Intact glycopeptide analysis of human prostate tissue reveals site-specific heterogeneity of protein glycosylation in prostate cancer.

Approximately 300,000 American men were diagnosed with prostate cancer in 2024. Existing screening approaches based on measuring levels of prostate-specific antigen in the blood lack specificity for prostate cancer. Studying the glycans attached to proteins has the potential to generate new biomarker candidates and/or increase the specificity of existing protein biomarkers, and studying protein glycosylation changes in prostate cancer could also add new information to our understanding of prostate cancer biology. Here, we present the analysis of N-glycoproteins in clinical prostate cancer tissue and patient-matched, non-cancerous adjacent tissue using LC-MS/MS-based intact N-linked glycopeptide analysis. This analysis allowed us to characterize protein N-linked glycosylation changes in prostate cancer at the glycoprotein, glycopeptide, and glycosite levels. Overall, 1894 unique N-glycosites on 7022 unique N-glycopeptides from 1354 unique glycoproteins were identified. Importantly, we observed an overall increase in glycoprotein, glycopeptide, and glycosite counts in prostate cancer tissue than non-cancerous tissue. We identified biological functions enriched in prostate cancer that relate to cancer development. Additionally, we characterized N-glycosite-specific changes in prostate cancer, demonstrating significant meta- and micro-heterogeneity in N-glycan composition in prostate cancer in comparison to non-cancerous tissue. Our findings support the idea that protein glycosylation is heavily impacted and aberrant in prostate cancer and provide examples of N-glycosite-specific changes that could be exploited for more specific markers of prostate cancer.