title: Differential analysis of N-glycoproteome between hepatocellular carcinoma and normal human liver tissues by combination of multiple protease digestion and solid phase based labeling.
abstract: BACKGROUND
  Dysregulation of glycoproteins is closely related with many diseases. Quantitative proteomics methods are powerful tools for the detection of glycoprotein alterations. However, in almost all quantitative glycoproteomics studies, trypsin is used as the only protease to digest proteins. This conventional method is unable to quantify N-glycosites in very short or long tryptic peptides and so comprehensive glycoproteomics analysis cannot be achieved.
  
  
  METHODS
  In this study, a comprehensive analysis of the difference of N-glycoproteome between hepatocellular carcinoma (HCC) and normal human liver tissues was performed by an integrated workflow combining the multiple protease digestion and solid phase based labeling. The quantified N-glycoproteins were analyzed by GoMiner to obtain a comparative view of cellular component, biological process and molecular function.
  
  
  CONCLUSIONS
  An integrated workflow was developed which enabled the processes of glycoprotein coupling, protease digestion and stable isotope labeling to be performed in one reaction vessel. This workflow was firstly evaluated by analyzing two aliquots of the same protein extract from normal human liver tissue. It was demonstrated that the multiple protease digestion improved the glycoproteome coverage and the quantification accuracy. This workflow was further applied to the differential analysis of N-glycoproteome of normal human liver tissue and that with hepatocellular carcinoma. A total of 2,329 N-glycosites on 1,052 N-glycoproteins were quantified. Among them, 858 N-glycosites were quantified from more than one digestion strategy with over 99% confidence and 1,104 N-glycosites were quantified from only one digestion strategy with over 95% confidence. By comparing the GoMiner results of the N-glycoproteins with and without significant changes, the percentage of membrane and secreted proteins and their featured biological processes were found to be significant different revealing that protein glycosylation may play the vital role in the development of HCC.
authors.affiliation: Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Sun
authors.affiliation: The Second Affiliated Hospital of Dalian Medical University, Dalian 116027, China.
authors.forename: Deguang
authors.initials: D
authors.lastname: Sun
authors.affiliation: Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Fangjun
authors.initials: F
authors.lastname: Wang
authors.affiliation: Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.affiliation: Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Zhang
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Xu
authors.affiliation: Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.affiliation: The Second Affiliated Hospital of Dalian Medical University, Dalian 116027, China.
authors.forename: Liming
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
keywords: Multiple protease digestion; N-glycoproteome; N-glycosite; Quantitative analysis
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