Systematic and site-specific analysis of N-glycoproteins on the cell surface by integrating bioorthogonal chemistry and MS-based proteomics.

Abstract

Glycoproteins on the cell surface are essential for various cellular activities including cell-cell communication and cell-matrix interaction. Alterations of glycosylation are correlated with many diseases such as cancer and infectious diseases. However, it is greatly challenging to systematically and site-specially analyze glycoproteins only located on cell surface because of the heterogeneity of glycans, the low abundance of many surface glycoproteins and the requirement of effective methods to separate surface glycoproteins. In this chapter, we briefly review existing mass spectrometry (MS)-based methods for global analysis of surface glycoproteins. Then we discuss an effective method integrating metabolic labeling, click and enzymatic reactions, and MS-based proteomics to comprehensively and site-specifically investigate cell surface N-glycoproteins. A detailed protocol for this method is also included. In combination with quantitative proteomics, we applied this method to quantify cell surface N-glycoproteins and study the relationship between cell invasiveness and N-sialoglycoproteins on the cell surface. Considering the importance of surface glycoproteins, this method can be extensively applied to advance glycoscience, which leads to a better understanding of the molecular mechanisms of human diseases, and the discovery of surface glycoproteins as biomarkers for disease detection.