title: Simultaneous analyses of N-linked and O-linked glycans of ovarian cancer cells using solid-phase chemoenzymatic method.
abstract: BACKGROUND
  Glycans play critical roles in a number of biological activities. Two common types of glycans, N-linked and O-linked, have been extensively analyzed in the last decades. N-glycans are typically released from glycoproteins by enzymes, while O-glycans are released from glycoproteins by chemical methods. It is important to identify and quantify both N- and O-linked glycans of glycoproteins to determine the changes of glycans.
  
  
  METHODS
  The effort has been dedicated to study glycans from ovarian cancer cells treated with O-linked glycosylation inhibitor qualitatively and quantitatively. We used a solid-phase chemoenzymatic approach to systematically identify and quantify N-glycans and O-glycans in the ovarian cancer cells. It consists of three steps: (1) immobilization of proteins from cells and derivatization of glycans to protect sialic acids; (2) release of N-glycans by PNGase F and quantification of N-glycans by isobaric tags; (3) release and quantification of O-glycans by β-elimination in the presence of 1-phenyl-3-methyl-5-pyrazolone (PMP).
  
  
  RESULTS
  We used ovarian cancer cell lines to study effect of O-linked glycosylation inhibitor on protein glycosylation. Results suggested that the inhibition of O-linked glycosylation reduced the levels of O-glycans. Interestingly, it appeared to increase N-glycan level in a lower dose of the O-linked glycosylation inhibitor. The sequential release and analyses of N-linked and O-linked glycans using chemoenzymatic approach are a platform for studying N-glycans and O-glycans in complex biological samples.
  
  
  CONCLUSIONS
  The solid-phase chemoenzymatic method was used to analyze both N-linked and O-linked glycans sequentially released from the ovarian cancer cells. The biological studies on O-linked glycosylation inhibition indicate the effects of O-glycosylation inhibition to glycan changes in both O-linked and N-linked glycan expression.
authors.affiliation: Department of Pathology, Johns Hopkins Medicine, Smith Bldg 4013, 400 N. Broadway, Baltimore, MD 21287 USA.
authors.forename: Shuang
authors.identifier: 0000-0001-7958-0594
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Pathology, Johns Hopkins Medicine, Smith Bldg 4013, 400 N. Broadway, Baltimore, MD 21287 USA.
authors.forename: Naseruddin
authors.initials: N
authors.lastname: Höti
authors.affiliation: Department of Pathology, Johns Hopkins Medicine, Smith Bldg 4013, 400 N. Broadway, Baltimore, MD 21287 USA.
authors.forename: Weiming
authors.initials: W
authors.lastname: Yang
authors.affiliation: Department of Pathology, Johns Hopkins Medicine, Smith Bldg 4013, 400 N. Broadway, Baltimore, MD 21287 USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Pathology, Johns Hopkins Medicine, Smith Bldg 4013, 400 N. Broadway, Baltimore, MD 21287 USA.
authors.forename: Lijun
authors.initials: L
authors.lastname: Chen
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland College Park, Rockville, MD 20850 USA.
authors.forename: Shuwei
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Pathology, Johns Hopkins Medicine, Smith Bldg 4013, 400 N. Broadway, Baltimore, MD 21287 USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
keywords: Chemoenzymatic; Glycomics; Glycoprotein; Solid phase