译：

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无标记定量交联/质谱法的重现性

定量交联/质谱法（QCLMS）是研究蛋白质和多亚基复合物构象变化的新兴方法。区分蛋白质构象需要可重复地鉴定和定量交联的肽。在这里，我们分析了使用双[磺基琥珀酰亚胺基]辛二酸酯的多次交联反应之间的变化（BS ³交联的人血清白蛋白（HSA），并评估如何通过LC-MS分析鉴定和定量可再现的交联肽。为了使QCLMS可供更广泛的研究社区使用，我们开发了一个工作流，该工作流集成了已建立的软件工具MaxQuant用于光谱预处理，Xi用于交联肽段识别，最后是Skyline用于定量（MS1过滤）。在我们的样品中鉴定出的221个唯一残基对中，随后在10个分析中定量了124个，变异系数（CV）值分别为14％（进样重复）和32％（反应重复）。因此，我们的结果证明了QCLMS的重现性与一般定量蛋白质组学的重现性相符，并且我们为基于MS1的交联肽定量建立了强大的工作流程。