Defining the Cell Surface Cysteinome Using Two-Step Enrichment Proteomics

The plasma membrane
proteome is a rich resource of functionally
important 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 low-density lipoprotein
(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（plasma membrane proteome）是一类富含功能关键且具有治疗相关性蛋白靶点的宝贵资源。细胞表面蛋白质组以高疏水性、高度糖基化、富含二硫键的序列以及整体丰度较低为显著特征，在现有成熟的蛋白质组学分析流程（包括我们团队自研的半胱氨酸化学蛋白质组学（cysteine chemoproteomics）平台）中仍未得到充分采样。本研究将细胞表面糖蛋白捕获（cell surface glycoprotein capture）技术与半胱氨酸化学蛋白质组学相结合，建立了一种两步富集法（two-stage enrichment method），可实现细胞表面半胱氨酸组（cell Surface Cysteinome）的化学蛋白质组学表征。我们开发的“Cys-Surf”平台可在1046种蛋白质中捕获超过2800个膜蛋白半胱氨酸残基，其中包含1907个此前未被批量蛋白质组学分析（bulk proteomic analysis）所检测到的残基。通过将Cys-Surf平台与同位素化学蛋白质组学检测相结合，我们共鉴定出821个可配体结合的半胱氨酸残基，其中既包含已知位点，也涵盖全新位点。Cys-Surf平台还可高效区分氧化还原敏感型半胱氨酸，包括那些易随激活状态变化而发生氧化态改变的半胱氨酸，以及对外源还原剂添加敏感的残基。为验证Cys-Surf平台鉴定功能关键半胱氨酸的能力，我们在低密度脂蛋白受体（low-density lipoprotein receptor, LDLR）中发现了一个氧化还原敏感型半胱氨酸，该残基可同时影响该蛋白的定位与低密度脂蛋白（low-density lipoprotein, LDL）颗粒的摄取过程。综上，以两步富集范式为核心特色的Cys-Surf平台，为解析质膜半胱氨酸组（plasma membrane cysteinome）的功能与治疗应用潜力提供了一种定制化研究策略。