Mass Spectrometry-Based Chemical and Enzymatic Methods for Global Analysis of Protein Glycosylation

Abstract

Glycosylation is one of the most common protein modifications, and it is essential for mammalian cell survival. It often determines protein folding and trafficking, and regulates nearly every extracellular activity, including cell-cell communication and cell-matrix interactions. Aberrant protein glycosylation events are hallmarks of human diseases such as cancer and infectious diseases. Therefore, glycoproteins can serve as effective biomarkers for disease detection and targets for drug and vaccine development. Despite the importance of glycoproteins, global analysis of protein glycosylation (either glycoproteins or glycans) in complex biological samples has been a daunting task, and here we mainly focus on glycoprotein analysis using mass spectrometry (MS)-based bottom-up proteomics. Although the emergence of MS-based proteomics has provided a great opportunity to analyze glycoproteins globally, the low abundance of many glycoproteins and the heterogeneity of glycans dramatically increase the technical difficulties. In order to overcome these obstacles, considerable progress has been made in recent years, which has contributed to comprehensive analysis of glycoproteins. In our lab, we developed effective MS-based chemical and enzymatic methods to (1) globally analyze glycoproteins in complex biological samples, (2) target glycoproteins specifically on the surface of human cells, (3) systematically quantify glycoprotein and surface glycoprotein dynamics (the abundance changes of glycoproteins as a function of time), and (4) selectively characterize glycoproteins with a particular and important glycan. In this Account, we first briefly describe the glycopeptide/protein enrichment methods in the literature and then discuss the developments of boronic acid-based methods to enrich glycopeptides for large-scale analysis of protein glycosylation. Boronic acids can form reversible covalent interactions with sugars, but the low binding affinity of normal boronic acid-based methods prevents us from capturing glycoproteins with low abundance, which often contain more valuable information. We enhanced the boronic acid-glycan interactions by using a boronic acid derivative (benzoboroxole) and conjugating it onto a dendrimer to allow synergistic interactions between the boronic acid derivative and sugars. The new method is capable of globally analyzing protein glycosylation with site and glycan structure information, especially for those with low abundance. In the next part, we discuss the combination of metabolic labeling, click chemistry and enzymatic reactions, and MS-based proteomics as a very powerful approach for surface glycoproteome analysis in human cells. The methods enable us to specifically identify surface glycoproteins and to quantify their abundance changes and dynamics together with quantitative proteomics. The last section of this Account focuses on chemical and enzymatic methods to study glycoproteins containing a particular and important glycan (the Tn antigen, i.e., O-GalNAc). Although not comprehensive, this Account provides an overview of chemical and enzymatic methods to characterize protein glycosylation in combination with MS-based proteomics. These methods will have extensive applications in the fields of biology and biomedicine, which will lead to a better understanding of glycoprotein functions and the molecular mechanisms of diseases. Eventually, glycoproteins will be identified as effective biomarkers for disease detection and drug targets for disease treatment.

Figure 1.

Principle of the boronic acid-based chemical enrichment method for comprehensive analysis of protein N-glycosylation. Reproduced with permission from ref . Copyright 2014 American Society for Biochemistry and Molecular Biology.

Figure 2.

(a) Structures of boronic acid derivatives tested for glycoproteomic analysis. (b) Numbers of unique N-glycopeptides identified with each derivative from the parallel experiments. (c) Structure of the dendrimer conjugated with benzoboroxole. (d) Example of the synergistic interactions between multiple benzoboroxole molecules in a dendrimer and several sugars within one glycan. (e) Effect of the number of synthesis cycles and corresponding dendrimer size on the enrichment of glycopeptides. (f) Effect of reaction time on identification of N-glycopeptides. Adapted with permission from ref . Copyright 2018 Nature.

Figure 3.

Comprehensive analysis of protein N-glycosylation in human cells using the DBA method. (a) Comparison of unique N-glycosylation sites identified in MCF7 cells in biologically duplicate experiments. (b) Comparison of glycosylation sites and glycoproteins identified with BA and DBA beads. (c) Abundance distributions of N-glycoproteins identified with the BA and DBA beads. (d) Overlap of N-glycoproteins in three types of cells. (e) Clustering results for 180 N-glycoproteins exclusively identified in Jurkat cells. (f) Distribution of membrane proteins among identified N-glycoproteins. Reproduced with permission from ref . Copyright 2018 Nature.

Figure 4.

Principle of the chemical deglycosylation to break the glycosidic bonds but not the amide bond in an N-glycopeptide. (b) After the chemical deglycosylation, the innermost GlcNAc serves as a common tag for MS analysis. (c, d) Comparisons of the (c) glycosylation sites and (d) glycoproteins identified in yeast using the chemical deglycosylation and Endo H methods. Reproduced from ref . Copyright 2014 American Chemical Society.

Figure 5.

(a) Principle of the site-specific identification of the surface N-sialoglycoproteome. (b−j) Microscope images of tagging sialoglycoprotein on the HEK 293T cell surface: (b, e, h) images of cells (scale bar is 20 μm); (c, f, i) fluorescence signals of labeled cells reacted with DBCO−Fluor545; (d) fluorescence signals of labeled cells bound to DBCO−sulfo−biotin, followed by streptavidin−FITC; (g) cells without the biotin tag treated with streptavidin−FITC show no green signal; (j) after surface sialoglycoproteins were tagged with DBCO−Fluor545, cells were further treated with DBCO−sulfo−biotin followed by streptavidin−FITC, but no green signals were detected. (k) Gel results for metabolic labeling and click chemistry. The control sample was from unlabeled cells. Reproduced with permission from ref 48. Copyright 2015 Royal Society of Chemistry.

Figure 6.

(a) Analysis of sialoglycoproteins in the secretome and whole-cell lysate. (b) Molecular function analysis of surface N-sialoglycoproteins. (c) Site locations of types I and II N-sialoglycoproteins based on the transmembrane domain. Reproduced with permission from ref . Copyright 2015 Royal Society of Chemistry.

Figure 7.

Experimental procedure for studying the dynamics of surface glycoproteins and measuring their half-lives. Reproduced with permission from ref . Copyright 2017 Royal Society of Chemistry.

Figure 8.

(a) Example MSdata showing peptide identification and quantification. (b) Distribution of the half-lives of surface glycoproteins. (c) Median half-lives of glycoproteins with different molecular functions. Reproduced with permission from ref . Copyright 2017 Royal Society of Chemistry.

Figure 9.

(a) Principle of enrichment of glycopeptides with the Tn antigen. (b) Comparison of experimental results from three biologically independent experiments. (c) Overlap of glycoproteins identified from the three experiments. Adapted with permission from ref . Copyright 2017 John Wiley and Sons.