质膜蛋白质组是功能和治疗相关的蛋白质靶标的丰富资源。以高疏水性而著称,重糖基化,富含二硫键的序列,总体丰度低,细胞表面蛋白质组仍然在已建立的蛋白质组管道中采样不足,包括我们自己的半胱氨酸化学蛋白质组学平台。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配对,以建立两阶段富集方法,使细胞SurfaceCysteinome的化学蛋白质组学分析成为可能。我们的“Cys-Surf”平台在1,046种蛋白质中捕获了>2,800种总膜蛋白半胱氨酸,包括之前未通过大量蛋白质组学分析捕获的1,907个残基。通过将Cys-Surf与同位素化学蛋白质组读数配对,我们发现了821个可连接的半胱氨酸,包括已知和新颖的网站。Cys-Surf还强烈描绘了对氧化还原敏感的半胱氨酸,包括对半胱氨酸氧化态的活化依赖性改变的半胱氨酸和对添加外源还原剂敏感的残基。举例说明Cys-Surf描绘功能上重要的半胱氨酸的能力,我们在低密度脂蛋白受体(LDLR)中发现了一个对氧化还原敏感的半胱氨酸,该半胱氨酸同时影响LDL颗粒的蛋白质定位和摄取.一起来看,Cys-Surf平台,以其两阶段富集范式而著称,代表了一种定制的方法来描绘质膜半胱氨酸体的功能和治疗潜力。
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.