Mass spectrometry-based structure-specific N-glycoproteomics and biomedical applications

Abstract

N-linked glycosylation is a common posttranslational modification of proteins that results in macroheterogeneity of the modification site. However, unlike simpler modifications, N-glycosylation introduces an additional layer of complexity with tens of thousands of possible structures arising from various dimensions, including different monosaccharide compositions, sequence structures, linking structures, isomerism, and three-dimensional conformations. This results in additional microheterogeneity of the modification site of N-glycosylation, i.e., the same N-glycosylation site can be modified with different glycans with a certain stoichiometric ratio. N-glycosylation regulates the structure and function of N-glycoproteins in a site- and structure-specific manner, and differential expression of N-glycosylation under disease conditions needs to be characterized through site- and structure-specific quantitative analysis. Numerous advanced methods ranging from sample preparation to mass spectrum analysis have been developed to distinguish N-glycan structures. Chemical derivatization of monosaccharides, online liquid chromatography separation and ion mobility spectrometry enable the physical differentiation of samples. Tandem mass spectrometry further analyzes the macro/microheterogeneity of intact N-glycopeptides through the analysis of fragment ions. Moreover, the development of search engines and AI-based software has enhanced our understanding of the dissociation patterns of intact N-glycopeptides and the clinical significance of differentially expressed intact N-glycopeptides. With the help of these modern methods, structure-specific N-glycoproteomics has become an important tool with extensive applications in the biomedical field.

1. Introduction

N-linked glycosylation, referred to as N-glycosylation, is a common posttranslational modification of proteins. For the glycosylation site, N-glycosylation occurs at the motif of N-X-S/T/C (X≠P), and approximately 80% of human proteins contain at least one such motif, which is called a putative N-glycoprotein [1,2]. A protein may contain more than one motif in its sequence, and an N-glycoprotein can be glycosylated by one or more N-glycans [3,4]. This macroheterogeneity in the N-glycosylation site parallels that of other small molecules, such as methylation, acetylation, and phosphorylation. In terms of the modification structure, unlike other small-molecule modifications, which involve only a single structure, N-glycosylation involves tens of thousands of structures, including monosaccharide compositions, sequence structures, linkage structures, positional isomers, stereo-conformations and other structural dimensions [5–8]. Consequently, N-glycosylation exhibits microheterogeneity, as one glycosite is commonly modified with different N-glycan structures at specific stoichiometric ratios [9,10].

N-glycosylation regulates the structure and function of N-glycoproteins in a site- and structure-specific manner [11,12]. For example, glycosylation at N71 on mutated programmed cell death protein 1 (PD-1) can decrease the suppression of chimeric antigen receptor T cells (CAR-T cells) to enhance the cytotoxicity and efficacy of immunotherapy [13]. N-glycosylation of N57 on PD-1 is necessary for its binding to PD-L1 [14]. AFP-L3, a glycosylation variant of alpha fetoprotein (AFP) with a specific core-fucosylated N-glycan structure (YY(F)M(MYLS)MYLS, where Y represents N-acetylglucosamine, M represents mannose, L represents galactose, F represents fucose and S represents sialic acid), exhibits greater sensitivity than AFP in the early diagnosis of hepatocellular carcinoma (HCC) [15]. The terminal fucose of N-glycan is highly involved in various biological processes, such as virus infection [16], the gut microbiome [17], and intestinal commensal bacteria [18]. Furthermore, terminal sialic acid is strongly associated with anti-inflammatory effects [19], allergies [20], and the gut microbiome [21]. The immunoglobulin G (IgG) modified with α2,6 sialic acid in the Fc fragment is an anti-inflammatory agent for arthritis treatment, whereas its α2,3 sialic acid is not. N-glycosylation under pathological conditions was characterized by site- and structure-specific quantitative analysis.

With the rapid development and widespread application of material preparation, highly efficient liquid chromatography separation, high-throughput tandem mass spectrometry analysis, intelligent bioinformatics and other technologies, omics technology based on mass spectrometry has been widely applied for qualitative and quantitative analysis of site- and structure-specific protein N-glycosylation under both normal physiological and abnormal pathological conditions.