title: An integrative strategy for quantitative analysis of the N-glycoproteome in complex biological samples.
abstract: BACKGROUND
  The complexity of protein glycosylation makes it difficult to characterize glycosylation patterns on a proteomic scale. In this study, we developed an integrated strategy for comparatively analyzing N-glycosylation/glycoproteins quantitatively from complex biological samples in a high-throughput manner. This strategy entailed separating and enriching glycopeptides/glycoproteins using lectin affinity chromatography, and then tandem labeling them with 18O/16O to generate a mass shift of 6 Da between the paired glycopeptides, and finally analyzing them with liquid chromatography-mass spectrometry (LC-MS) and the automatic quantitative method we developed based on Mascot Distiller.
  
  
  RESULTS
  The accuracy and repeatability of this strategy were first verified using standard glycoproteins; linearity was maintained within a range of 1:10-10:1. The peptide concentration ratios obtained by the self-build quantitative method were similar to both the manually calculated and theoretical values, with a standard deviation (SD) of 0.023-0.186 for glycopeptides. The feasibility of the strategy was further confirmed with serum from hepatocellular carcinoma (HCC) patients and healthy individuals; the expression of 44 glycopeptides and 30 glycoproteins were significantly different between HCC patient and control serum.
  
  
  CONCLUSIONS
  This strategy is accurate, repeatable, and efficient, and may be a useful tool for identification of disease-related N-glycosylation/glycoprotein changes.
authors.forename: Ji
authors.initials: J
authors.lastname: Wang
authors.forename: Chuang
authors.initials: C
authors.lastname: Zhou
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Jun
authors.initials: J
authors.lastname: Yao
authors.forename: Haojie
authors.initials: H
authors.lastname: Lu
authors.forename: Qiongzhu
authors.initials: Q
authors.lastname: Dong
authors.affiliation: Department of Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China. zhou1997@gmail.com.
authors.forename: Haijun
authors.initials: H
authors.lastname: Zhou
authors.forename: Lunxiu
authors.initials: L
authors.lastname: Qin