Protein glycosylation is a class of prevalent modification of proteins involved in multiple biological functions [1, 2, 3]. Protein glycosylation is a nontemplated process, orchestrated by numerous enzymes, such as glycosyltransferases and glycosidases. In cells, the coordination between glycosyltransferases, glycosidases, and glycan transporters regulate the glycosylation process and the function of glycoproteins, which affects biological processes [4,5], such as cellular development [6], host–pathogen interaction [7], and immunity (Figure 1a) [8]. This modification is highly complex because diverse glycans could covalently attach to various amino acids through different configurations. Two major types of protein glycosylation have been studied: N-glycosylation and O-glycosylation. N-glycosylation refers to glycans linked to the nitrogen of asparagine (Asn). N-glycosylation mainly includes oligomannose-type, complex-type, and hybrid-type glycans sharing the common pentasaccharide core structure (Figure 1b). l-fucose, especially when attached to the innermost N-acetylglucosamine (GlcNAc), results in the core-fucose N-linked glycans. There are mainly nine types of O-glycosylation in vertebrates, which refers to glycans bound to oxygen of different amino acids, including serine/threonine (Ser/Thr), tyrosine, hydroxylysine, and hydroxyproline [9]. O-GlcNAcylation is a famous modification referring to a single monosaccharide (GlcNAc) bound to Ser/Thr. Additionally, mucin-type O-glycosylation contains different core structures and versatile extended composition on N-acetylgalactosamine (GalNAc), linked to Ser/Thr on proteins (Figure 1b).

Aberrant protein glycosylation is associated with numerous diseases [2,3,10,11]. For example, tumor-associated glycans are novel clinical biomarkers for cancers and offer the promise of therapeutic intervention by targeting specific oligosaccharide antigens [12,13]. The detection and quantification of protein glycosylation is critical for unraveling the role of glycosylation under both physiological and pathophysiological conditions [5]. However, the complexity and heterogeneity of protein glycosylation make the study of this modification an analytical challenge. Lectin and antibodies are conventional methods for detecting protein glycosylation. However, generating antibodies against certain glycans can be challenging. Antibodies and lectins often rely on avidity for enrichment, but they are associated with drawbacks including low affinity and cross-reactivity [14]. Metabolic labeling strategy is a powerful tool for glycosylation analysis depending on the metabolic process to replace a specific building block of glycoproteins with modified sugar analogs [15,16]. While metabolic labeling is a relatively simple process, it may not accurately reflect endogenous levels of glycosylation due to competition with native substrates. The metabolic labeling method is not applicable for analyzing human samples but is suitable for analyzing global glycosylation that contains specific building blocks. Chemoenzymatic labeling strategy is an alternative method for glycoprotein analysis, in which the recombinant synthetase (e.g., glycosyltransferases) selectively transfers tagged donors (e.g., sugar nucleotide) to a specific glycan structure (acceptor) on proteins [16,17]. Chemoenzymatic approaches rely on the promiscuity of recombinant enzymes toward donors and their strict acceptor selectivity, which is suitable for profiling specific glycan structures. Then, the tagged sugar, introduced by metabolic or enzymatic methods, can be subsequently captured by detection reporters or probes through bioorthogonal reaction [18]. These potent tools enable the systematic labeling, imaging, enrichment and analysis of glycoproteins [17], as well as further glycoengineering to probe glycan binding proteins [19], construct antibodies on cell surfaces [20] and decipher cell–cell interactions [21]. This review will highlight recent chemoenzymatic labeling strategies used in enrichment and site-specific profiling for glycoproteomic analysis. Many excellent review papers have been published for general information on chemoenzymatic glycan labeling and proteomic analysis [15,22, 23, 24∗, 25].

Mass spectrometry (MS)-based glycoproteomics is a powerful approach for identifying glycoproteins and glycosites. Due to the low abundance of glycosylation, the selective enrichment process is essential for sensitive glycoproteomic analysis and benefits the in-depth study of complex samples [24]. Various enrichment methods have been developed to dramatically improve the coverage of glycoproteomics in different samples [22]. Enrichment methods by exploiting the noncovalent affinity interactions have been widely used for glycopeptide enrichment, such as hydrophilic interaction liquid chromatography (HILIC) [26], lectin/antibody beads, inactivated enzymes [27], porous graphitic carbon (PGC), immobilized metal affinity chromatography (IMAC) and so on [25]. Furthermore, reversible covalent interactions between chemical groups and glycan moieties allow efficient enrichment of glycopeptides for MS analysis, such as boronic acid derivatives [28] and hydrazide [29]. The tagged glycan-modified substrates from metabolic and chemoenzymatic labeling methods are reacted with biotinylated probes or reporters. Enrichment usually depends on a remarkably high affinity between biotin and streptavidin beads [30], then the enriched glycopeptides are subjected to cleavage by the appropriate reactive treatment with cleavable or enzymatic groups [31,32].