Kalxdorf Mathias, Gade Stephan, Eberl H Christian, Bantscheff Marcus
From ‡Cellzome, A GSK Company, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
From ‡Cellzome, A GSK Company, Meyerhofstrasse 1, 69117 Heidelberg, Germany
Mol Cell Proteomics. 2017 May;16(5):770-785. doi: 10.1074/mcp.M116.063859. Epub 2017 Mar 23.
The plasma membrane proteome plays a crucial role in inter- and intracellular signaling, cell survival, and cell identity. As such, it is a prominent target for pharmacological intervention. The relatively low abundance of this subproteome in conjunction with challenging extractability and solubility still hampers its comprehensive analysis. Here, we combined a chemical glycoprotein-tagging strategy with mass spectrometry to enable comprehensive analysis of the cell-surface glycoproteome. To benchmark this workflow and to provide guidance for cell line selection for functional experiments, we generated an inventory of the -linked cell-surface glycoproteomes of 15 standard laboratory human cell lines and three primary lymphocytic cell types. On average, about 900 plasma membrane and secreted proteins were identified per experiment, including more than 300 transporters and ion channels. Primary cells displayed distinct expression of surface markers and transporters underpinning the importance of carefully validating model cell lines selected for the study of cell surface-mediated processes. To monitor dynamic changes of the cell-surface proteome in a highly multiplexed experiment, we employed an isobaric mass tag-based chemical labeling strategy. This enabled the time-resolved analysis of plasma membrane protein presentation during differentiation of the monocytic suspension cell line THP-1 into macrophage-like adherent cells. Time-dependent changes observed in membrane protein presentation reflect functional remodeling during the phenotypic transition in three distinct phases: rapid surface presentation and secretion of proteins from intracellular pools concurrent with rapid internalization of no longer needed proteins and finally delayed presentation of newly synthesized macrophage markers. Perturbation of this process using marketed receptor tyrosine kinase inhibitors revealed dasatinib to severely compromise macrophage differentiation due to an off-target activity. This finding suggests that dynamic processes can be highly vulnerable to drug treatment and should be monitored more rigorously to identify adverse drug effects.
质膜蛋白质组在细胞间和细胞内信号传导、细胞存活及细胞特性方面发挥着关键作用。因此，它是药物干预的一个重要靶点。该亚蛋白质组丰度相对较低，再加上提取和溶解性方面的挑战，仍然阻碍了对其进行全面分析。在此，我们将化学糖蛋白标记策略与质谱联用，以实现对细胞表面糖蛋白组的全面分析。为了评估该工作流程并为功能实验的细胞系选择提供指导，我们编制了一份15种标准实验室人类细胞系和三种原代淋巴细胞类型的N-连接细胞表面糖蛋白组清单。每次实验平均鉴定出约900种质膜和分泌蛋白，其中包括300多种转运蛋白和离子通道。原代细胞显示出表面标志物和转运蛋白的独特表达，这突出了仔细验证为研究细胞表面介导过程而选择的模型细胞系的重要性。为了在一个高度多重化的实验中监测细胞表面蛋白质组的动态变化，我们采用了基于等压质量标签的化学标记策略。这使得我们能够在单核细胞悬浮细胞系THP-1分化为巨噬细胞样贴壁细胞的过程中对质膜蛋白表达进行时间分辨分析。在膜蛋白表达中观察到的时间依赖性变化反映了表型转变过程中三个不同阶段的功能重塑：从细胞内池快速表面表达和分泌蛋白质，同时不再需要的蛋白质快速内化，最后是新合成的巨噬细胞标志物的延迟表达。使用市售的受体酪氨酸激酶抑制剂对这一过程进行干扰后发现，达沙替尼由于脱靶活性而严重损害巨噬细胞分化。这一发现表明，动态过程可能对药物治疗高度敏感，应更严格地进行监测以识别药物不良反应。
