Distinct urinary glycoprotein signatures in prostate cancer patients

Abstract

Novel biomarkers are needed to complement prostate specific antigen (PSA) in prostate cancer (PCa) diagnostic screening programs. Glycoproteins represent a hitherto largely untapped resource with a great potential as specific and sensitive tumor biomarkers due to their abundance in bodily fluids and their dynamic and cancer-associated glycosylation. However, quantitative glycoproteomics strategies to detect potential glycoprotein cancer markers from complex biospecimen are only just emerging. Here, we describe a glycoproteomics strategy for deep quantitative mapping of N- and O-glycoproteins in urine with a view to investigate the diagnostic value of the glycoproteome to discriminate PCa from benign prostatic hyperplasia (BPH), two conditions that remain difficult to clinically stratify. Total protein extracts were obtained, concentrated and digested from urine of six PCa patients (Gleason score 7) and six BPH patients. The resulting peptide mixtures were TMT-labeled and mixed prior to a multi-faceted sample processing including hydrophilic interaction liquid chromatography (HILIC) and titanium dioxide SPE based enrichment, endo-/exoglycosidase treatment and HILIC-HPLC pre-fractionation. The isolated N- and O-glycopeptides were detected and quantified using high resolution mass spectrometry. We accurately quantified 729 N-glycoproteins spanning 1,310 unique N-glycosylation sites and observed 954 and 965 unique intact N- and O-glycopeptides, respectively, across the two disease conditions. Importantly, a panel of 56 intact N-glycopeptides perfectly discriminated PCa and BPH (ROC: AUC = 1). This study has generated a panel of intact glycopeptides that has a potential for PCa detection.

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

“…The highest PMG expression in PCa was observed in PCa grade 3 tissues (Gleason score 7) (Figure G). Although trends indicating a PCa grade‐specific expression of PMG were observed this could not be statistically supported likely due to a gradual rather than an abrupt aberration in the protein and/or glycosylation machinery during PCa progression as we have reported on before …”

“…Tissue samples were obtained from multiple cancer patient cohorts or from control individuals suffering from a range of other non‐cancer conditions at various hospitals and clinics as described in Table (see also Table S2, Supporting Information, for further details of the investigated tissue samples). Many of these tissue samples have been described in recent publications . In brief, tumorigenic and non‐tumorigenic tissues were collected via surgery by trained clinicians after written and informed patient consent were obtained, see Table and Supporting Information for details of ethics.…”

Tissue Proteome Signatures Associated with Five Grades of Prostate Cancer and Benign Prostatic Hyperplasia

“…We also compared the differentially expressed proteins between BPH and Gleason 7 (grades 2 and 3) with the differentially expressed proteins found in our previous published urinary proteome from BPH and Gleason 7‐PCa patients . 85 proteins were found in the tissues with a significant difference between BPH and Gleason 7‐PCa (FDR<0.05); amongst them 40 and 45 was down‐ and upregulated in PCa compared to BPH, respectively.…”

Glycans as Biomarkers in Prostate Cancer

“…In prostate cancer, β-1,6-GlcNAc tri-branched and tetra-branched N-glycans are linked to both metastasis in xenograft models and disease-free survival in patients [73]. Serum N-glycan signatures have shown promise as diagnostic and predictive biomarkers in prostate cancer [91]. Changes to branched N-glycans can help distinguish BPH and prostate cancer [43,92], and increased serum triantennary and tetraantennary N-glycans have clinical utility to predict castrate-resistant prostate cancer [74].…”