Pyridinium-Based Strategy for a Bioorthogonal Conjugation-Assisted Purification Method for Profiling Cell Surface Proteome

Cell surface proteins (CSPs) are valuable targets for
therapeutic
agents, but achieving highly selective CSP enrichment in cellular
physiology remains a technical challenge. To address this challenge,
we propose a newly developed sulfo-pyridinium ester (SPE) cross-linking
probe, followed by two-step imaging and enrichment. The SPE probe
showed higher efficiency in labeling proteins than similar NHS esters
at the level of cell lysates and demonstrated specificity for Lys
in competitive experiments. More importantly, this probe could selectively
label the cell membranes in cell imaging with only negligible labeling
of the intracellular compartment. Moreover, we successfully performed
this strategy on MCF-7 live cells to label 425 unique CSPs from 1162
labeled proteins. Finally, we employed our probe to label the CSPs
of insulin-cultured MCF-7, revealing several cell surface targets
of key functional biomarkers and insulin-associated pathogenesis.
The above results demonstrate that the SPE method provides a promising
tool for the selective labeling of cell surface proteins and monitoring
transient cell surface events.

细胞表面蛋白（CSPs）是治疗剂的宝贵靶点，然而在细胞生理学中实现高度选择性的CSP富集仍然是一项技术挑战。为应对这一挑战，我们提出了一种新开发的磺基吡啶酯（SPE）交联探针，随后进行两步成像和富集。SPE探针在细胞裂解物水平上标记蛋白质的效率高于类似的NHS酯，并在竞争实验中显示出对赖氨酸的特异性。更重要的是，该探针能够在细胞成像中仅对细胞膜进行选择性标记，而对细胞内区室几乎无标记。此外，我们成功将该策略应用于MCF-7活细胞，标记了来自1162个标记蛋白的425个独特的CSPs。最后，我们利用该探针标记了胰岛素培养的MCF-7细胞表面的CSPs，揭示了多个关键功能生物标志物和胰岛素相关发病机制的相关细胞表面靶点。上述结果表明，SPE方法为细胞表面蛋白的选择性标记和监测瞬态细胞表面事件提供了一种有前景的工具。