Cell surface proteins perform critical functions related to immune response, signal transduction, cell–cell interactions, and cell migration. Expression of specific cell surface proteins can determine cell-type identity, and can be altered in diseases including infections, cancer and genetic disorders. Identification of the cell surface proteome remains a challenge despite several enrichment methods exploiting their biochemical and biophysical properties. Here, we report a novel method for enrichment of proteins localized to cell surface. We developed this new approach designated surface Biotinylation Site Identification Technology (sBioSITe) by adapting our previously published method for direct identification of biotinylated peptides. In this strategy, the primary amine groups of lysines on proteins on the surface of live cells are first labeled with biotin, and subsequently, biotinylated peptides are enriched by anti-biotin antibodies and analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). By direct detection of biotinylated lysines from PC-3, a prostate cancer cell line, using sBioSITe, we identified 5851 peptides biotinylated on the cell surface that were derived from 1409 proteins. Of these proteins, 533 were previously shown or predicted to be localized to the cell surface or secreted extracellularly. Several of the identified cell surface markers have known associations with prostate cancer and metastasis including CD59, 4F2 cell-surface antigen heavy chain (SLC3A2) and adhesion G protein-coupled receptor E5 (CD97). Importantly, we identified several biotinylated peptides derived from plectin and nucleolin, both of which are not annotated in surface proteome databases but have been shown to have aberrant surface localization in certain cancers highlighting the utility of this method. Detection of biotinylation sites on cell surface proteins using sBioSITe provides a reliable method for identifying cell surface proteins. This strategy complements existing methods for detection of cell surface expressed proteins especially in discovery-based proteomics approaches.

中文翻译：

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sBioSITe 通过直接富集生物素化肽实现细胞表面蛋白质组的灵敏鉴定

细胞表面蛋白执行与免疫反应、信号转导、细胞间相互作用和细胞迁移相关的关键功能。特定细胞表面蛋白的表达可以决定细胞类型的特性，并且可以在感染、癌症和遗传性疾病等疾病中发生改变。尽管有多种富集方法利用了细胞表面蛋白质组的生化和生物物理特性，但细胞表面蛋白质组的鉴定仍然是一个挑战。在这里，我们报告了一种富集细胞表面蛋白质的新方法。我们通过调整我们之前发布的直接识别生物素化肽的方法，开发了这种称为表面生物素化位点识别技术（sBioSITe）的新方法。在该策略中，首先用生物素标记活细胞表面蛋白质上的赖氨酸的伯胺基团，随后通过抗生物素抗体富集生物素化的肽，并通过液相色谱-串联质谱法（LC-MS/多发性硬化症）。通过使用 sBioSITe 直接检测前列腺癌细胞系 PC-3 中的生物素化赖氨酸，我们鉴定了细胞表面上源自 1409 种蛋白质的 5851 种生物素化肽。在这些蛋白质中，有 533 种先前被证明或预测位于细胞表面或分泌到细胞外。一些已鉴定的细胞表面标记物已知与前列腺癌和转移相关，包括 CD59、4F2 细胞表面抗原重链 (SLC3A2) 和粘附 G 蛋白偶联受体 E5 (CD97)。重要的是，我们鉴定了源自凝集素和核仁素的几种生物素化肽，这两种肽均未在表面蛋白质组数据库中注释，但已被证明在某些癌症中具有异常的表面定位，突出了该方法的实用性。使用 sBioSITe 检测细胞表面蛋白上的生物素化位点为识别细胞表面蛋白提供了可靠的方法。该策略补充了检测细胞表面表达蛋白质的现有方法，特别是在基于发现的蛋白质组学方法中。