Müller Fränze, Fischer Lutz, Chen Zhuo Angel, Auchynnikava Tania, Rappsilber Juri
Chair of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, 13355, Berlin, Germany.
Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland, EH9 3BF, UK.
J Am Soc Mass Spectrom. 2018 Feb;29(2):405-412. doi: 10.1007/s13361-017-1837-2. Epub 2017 Dec 18.
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 (BS)-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. Graphical Abstract ᅟ.
定量交联/质谱分析（QCLMS）是一种用于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肽段。在此，我们分析了使用辛二酸双[磺基琥珀酰亚胺酯]（BS）交联的人血清白蛋白（HSA）的多个交联反应之间的差异，并评估了通过液相色谱-质谱分析可重复地鉴定和定量交联肽段的程度。为了使更广泛的研究群体能够使用QCLMS，我们开发了一种工作流程，该流程整合了用于光谱预处理的成熟软件工具MaxQuant、用于交联肽段鉴定的Xi以及最终用于定量（MS1过滤）的Skyline。在我们样品中鉴定出的221个独特残基对中，有124个随后在10次分析中进行了定量，注射复制品的变异系数（CV）值为14%，反应复制品的变异系数（CV）值为32%。因此，我们的结果表明QCLMS的重现性与一般定量蛋白质组学的重现性一致，并且我们建立了一种用于基于MS1的交联肽段定量的稳健工作流程。图形摘要ᅟ。
