AbstractIntroductionProstate‐specific membrane antigen (PSMA) is a US Food and Drug Administration‐approved theranostic target for prostate cancer (PCa). Although PSMA is known to be glycosylated, the composition and functional roles of its N‐linked glycoforms have not been fully characterized.MethodsPSMA was isolated from pooled seminal plasma from low‐risk grade Groups 1 and 2 PCa patients. Intact glycopeptides were analyzed by mass spectrometry to identify site‐specific glycoforms.ResultsWe observed a rich distribution of PSMA glycoforms in seminal plasma from low and low‐intermediate‐risk PCa patients. Some interesting generalities can be drawn based on the predicted topology of PSMA on the plasma membrane. The glycoforms at ASN‐459, ASN‐476, and ASN‐638 residues that are located at the basal domain facing the plasma membrane in cells, are predominantly high mannose glycans. ASN‐76 which is located in the interdomain region adjacent to the apical domain of the protein shows a mixture of high mannose glycans and complex glycans, whereas ASN‐121, ASN‐195 and ASN‐336 that are located and are exposed at the apical domain of the protein predominantly possess complex sialylated and fucosylated N‐linked glycans. These highly accessible glycosites display the greatest diversity in isoforms across the patient samples.ConclusionsOur study provides novel qualitative insights into PSMA glycoforms that are present in the seminal fluid of PCa patients. The presence of a rich diversity of glycoforms in seminal plasma provides untapped potential for glycoprotein biomarker discovery and as a clinical sample for noninvasive diagnostics of male urological disorders and diseases including PCa. Specifically, our glycomics approach will be critical in uncovering PSMA glycoforms with utility in staging and risk stratification of PCa.