Basic pH reversed-phase liquid chromatography (bRPLC) in combination with tip-based strong cation exchange (SCX-Tip), ReST, an efficient approach for large-scale cross-linked peptide analysis.

Chemical cross-linking in combination with mass spectrometry (XL-MS) has emerged as a useful method for structural elucidation of proteins and protein complexes. Due to the low stoichiometry of cross-linked peptides, a specific enrichment method is always necessary prior to LC-MS/MS analysis, especially for complex samples. Currently, strong cation exchange chromatography (SCX), size exclusion chromatography (SEC), and affinity tag-based enrichment are among the widely used enrichment strategies. Herein, we present a two-dimensional strategy combining basic pH reversed-phase liquid chromatography (bRPLC) fractionation and tip-based SCX (SCX-Tip) enrichment, termed ReST, for the characterization of cross-linked peptides. We revealed the unbiased separation effects of the bRPLC in the cross-linked peptide fractionation. We optimized the enrichment conditions of SCX-Tip using well-designed cross-linked peptides. Taking advantage of the high resolution of bRPLC separation and the high enrichment efficiency of SCX-Tip, we were able to identify 43.6% more cross-linked peptides than the conventional SCX approach. The presented ReST is a simple and efficient approach for proteome-scale protein-protein interaction studies.