Abstract 5662: N-linked glycosylation site mapping in prostate cancer and matched normal tissue: Defining glycan microheterogeneity

• Published in: Cancer research (Chicago, Ill.) Vol. 78; no. 13_Supplement; p. 5662
• Main Authors: Totten, Sarah Michelle, Tanimoto, Cheylene, Bermudez, Abel, Hembree, Amy, Brooks, James D., Pitteri, Sharon J.
• Format: Journal Article
• Language: English
• Published: 01.07.2018
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2018-5662

Abstract The primary goal of this study is to map specific glycan structures occupying glycosylation sites on proteins from prostate cancer tissue in men treated with radical prostatectomies, and to compare detailed glycosylation patterns in malignant tissue to that of matched normal tissue. Glycosylation is a common, yet complex, post-translational modification known to play an integral role in a number of biologic processes. It has become well recognized that glycosylation is aberrantly expressed in a variety of cancers, including prostate cancer, making glycosylation an attractive source for potential biomarkers and therapeutics targets. Although altered expression of glycosylation in prostate cancer is well reported in literature, detailed characterization of the microheterogeneity of glycan structures occupying specific sites on glycoproteins derived from cancer tissue is largely unknown. Defining glycosylation in a site-specific manner is key to understanding glycosylation's involvement in cellular functions that promote tumorigenesis, invasion, and metastasis. Elucidating alterations among specific glycoforms may reveal novel biomarkers with increased specificity and clinical utility for prostate cancer diagnosis. Using state-of-the-art mass spectrometry, this study represents the most comprehensive, site-specific glycosylation analysis of primary prostate tissues comparing malignant and normal states, and builds on our previously described global glycoproteomic analysis. In this study, intact glycopeptide analysis was performed on prostate cancer and matched normal prostate tissue from eleven men with high-grade prostate cancer post prostatectomy. Proteins were extracted from tissue lysates, reduced, alkylated, and digested with trypsin. The resulting digest was enriched for glycopeptides using strong anion exchange in electrostatic repulsion hydrophilic interaction liquid chromatography mode. Using this technique, releasing the glycan is not needed for elution and glycopeptides remain intact for site-specific analysis. Enriched fractions were analyzed by LC-MS/MS, using higher-energy collision-induced dissociation for glycan and peptide backbone fragmentation. Protein Metrics Byonic software was used to make protein and glycopeptide identifications, which were subsequently confirmed by manual expertise. Preliminary results reveal an average of 125 glycopeptides identifications per patient across 70 unique glycoproteins. Glycopeptide identifications provide peptide sequence, glycosylation site localization, and glycan composition. Among annotated proteins with mapped glycosylation sites were prostatic acid phosphatase, nidogen, zinc-alpha-2-glycoprotein, integrin alpha-1, hemopexin, and hypoxia upregulated protein 1. Commonly identified glycan structures include multiply sialylated and fucosylated species. Citation Format: Sarah Michelle Totten, Cheylene Tanimoto, Abel Bermudez, Amy Hembree, James D. Brooks, Sharon J. Pitteri. N-linked glycosylation site mapping in prostate cancer and matched normal tissue: Defining glycan microheterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5662.