Hypoxic tumour tissue is known to be resistant to treatment and is associated with a poor clinical prognosis. There are several reasons why this might be, including the capacity of hypoxia to drive genomic instability and alter DNA damage repair pathways. Head and neck squamous cell carcinoma (HNSCC) has been found to be a complex group of malignancies, with notable features that include a marked effect of hypoxia on treatment outcomes in standard-of-care chemoradiation and evidence for hypoxia-induced immunosuppression. UT-SCC-54C, a HNSCC line, was used as a representative to characterise the proteome-wide changes under hypoxic culture.
In order to generate a comprehensive peptide mass spectral library, whole-cell lysates from log-phase cultures growing under standard (aerobic) culture conditions were collected, digested with trypsin, and fractionated by strong cation exchange column for deep protein coverage to facilitate protein identification. Each fraction was spiked with iRT standards (retention time calibrant) and analysed in a data-dependent acquisition mode. Cells cultured under several chronic hypoxic conditions were then analysed together with those cultured under standard conditions using a TripleTOF system in data-independent acquisition mode (SWATH) to obtain quantitative data.
More than 6,000 individual proteins were identified and this formed the basis of the mass spectral library for the cell line. This spectral library was further refined to a peptide ion library for SWATH data extraction (using Skyline). The SWATH-MS data identified about 3,700 proteins and 12,800 peptides in each sample (condition), suggesting good protein coverage depth. Statistical analysis using Tukey Median Polish analysis (MSStats) revealed some key protein changes under chronic hypoxia (compared to air). As expected, proteins involved in HIF-1 signalling pathways (e.g. ALDOA, PDK1, PGK1 and SLC2A1) were significantly increased. Interestingly, several proteins with potential roles in genomic stability (c7orf49 and UHRF1) were downregulated under hypoxia and these are currently being investigated.