Yan Tianyang, Boatner Lisa M, Cui Liujuan, Tontonoz Peter, Backus Keriann M
Department of Biological Chemistry, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095 (USA).
Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095 (USA).
bioRxiv. 2023 Oct 19:2023.10.17.562832. doi: 10.1101/2023.10.17.562832.
The plasma membrane proteome is a rich resource of functional and therapeutically relevant protein targets. Distinguished by high hydrophobicity, heavy glycosylation, disulfide-rich sequences, and low overall abundance, the cell surface proteome remains undersampled in established proteomic pipelines, including our own cysteine chemoproteomics platforms. Here we paired cell surface glycoprotein capture with cysteine chemoproteomics to establish a two-stage enrichment method that enables chemoproteomic profiling of cell Surface Cysteinome. Our "Cys-Surf" platform captures >2,800 total membrane protein cysteines in 1,046 proteins, including 1,907 residues not previously captured by bulk proteomic analysis. By pairing Cys-Surf with an isotopic chemoproteomic readout, we uncovered 821 total ligandable cysteines, including known and novel sites. Cys-Surf also robustly delineates redox-sensitive cysteines, including cysteines prone to activation-dependent changes to cysteine oxidation state and residues sensitive to addition of exogenous reductants. Exemplifying the capacity of Cys-Surf to delineate functionally important cysteines, we identified a redox sensitive cysteine in the low-density lipoprotein receptor (LDLR) that impacts both the protein localization and uptake of LDL particles. Taken together, the Cys-Surf platform, distinguished by its two-stage enrichment paradigm, represents a tailored approach to delineate the functional and therapeutic potential of the plasma membrane cysteinome.
质膜蛋白质组是功能和治疗相关蛋白质靶点的丰富资源。细胞表面蛋白质组以高疏水性、高度糖基化、富含二硫键序列和总体丰度低为特征，在包括我们自己的半胱氨酸化学蛋白质组学平台在内的既定蛋白质组学流程中，其采样仍然不足。在这里，我们将细胞表面糖蛋白捕获与半胱氨酸化学蛋白质组学相结合，建立了一种两阶段富集方法，能够对细胞表面半胱氨酸组进行化学蛋白质组分析。我们的“Cys-Surf”平台在1046种蛋白质中捕获了超过2800个总膜蛋白半胱氨酸，其中包括1907个以前通过整体蛋白质组分析未捕获的残基。通过将Cys-Surf与同位素化学蛋白质组学读数相结合，我们发现了821个可配体半胱氨酸，包括已知和新发现的位点。Cys-Surf还能有力地描绘氧化还原敏感的半胱氨酸，包括易于发生依赖激活的半胱氨酸氧化态变化的半胱氨酸以及对外源还原剂添加敏感的残基。作为Cys-Surf描绘功能重要半胱氨酸能力的例证，我们在低密度脂蛋白受体（LDLR）中鉴定出一个氧化还原敏感的半胱氨酸，它影响蛋白质定位和LDL颗粒的摄取。综上所述，以其两阶段富集模式为特征的Cys-Surf平台代表了一种定制方法，用于描绘质膜半胱氨酸组的功能和治疗潜力。
