Abstract
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. Methods: 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. Results: 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. Conclusions: 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.
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. Methods: 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. Results: 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. Conclusions: 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.
| Original language | English |
|---|---|
| Article number | 36 |
| Number of pages | 18 |
| Journal | Proteomes |
| Volume | 13 |
| Publication status | Published - 5 Aug 2025 |
