Proteomics

Dataset Information

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Reverse-phase liquid chromatography (RPLC) in Combination with Tip-Based Strong Cation Exchange (SCX-Tip) Fractionation, ReST, an Alternative Approach for Cross-linked Peptides analysis


ABSTRACT: Chemical cross-linking in combination with mass spectrometry (XL-MS) has emerged as a useful method for structural elucidation of proteins and protein complexes. Efficient enrichment procedures are necessary to analyze cross-linked products due to their relatively low abundance. 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 enrichment strategy combining basic RPLC (bRPLC) fractionation and tip-based SCX (SCX-Tip) enrichment, termed ReST method, 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 for cross-linked peptides using well-designed cross-linked peptides. Taking advantage of the high resolution of bRPLC fractionation and the high enrichment efficiency of SCX-Tip, we were able to identify 43.6% more cross-links than the conventional SCX approach. The presented ReST approach is an effective fractionation method for XL-MS analysis, and could be beneficial for protoeme-scale protein-protein interaction studies. Moreover, more stringent data filtering was proposed in order to achieve the high confidence of cross-linked peptide identification.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Zhen Wu  

LAB HEAD: Xumin Zhang

PROVIDER: PXD023817 | Pride | 2022-06-16

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
20200510_HeLa_RP_Re1.pdResult Other
20200510_HeLa_RP_Re2.pdResult Other
20200510_HeLa_RP_Re3.pdResult Other
20200510_HeLa_RP_SCX_Re1.pdResult Other
20200510_HeLa_RP_SCX_Re2.pdResult Other
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Publications

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.

Wu Zhen Z   Li Jiaqi J   Huang Lin L   Zhang Xumin X  

Analytica chimica acta 20210707


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  ...[more]

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