title: High-throughput determination of the site-specific N-sialoglycan occupancy rates by differential oxidation of glycoproteins followed with quantitative glycoproteomics analysis.
abstract: Sialylated glycoproteins, which play important roles in tumor progression, have been extensively analyzed for the discovery of potential biomarkers for cancer diagnosis and prognosis. The site-specific N-sialoglycan occupancy rates of glycoproteins reflect the activities of glycosyltransferases and glycosidases in vivo and could be novel disease biomarkers. However, a high-throughput method to determine the N-sialoglycan occupancy rates is not available. On the basis of the fact that dihydroxy of sialic acid of glycan chains in glycoproteins can be specifically oxidized to aldehyde in mild periodate concentration while all types of glycan chains can be oxidized in high periodate concentration, we developed a modified protein-level hydrazide chemistry method for the determination of the N-sialoglycan occupancy rates. This method was first applied to determine the N-sialoglycan occupancy rates of two glycosites on human transferrin. These two sites were found to be fully sialylated and the N-sialoglycan occupancy rates were found to under significant decrease after the neuraminidase treatment. This method was then applied to analyze N-sialoglycan occupancy rates in proteome samples. We determined 496 and 632 site-specific N-sialoglycan occupancy rates on 334 and 394 proteins from hepatocellular carcinoma (HCC) and normal human liver tissues, respectively. By comparing the N-sialoglycan occupancy rates between the above two samples, we determined 76 N-sialoglycosites with more than a 2-fold change. This method was demonstrated to be an effective and high-throughput method for the analysis of the N-sialoglycan occupancy rates.
authors.affiliation: CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Zhang
authors.initials: Z
authors.lastname: Zhang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Sun
authors.forename: Jun
authors.initials: J
authors.lastname: Zhu
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Guang
authors.initials: G
authors.lastname: Huang
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D000447:Aldehydes / Q000737:chemistry; D000595:Amino Acid Sequence; D006528:Carcinoma, Hepatocellular / Q000737:chemistry* / Q000175:diagnosis; D016022:Case-Control Studies; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D008113:Liver Neoplasms / Q000737:chemistry* / Q000175:diagnosis; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D009363:Neoplasm Proteins / Q000032:analysis / Q000737:chemistry*; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000737:chemistry*; D020543:Proteome / Q000032:analysis / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014168:Transferrin / Q000737:chemistry
chemical_list: D000447:Aldehydes; D006023:Glycoproteins; D006834:Hydrazines; D009363:Neoplasm Proteins; D011134:Polysaccharides; D020543:Proteome; D014168:Transferrin; D019158:N-Acetylneuraminic Acid