Discovery Proteomics, Amgen Research, South San Francisco, California 94080, United States.
Inflammation, Amgen Research, South San Francisco, California 94080, United States.
J Proteome Res. 2024 Feb 2;23(2):618-632. doi: 10.1021/acs.jproteome.3c00432. Epub 2024 Jan 16.
Cell surface proteins represent an important class of molecules for therapeutic targeting and cellular phenotyping. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, post-translational modifications, hydrophobic regions, and processing requirements. To improve their identification, we optimized a Cell-Surface Capture (CSC) workflow that incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), nonenzymatic deamidation (digestion and deglycosylation buffers), and data acquisition methods (DDA, DIA, and TMT). Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid, SDS/urea-based lysis buffers, single-pot solid-phased-enhanced sample-preparation (SP3), and streptavidin magnetic beads for maximal surfaceome coverage. Notably, with semiautomated processing, sample handling was simplified and between ∼600 and 900 cell surface N-glycoproteins were identified from only 25-200 μg of HeLa protein. CSC also revealed significant differences between monolayer cultures and tumor xenografts of murine CT26 colon adenocarcinoma samples that may aid in target identification for drug development. Overall, the improved efficiency of the magnetic-based CSC workflow identified both previously reported and novel N-glycosites with less material and high reproducibility that should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.
细胞表面蛋白是治疗靶点和细胞表型分析的重要分子类别。然而，由于其丰度低、翻译后修饰、疏水区和加工要求，通过基于质谱的蛋白质组学方法对其进行富集和检测仍然具有挑战性。为了提高它们的鉴定效率，我们优化了一种包含基于磁珠的处理的细胞表面捕获（CSC）工作流程。使用这种方法，我们评估了标记条件（生物素标签和催化剂）、富集特异性（链霉亲和素珠）、漏切（裂解缓冲液）、非酶脱酰胺（消化和去糖基化缓冲液）以及数据采集方法（DDA、DIA 和 TMT）。我们的研究结果支持使用烷氧基胺-PEG4-生物素加 5-甲氧基-邻氨基苯甲酸、SDS/尿素基裂解缓冲液、单步固相增强样品制备（SP3）和链霉亲和素磁珠来实现最大的表面组覆盖。值得注意的是，通过半自动处理，简化了样品处理过程，仅从 25-200μg 的 HeLa 蛋白中就鉴定出了约 600 到 900 个细胞表面 N-糖蛋白。CSC 还揭示了单层培养物和肿瘤异种移植物之间的显著差异，这可能有助于为药物开发确定靶标。总的来说，基于磁珠的 CSC 工作流程的改进效率，使用更少的材料和更高的重复性，鉴定了先前报道和新的 N-糖基化位点，这应该有助于通过提供以前未记录的细胞表型的见解来推进表面组学领域的发展。
