Advances in mass spectrometry-based glycoproteomics

Abstract

Protein glycosylation, an important PTM, plays an essential role in a wide range of biological processes such as immune response, intercellular signaling, inflammation, and host-pathogen interaction. Aberrant glycosylation has been correlated with various diseases. However, studying protein glycosylation remains challenging because of low abundance, microheterogeneities of glycosylation sites, and poor ionization efficiency of glycopeptides. Therefore, the development of sensitive and accurate approaches to characterize protein glycosylation is crucial. The identification and characterization of protein glycosylation by MS is referred to as the field of glycoproteomics. Methods such as enrichment, metabolic labeling, and derivatization of glycopeptides in conjunction with different MS techniques and bioinformatics tools, have been developed to achieve an unequivocal quantitative and qualitative characterization of glycoproteins. This review summarizes the recent developments in the field of glycoproteomics over the past 6 years (2012 to 2018).

Keywords: Derivatization; Enrichment; Glycoproteins; Glycosylation; Metabolic labeling.

Figure 1.

Workflow for recent developments on MS-based glycoproteomics.

Figure 2.

MALDI-TOF-MS analyses of sialylated glycopeptides by dimethylamine reaction under EDC and HOBt at 60° C for 3 hours. Reprinted with permission from [69].

Figure 3.

MSⁿ analyses of structure and linkage isomers of permethylated glycopeptides using LTQ Orbitrap Fusion Tribrid mass spectrometer. (A) MSⁿ identification of permethylated structural isomers among the high mannose glycoforms of RNase B. (B) MSⁿ identification of permethylated sialylated α 2,3 and α 2,6-linkage isomers of same N-glycopeptide from fetuin sample. α 2,3-linkage of O-glycan was also verified. Reprinted with permission from [77].

Figure 4.

UVPD of a doubly deprotonated O-linked glycopeptide anion from kappa-casein: (A) UVPD spectrum; (B) zoom-in of low m/z regions of spectrum shown in A; (C) Zoom-in of high m/z range of UVPD spectrum shown in A. Reprinted with permission from [111].

Figure 5.

Electron transfer MS analysis coupled with Ion mobility and vibrational activation (ET-IM-VA) of the N-glycopeptide derived from coral tree lectin. The IM-MS heat map is shown in (A). The CID spectrum is given in (B), while the ETD spectrum extracted is given in (C). Experimental sequence is summarized in insets in A-C. Fragmentation behaviors of the glycopeptide in ETD and CID are depicted in (D). Reprinted with permission from [106].

Figure 6.

(A) The principle of the site-specific identification of the cell surface N sialoglycoproteome by integrating metabolic labeling, copper-free click chemistry and MS-based proteomics techniques. (B) Experimental procedure of cell surface N-sialoglycoproteome site-specific identification on breast cancer cell line MDA-MB-231 and MCF-7. (C) Comparison of cell surface N-sialoglycosylation sites between two cell lines. (D) Cell surface N-sialoglycoproteins identified in two cell lines. (E) Glycosylation site location of the type I and II N-sialoglycoproteins. Reprinted with permission from [151].