Background
Dysregulation of glycoproteins is closely related with many diseases. Quantitative proteomics
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.
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.