title: Glycoproteomics: identifying the glycosylation of prostate specific antigen at normal and high isoelectric points by LC-MS/MS.
abstract: Prostate specific antigen (PSA) is currently used as a biomarker to diagnose prostate cancer. PSA testing has been widely used to detect and screen prostate cancer. However, in the diagnostic gray zone, the PSA test does not clearly distinguish between benign prostate hypertrophy and prostate cancer due to their overlap. To develop more specific and sensitive candidate biomarkers for prostate cancer, an in-depth understanding of the biochemical characteristics of PSA (such as glycosylation) is needed. PSA has a single glycosylation site at Asn69, with glycans constituting approximately 8% of the protein by weight. Here, we report the comprehensive identification and quantitation of N-glycans from two PSA isoforms using LC-MS/MS. There were 56 N-glycans associated with PSA, whereas 57 N-glycans were observed in the case of the PSA-high isoelectric point (pI) isoform (PSAH). Three sulfated/phosphorylated glycopeptides were detected, the identification of which was supported by tandem MS data. One of these sulfated/phosphorylated N-glycans, HexNAc5Hex4dHex1s/p1 was identified in both PSA and PSAH at relative intensities of 0.52 and 0.28%, respectively. Quantitatively, the variations were monitored between these two isoforms. Because we were one of the laboratories participating in the 2012 ABRF Glycoprotein Research Group (gPRG) study, those results were compared to that presented in this study. Our qualitative and quantitative results summarized here were comparable to those that were summarized in the interlaboratory study.
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University , Lubbock, Texas 79409, United States.
authors.forename: Ehwang
authors.initials: E
authors.lastname: Song
authors.forename: Anoop
authors.initials: A
authors.lastname: Mayampurath
authors.forename: Chuan-Yih
authors.initials: CY
authors.lastname: Yu
authors.forename: Haixu
authors.initials: H
authors.lastname: Tang
authors.forename: Yehia
authors.initials: Y
authors.lastname: Mechref
mesh_terms: D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007526:Isoelectric Point; D007610:Kallikreins / Q000378:metabolism*; D008969:Molecular Sequence Data; D017430:Prostate-Specific Antigen / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000378:metabolism; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D006020:Glycopeptides; D020543:Proteome; C549870:KLK3 protein, human; D007610:Kallikreins; D017430:Prostate-Specific Antigen
keywords: LC−MS/MS; N-linked glycosylation; PSA; Prostate specific antigen; glycopeptide; glycoproteomics