PhoXplex: Combining Phospho-enrichable Cross-Linking with Isobaric Labeling for Quantitative Proteome-Wide Mapping of Protein Interfaces

被引:1
|
作者
Ramazanova, Runa D. Hoenger [1 ]
Roumeliotis, Theodoros I. [1 ]
Wright, James C. [1 ]
Choudhary, Jyoti S. [1 ]
机构
[1] Inst Canc Res, Chester Beatty Labs, Funct Prote Team, London SW3 6JB, England
基金
英国惠康基金; 英国医学研究理事会;
关键词
cross-linking mass spectrometry (XL-MS); enrichablecross-linker; PhoX; TMT; cancer cell lines; mutations; protein interactions; MASS-SPECTROMETRY; STRUCTURAL-ANALYSIS; CANCER;
D O I
10.1021/acs.jproteome.4c00567
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Integrating cross-linking mass spectrometry (XL-MS) into structural biology workflows provides valuable information about the spatial arrangement of amino acid stretches, which can guide elucidation of protein assembly architecture. Additionally, the combination of XL-MS with peptide quantitation techniques is a powerful approach to delineate protein interface dynamics across diverse conditions. While XL-MS is increasingly effective with isolated proteins or small complexes, its application to whole-cell samples poses technical challenges related to analysis depth and throughput. The use of enrichable cross-linkers has greatly improved the detectability of protein interfaces in a proteome-wide scale, facilitating global protein-protein interaction mapping. Therefore, bringing together enrichable cross-linking and multiplexed peptide quantification is an appealing approach to enable comparative characterization of structural attributes of proteins and protein interactions. Here, we combined phospho-enrichable cross-linking with TMT labeling to develop a streamline workflow (PhoXplex) for the detection of differential structural features across a panel of cell lines in a global scale. We achieved deep coverage with quantification of over 9000 cross-links and long loop-links in total including potentially novel interactions. Overlaying AlphaFold predictions and disorder protein annotations enables exploration of the quantitative cross-linking data set, to reveal possible associations between mutations and protein structures. Lastly, we discuss current shortcomings and perspectives for deep whole-cell profiling of protein interfaces at large-scale.
引用
收藏
页码:5209 / 5220
页数:12
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