Characterization of the Glycosylation Site of Human PSA Prompted by Missense Mutation using LC–MS/MS

Abstract

Prostate specific antigen (PSA) is currently used as a diagnostic biomarker for prostate cancer. It is a glycoprotein possessing a single glycosylation site at N69. During our previous study of PSA N69 glycosylation, additional glycopeptides were observed in the PSA sample that were not previously reported and did not match glycopeptides of impure glycoproteins existed in the sample. This extra glycosylation site of PSA is associated with mutation in KLK3 genes. Among single nucleotide polymorphisms (SNPs) of KLKs families, the rs61752561 in KLK3 genes is an unusual missense mutation resulting in the conversion of D102 to N in PSA amino acid sequence. Accordingly, a new N-linked glycosylation site is created with an N102MS motif. Here, we report the first qualitative and quantitative glycoproteomic study of PSA N102 glycosylation site by LC-MS/MS. We successfully applied tandem MS to verify the amino acid sequence possessing N102 glycosylation site and associated glycoforms of PSA samples acquired from different suppliers. A total of 21, 7, and 16 glycoforms were detected for LeeBio, Sigma, and EMD PSA samples, respectively. Interestingly, fucosylated glycopeptides were not detected on N102. Among the 3 PSA samples, HexNAc2Hex5 was the predominant glycoform at N102 while HexNAc4Hex5Fuc1NeuAc1 or HexNAc4Hex5Fuc1NeuAc2 were the primary glycoforms at N69.

Recent advances in mass spectrometric analysis of glycoproteins

“…The identification and quantitation of a mutant glycosylation site (N102) of PSA were achieved for the first time, using C 18 ‐LC−MS analysis with different fragmentation methods (CID/HCD and ETD) . The amino acid sequences and associated glycoforms of three different PSA samples were successfully investigated, and it was demonstrated that HexNAc2Hex5 is the primary glycoform at N102, whereas HexNAc4Hex5Fuc1NeuAc1 or HexNAc4Hex5Fuc1NeuAc2 is the predominant form at N69 (Fig.…”

“…The fragments indicating N102 glycosylation and the carbamidomethylation of methionine are highlighted in yellow. Reprint with permission from .…”

Characterization of isomeric glycan structures by LC‐MS/MS

“…Glycomics changes associated with trumatic brain injury were also assessed using the above LC‐MS/MS analysis of permethylated glycans . Glycans derived from glioma stem cell xenografts , neuroblastoma and breast cancer cell lines, IgG , prostate‐specific antigen , and human milk, bovine milk, and goat milk were also analyzed using RPLC‐MS/MS of reduced and permethylated glycans.…”

Isotope Targeted Glycoproteomics (IsoTaG) to Characterize Intact, Metabolically Labeled Glycopeptides from Complex Proteomes

“…The challenge of confident assignment by database searching has necessitated simplification of the database by either reduction of the glycan [e.g., truncation of the glycan in a complex proteome (Steentoft et al, 2011; Zielinska et al)], or reduction of the proteome [e.g., glycoprotein immunoprecipitation (Song et al, 2015; Song et al, 2014)]. Methods to survey the intact glycoproteome from complex samples are assisted by advances in MS methods and database searching (Medzihradszky et al, 2015; Trinidad et al, 2013; Wu et al, 2014).…”