| Chemical cross-linking coupled with mass spectrometry (CXMS) is an important tool to analyze protein structures and protein-protein interactions. In the last five years, CXMS has made great progress in both methods and applications. In terms of methods, on the one hand, cleavable cross-linkers and new separation and enrichment methods have shown good prospects, and on the other hand, more efficient cross-linked peptide search engines and quality control methods provide powerful tools for CXMS data analysis. In terms of applications, on the one hand, CXMS combined with cryo-electron microscopy has determined a large number of protein structures, and on the other hand, CXMS has shown the potential to analyze protein-protein interaction networks at a proteome scale. The intensive research on CXMS in methods and applications reflect the important role of this technology. Here we review the various aspects of CXMS, including cross-linker selection, cross-linking reaction, protein digestion, separation and enrichment, data acquisition, cross-linked peptide identification, quality control, and application, and mainly focus on progress in the last five years. Lastly, we discuss the challenges and opportunities of CXMS in the future. In section 1, we review cross-linkers from the aspects of reactive group and spacer arm. In section 2, we give tips for the cross-linking reaction. In section 3, we describe the sequential digestion strategy for cross-linked proteins. In section 4, we elaborate enrichment methods for cross-linked peptides, including affinity purification, chromatographic separation, and ion mobility mass spectrometry. In section 5, we elaborate data acquisition methods for cross-linked peptides, and compare three methods for MS-cleavable cross-linked peptides. In section 6, we elaborate search engines for cross-linked peptide identification. In section 7, we describe quality control methods for cross-linked peptide identification. In section 8, we review applications of CXMS and list some proteome-wide CXMS studies. In section 9, we conclude the paper and discuss the challenges and opportunities of CXMS in the future. |
