In-Depth Glycoproteomic Assay of Urinary Prostatic Acid Phosphatase

Abstract

Prostate-specific antigen (PSA) is a well-known clinical biomarker in prostate cancer (PCa) diagnosis, but a better test is still needed, as the serum-level-based PSA quantification exhibits limited specificity and comes with poor predictive value. Prior to PSA, prostatic acid phosphatase (PAP) was used, but it was replaced by PSA because PSA improved the early detection of PCa. Upon revisiting PAP and its glycosylation specifically, it appears to be a promising new biomarker candidate. Namely, previous studies have indicated that PAP glycoforms differ between PCa and non-PCa individuals. However, an in-depth characterization of PAP glycosylation is still lacking. In this study, we established an in-depth glycoproteomic assay for urinary PAP by characterizing both the micro- and macroheterogeneity of the PAP glycoprofile. For this purpose, PAP samples were analyzed by capillary electrophoresis coupled to mass spectrometry after affinity purification from urine and proteolytic digestion. The developed urinary PAP assay was applied on a pooled DRE (digital rectal examination) urine sample from nine individuals. Three glycosylation sites were characterized, namely N₉₄, N₂₂₀, and N₃₃₃, via N-glycopeptide analysis. Taking sialic acid linkage isomers into account, a total of 63, 27, and 4 N-glycan structures were identified, respectively. The presented PAP glycoproteomic assay will enable the determination of potential glycomic biomarkers for the early detection and prognosis of PCa in cohort studies.

Figure 1

In-depth urinary PAP glycoproteomic workflow. PAP was enriched from DRE urine by immunoaffinity capturing overnight. After capturing, PAP was proteolytically digested and directly subjected to CE-MS measurements. Three glycosylation sites were identified and characterized in an in-depth manner.

Figure 2

Extracted ion electropherograms (EIEs) of the most abundant glycans on DRE urinary PAP on (a) site N₉₄, (b) site N₂₂₀ and (c) site N₃₃₃. The glycan structure with a yellow background color in part (b) highlights the ketodeoxynononic acid (Kdn)-containing glycan identified on PAP. Three isomers of the Kdn-containing glycan were observed. PEP: peptide backbone. Asterisk indicates non-glycopeptide analytes. Peptide backbone of site N₉₄ is FLN₉₄ESYK. Peptide backbone of site N₂₂₀ is VYDPLYCESVHN₂₂₀FTLPSWATEDTMTK. Peptide backbone of site N₃₃₃ is N₃₃₃ETQHEPYPLMLPGCSPSCPLER.

Figure 3

Intra- and interday validation of the developed prostatic acid phosphatase (PAP) glycoproteomic assay, showing the relative abundance of the 10 most abundant glycopeptides per glycosylation sites. The seminal plasma PAP standard was spiked into a female urine pool (FUP), and an in-solution digestion was performed and used to test the repeatability (intraday, n = 4) and intermediate precision (interday, n = 12) of the PAP glycoproteomic assay by CE-MS. For site N₃₃₃, the average relative standard deviation (AVG RSD) shows the top three analytes.

Figure 4

Identification of Kdn-containing N-glycans in seminal plasma PAP glycopeptides via tandem MS using CE-MS. (a, b) Tandem MS spectra of glycopeptides H6N5F1S3 and H6N5F1S2K1 on the N-glycosylation site N₂₂₀ with peptide backbone VYDPLYCESVHNFTLPSWATEDTMTK. H: hexose. N: N-acetylglucosamine. F: fucose. S: N-acetylneuraminic acid (Neu5Ac). K: ketodeoxynononic acid (Kdn). Pep: peptide backbone. Green background highlights the significant ions H1N1K1 for Kdn identification. Blue diamonds indicate the parent ions (quadruply charged) of the glycopeptides. The assignment of glycan structures is based on tandem MS spectra.