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.