State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai, 200438, China.
State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai, 200438, China.
Anal Chim Acta. 2021 Sep 22;1179:338838. doi: 10.1016/j.aca.2021.338838. Epub 2021 Jul 7.
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.
化学交联结合质谱（XL-MS）已成为一种用于蛋白质和蛋白质复合物结构解析的有用方法。由于交联肽的化学计量低，在进行 LC-MS/MS 分析之前，通常需要进行特定的富集方法，特别是对于复杂的样品。目前，强阳离子交换色谱（SCX）、尺寸排阻色谱（SEC）和基于亲和标签的富集是广泛使用的富集策略之一。在此，我们提出了一种二维策略，结合碱性 pH 值反相液相色谱（bRPLC）分级和基于尖端的 SCX（SCX-Tip）富集，称为 ReST，用于交联肽的表征。我们揭示了 bRPLC 在交联肽分级中的无偏分离效果。我们使用精心设计的交联肽优化了 SCX-Tip 的富集条件。利用 bRPLC 分离的高分辨率和 SCX-Tip 的高富集效率，我们能够鉴定出比传统 SCX 方法多 43.6%的交联肽。所提出的 ReST 是一种用于蛋白质组规模蛋白质-蛋白质相互作用研究的简单有效的方法。
