Cell-surface tethered promiscuous biotinylators enable comparative small-scale surface proteomic analysis of human extracellular vesicles and cells.
Cell-surface tethered promiscuous biotinylators enable comparative small-scale surface proteomic analysis of human extracellular vesicles and cells.
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DOI：
10.7554/elife.73982
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发表时间：
2022-03-08
期刊：
影响因子：
7.7
通讯作者：
Wells JA
中科院分区：
文献类型：
--
作者：
Kirkemo LL;Elledge SK;Yang J;Byrnes JR;Glasgow JE;Blelloch R;Wells JA
癌症和健康细胞之间细胞表面蛋白质组差异的表征是识别新诊断和治疗靶点的宝贵方法。然而，蛋白质组学表面蛋白的选择性取样需要大量样品（>10e6 个细胞）和较长的标记时间。这些限制妨碍了对材料有限的生物样品的分析或快速表面蛋白质组变化的捕获。在这里，我们提出了两种将外源过氧化物酶（APEX2 和 HRP）直接连接到细胞上的标记方法，从而无需对细胞进行工程改造即可实现快速、小规模的细胞表面生物素化。我们使用了一种新型的脂质化 DNA 束缚 APEX2 (DNA-APEX2)，将其添加到细胞中后会促进细胞不可知的膜近端标记。或者，我们采用与麦芽凝集素 (WGA-HRP) 聚糖结合结构域融合的辣根过氧化物酶 (HRP)。这种方法产生了一种快速且商业上廉价的方法，可以直接标记表面含有常见 N-乙酰葡糖胺 (GlcNAc) 和唾液酸聚糖的细胞。简便的 WGA-HRP 方法允许细胞样品的高表面覆盖，并首次实现了细胞和细胞源性小细胞外囊泡 (EV) 的表面蛋白质组比较表征，从而实现了 953 细胞和 EV 表面注释蛋白的稳健定量。我们鉴定了新认识的 EV 富集标记子集，以及在 Myc 癌基因转化的前列腺癌 EV 上独特上调的蛋白质。这两种细胞系酶表面生物素化方法对于在一系列材料有限的样品类型中快速、直接标记表面蛋白非常有利。
Characterization of cell surface proteome differences between cancer and healthy cells is a valuable approach for the identification of novel diagnostic and therapeutic targets. However, selective sampling of surface proteins for proteomics requires large samples (>10e6 cells) and long labeling times. These limitations preclude analysis of material-limited biological samples or the capture of rapid surface proteomic changes. Here, we present two labeling approaches to tether exogenous peroxidases (APEX2 and HRP) directly to cells, enabling rapid, small-scale cell surface biotinylation without the need to engineer cells. We used a novel lipidated DNA-tethered APEX2 (DNA-APEX2), which upon addition to cells promoted cell agnostic membrane-proximal labeling. Alternatively, we employed horseradish peroxidase (HRP) fused to the glycan-binding domain of wheat germ agglutinin (WGA-HRP). This approach yielded a rapid and commercially inexpensive means to directly label cells containing common N-Acetylglucosamine (GlcNAc) and sialic acid glycans on their surface. The facile WGA-HRP method permitted high surface coverage of cellular samples and enabled the first comparative surface proteome characterization of cells and cell-derived small extracellular vesicles (EVs), leading to the robust quantification of 953 cell and EV surface annotated proteins. We identified a newly recognized subset of EV-enriched markers, as well as proteins that are uniquely upregulated on Myc oncogene-transformed prostate cancer EVs. These two cell-tethered enzyme surface biotinylation approaches are highly advantageous for rapidly and directly labeling surface proteins across a range of material-limited sample types.