Enzymatic Tagging of Glycoproteins on the Cell Surface for Their Global and Site-Specific Analysis with Mass Spectrometry

Abstract

The cell surface is normally covered with sugars that are bound to lipids or proteins. Surface glycoproteins play critically important roles in many cellular events, including cell-cell communications, cell-matrix interactions, and response to environmental cues. Aberrant protein glycosylation on the cell surface is often a hallmark of human diseases such as cancer and infectious diseases. Global analysis of surface glycoproteins will result in a better understanding of glycoprotein functions and the molecular mechanisms of diseases and the discovery of surface glycoproteins as biomarkers and drug targets. Here, an enzyme is exploited to tag surface glycoproteins, generating a chemical handle for their selective enrichment prior to mass spectrometric (MS) analysis. The enzymatic reaction is very efficient, and the reaction conditions are mild, which are well-suited for surface glycoprotein tagging. For biologically triplicate experiments, on average 953 N-glycosylation sites on 393 surface glycoproteins per experiment were identified in MCF7 cells. Integrating chemical and enzymatic reactions with MS-based proteomics, the current method is highly effective to globally and site-specifically analyze glycoproteins only located on the cell surface. Considering the importance of surface glycoproteins, this method is expected to have extensive applications to advance glycoscience.

Figure 1

Principle of site-specific and comprehensive analysis of N-glycoproteins on the cell surface by integrating enzymatic and chemical reactions with MS-based proteomics.

Figure 2

(A) Tandem mass spectrum of CEAAN#GTEACVCGGAFVGPR (# represents the glycosylation site). (B) Comparison of N-glycosylation sites and (C) N-glycoproteins identified on the cell surface of MCF7 cells in biologically triplicate experiments.

Figure 3

Classification of cell surface N-glycoproteins identified in MCF7 cells (A) and clustering of cell surface N-glycoproteins identified in MCF7 cells based on biological process (B).

Figure 4

Identification of N-glycosylation sites (A) and N-glycoproteins (B) on the cell surface of Jurkat cells with and without the neuraminidase treatment.

Figure 5

Procedure of quantification experiment with BFA treatment (A) and quantification results of surface N-glycoproteins (B) and N-glycopeptides (C) in MCF7 cells with BFA treatment.