科研之友
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摘要
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 O-18/O-16 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.
