An integrative strategy for quantitative analysis of the N-glycoproteome in complex biological samples

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. © 2014 wang et al.; licensee biomed central ltd.