Site-Specific Intact N-Linked Glycopeptide Characterization of Prostate-Specific Membrane Antigen from Metastatic Prostate Cancer Cells

Abstract

The composition of N-linked glycans that are conjugated to the prostate-specific membrane antigen (PSMA) and their functional significance in prostate cancer progression have not been fully characterized. PSMA was isolated from two metastatic prostate cancer cell lines, LNCaP and MDAPCa2b, which have different tissue tropism and localization. Isolated PSMA was trypsin-digested, and intact glycopeptides were subjected to LC-HCD-EThcD-MS/MS analysis on a Tribrid Orbitrap Fusion Lumos mass spectrometer. Differential qualitative and quantitative analysis of site-specific N-glycopeptides was performed using Byonic and Byologic software. Comparative quantitative analysis demonstrates that multiple glycopeptides at asparagine residues 51, 76, 121, 195, 336, 459, 476, and 638 were in significantly different abundance in the two cell lines ( p < 0.05). Biochemical analysis using endoglycosidase treatment and lectin capture confirm the MS and site occupancy data. The data demonstrate the effectiveness of the strategy for comprehensive analysis of PSMA glycopeptides. This approach will form the basis of ongoing experiments to identify site-specific glycan changes in PSMA isolated from disease-stratified clinical samples to uncover targets that may be associated with disease progression and metastatic phenotypes.

Prostate‐specific membrane antigen (PSMA) glycoforms in prostate cancer patients seminal plasma

“…Coomassie-stained PSMA bands were cut across the entire length of the preparative gel and subjected to in-gel trypsin digestion as we have previously described. 24,26 Briefly, excised bands were cut into 1 mm 2 cubes and destained in 50 mM ammonium bicarbonate in 50% acetonitrile before dehydration using 100% acetonitrile. In situ reduction and alkylation were achieved by incubating the gel pieces in 10 mM dithiothreitol in 50 mM ammonium bicarbonate at 56°C for 1 h followed by 55 mM iodoacetamide for 45 min at room temperature in darkness.…”

“…The reduced and alkylated proteins were trypsin digested at 37°C for 18 h. Digestion of PSMA with trypsin generates eight single N-linked glycopeptides and a single peptide that contains two N-linked glycosylation sites. Generated tryptic peptides were extracted as previously described, [24][25][26] then dried in a SpeedVac before glycopeptide enrichment using hydrophilic interaction liquid chromatography (HILIC) TopTips (Glygen). A portion of the nonenriched peptides were also analyzed to confirm the identity and total peptide sequence coverage of all PSMA peptides.…”

“…The instrument set-up and analytic condition were as we have recently described. 24 Briefly, 2 µg of peptides from each sample were injected online into a C18 PepMap precolumn (Cat. # 164946; Thermo Fisher Scientific) using 98% buffer A (2% acetonitrile containing 0.1% FA) connected in line with 500 mm × 75 µm C18 PepMap analytic column (Cat.…”

“…This was followed by a 140 min separation using a binary gradient buffer system (buffer A, 0.1% FA, and buffer B 0.1% [v/v] FA in 80% acetonitrile) under the following conditions as we have previously described, flow rate of 250 nL/min, isocratic 8% solvent B over 8 min, 8%−12% solvent B over 5 min; 12%-30% solvent B over 100 min; 30%-60% solvent B over 20 min; 60%-98% solvent B over 5 min; isocratic 98% solvent B over 10 min. 24 The glycopeptides samples derived from the two pooled seminal plasma cohorts were analyzed in quadruplicate technical replicate injections generating a sum of four spectral data files for each group. To minimize carryover, each sample injection was proceeded by two column washes in the sequence runs using blank buffer A injections.…”

“…22 We and others have performed initial characterization of the composition of N-linked glycans that are conjugated to PSMA isolated from human cells and tissues. 23,24 Although PSMA expression levels correlate with PCa severity, the roles of its conjugated N-linked glycoforms in aggressive disease progression and their potential utility as diagnostic markers, prognostic markers, and therapeutic targets have not been fully characterized.…”