Cell Surface Proteomics Reveals Hypoxia-Regulated Pathways in Cervical and Bladder Cancer.

Background Plasma membrane proteins (PMPs) play key roles in cell signalling, adhesion, and trafficking, and are attractive therapeutic targets in cancer due to their surface accessibility. However, their typically low abundance limits detection by conventional proteomic approaches. To improve PMP detection, we employed a surface proteomics workflow combining cell surface biotinylation and affinity purification prior to LC-MS/MS analysis in cervical (SiHa) and bladder (UMUC3) cancer cell lines cultured under normoxic (21% O2) or hypoxic (0.1% O2) conditions. In SiHa cells, 43 hypoxia-upregulated proteins were identified exclusively in the biotin-enriched fraction, including ITGB2, ITGA7, AXL, MET, JAG2, and CAV1/CAV2. In UMUC3 cells, 32 unique upregulated PMPs were detected, including CD55, ADGRB1, SLC9A1, NECTIN3, and ACTG1. These proteins were not observed in corresponding whole-cell lysates and are associated with extracellular matrix remodelling, immune modulation, and ion transport. Biotinylation enhanced the detection of membrane-associated pathways such as ECM organisation, integrin signalling, and PI3K-Akt activation. Protein-protein interaction analysis revealed links between membrane receptors and intracellular stress regulators, including mitochondrial proteins. These findings demonstrate that surface biotinylation improves the sensitivity and selectivity of plasma membrane proteomics under hypoxia, revealing hypoxia-responsive proteins and pathways not captured by standard whole-cell analysis.