A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry

Chemical cross-linking mass spectrometry (XL-MS) provides protein structural information by identifying covalently linked proximal amino acid residues on protein surfaces. The information gained by this technique is complementary to other structural biology methods such as x-ray crystallography, NMR and cryo-electron microscopy[1]. The extension of traditional quantitative proteomics methods with chemical cross-linking can provide information on the structural dynamics of protein structures and protein complexes. The identification and quantitation of cross-linked peptides remains challenging for the general community, requiring specialized expertise ultimately limiting more widespread adoption of the technique. We describe a general method for targeted quantitative mass spectrometric analysis of cross-linked peptide pairs. We report the adaptation of the widely used, open source software package Skyline, for the analysis of quantitative XL-MS data as a means for data analysis and sharing of methods. We demonstrate the utility and robustness of the method with a cross-laboratory study and present data that is supported by and validates previously published data on quantified cross-linked peptide pairs. This advance provides an easy to use resource so that any lab with access to a LC-MS system capable of performing targeted quantitative analysis can quickly and accurately measure dynamic changes in protein structure and protein interactions.

化学交联质谱技术（Chemical cross-linking mass spectrometry, XL-MS）通过鉴定蛋白质表面共价结合的邻近氨基酸残基，获取蛋白质结构信息。该技术所获信息与X射线晶体学、核磁共振波谱（NMR）及冷冻电子显微镜等其他结构生物学方法形成互补[1]。借助化学交联技术对传统定量蛋白质组学方法进行拓展，可获取蛋白质结构及蛋白质复合物的结构动态信息。但交联肽段的鉴定与定量仍对广大研究者构成挑战，需具备专业技术能力，最终制约了该技术的更广泛应用。本研究提出一种针对交联肽段对的靶向定量质谱分析通用方法。本研究将广泛使用的开源软件包Skyline适配于定量XL-MS数据分析，以此作为数据分析与方法共享的途径。本研究通过跨实验室研究验证了该方法的实用性与稳定性，并提供了可支撑并验证已发表定量交联肽段对数据的实验结果。此项进展提供了一款易用的工具，使得任何配备可开展靶向定量分析的液相色谱-质谱（Liquid Chromatography-Mass Spectrometry, LC-MS）系统的实验室，均可快速且精准地测定蛋白质结构与蛋白质相互作用的动态变化。