title: O Labeling for a Quantitative Proteomic Analysis of Glycoproteins in Hepatocellular Carcinoma.
abstract: INTRODUCTION: Quantitative proteomics using tandem mass spectrometry is an attractive approach for identification of potential cancer biomarkers. Fractionation of complex tissue samples into subproteomes prior to mass spectrometric analyses increases the likelihood of identifying cancer-specific proteins that might be present in low abundance. In this regard, glycosylated proteins are an interesting class of proteins that are already established as biomarkers for several cancers.  MATERIALS AND METHODS: In this study, we carried out proteomic profiling of tumor and adjacent non-cancer liver tissues from hepatocellular carcinoma (HCC) patients. Glycoprotein enrichment from liver samples using lectin affinity chromatography and subsequent (18)O/(16)O labeling of peptides allowed us to obtain relative abundance levels of lectin-bound proteins. As a complementary approach, we also examined the relative expression of proteins in HCC without glycoprotein enrichment. Lectin affinity enrichment was found to be advantageous to quantitate several interesting proteins, which were not detected in the whole proteome screening approach. We identified and quantitated over 200 proteins from the lectin-based approach. Interesting among these were fetuin, cysteine-rich protein 1, serpin peptidase inhibitor, leucine-rich alpha-2-glycoprotein 1, melanoma cell adhesion molecule, and heparan sulfate proteoglycan-2. Using lectin affinity followed by PNGase F digestion coupled to (18)O labeling, we identified 34 glycosylation sites with consensus sequence N-X-T/S. Western blotting and immunohistochemical staining were carried out for several proteins to confirm mass spectrometry results.  CONCLUSION: This study indicates that quantitative proteomic profiling of tumor tissue versus non-cancerous tissue is a promising approach for the identification of potential biomarkers for HCC.
authors.affiliation: Institute of Bioinformatics, International Technology Park, Bangalore, 560066, India; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Raghothama
authors.initials: R
authors.lastname: Chaerkady
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Thuluvath
authors.forename: Min-Sik
authors.initials: MS
authors.lastname: Kim
authors.forename: Anuradha
authors.initials: A
authors.lastname: Nalli
authors.forename: Perumal
authors.initials: P
authors.lastname: Vivekanandan
authors.forename: Jessica
authors.initials: J
authors.lastname: Simmers
authors.forename: Michael
authors.initials: M
authors.lastname: Torbenson
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Pandey