Tandem Mass Spectrometry for Structural Characterization of Glycopeptides

Overview of Glycopeptide Structure Analysis

Glycosylation is a post-translational modification of protein (peptide) molecules that occurs within organisms, typically in the Golgi apparatus and endoplasmic reticulum. Due to the diversity of glycosylation sites and glycan structures, glycan moieties have various effects on the modified protein (peptide) molecules, including manipulation of protein folding, protection of enzyme cleavage regions, and direct interaction with carbohydrate recognition receptors such as lectins. As glycosylated proteins are responsive to changes in steady states, they have become important target substances in biomarker research based on genomic studies. Research on glycoproteins extracted from animal and plant tissues or on glycopeptides derived from their hydrolysis has also become increasingly extensive in terms of functionality and biological activity. For example, by extracting functional substances from food raw materials and studying their effective bioactive components while analyzing structure-activity relationships, they can be applied in various fields such as food, biomedicine, and cosmetics, thereby increasing the added value of food raw material processing. In-depth studies of the structures of extensively existing glycosylated proteins imply the need for analytical tools with high sensitivity and high throughput to characterize the structural features of protein portions and glycan moieties in detail.

Main classes of glycoconjugates of the human cellular glycome (Schjoldager et al., 2020).

Glycosylation of proteins (peptides) is involved in immune defense, cell adhesion, cell recognition, and pathogen binding. It is a prevalent protein co-modification and post-translational modification within organisms. In polysaccharide modification of proteins within organisms, there are certain patterns. The most common linkage is through an N-glycosidic bond to asparagine, or through an O-glycosidic bond to serine or threonine residues of the protein peptide backbone, forming O-glycan structures that are not constrained by fixed peptide sequences, thus exhibiting diverse biological activities. Protein side chains can also contain C-glycans or S-glycans. The diversity of polysaccharides is caused by the different numbers, types, and linkage patterns of monosaccharide units composing them, which in turn leads to the diversity of protein glycosylation forms and the property diversity of glycoproteins, such as macroheterogeneity with or without site-specific binding, and microheterogeneity caused by polysaccharide structures. It is the detailed features of these structural characteristics that affect the biological activity of glycoproteins (peptides).

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Tandem Mass Spectrometry Fragmentation Techniques

In the process of studying the activity of glycoproteins (glycopeptides), structural identification is crucial. Mass spectrometry (MS), due to its fast analysis speed and high sensitivity, is a mature analytical method suitable for glycoprotein research in the field of proteomics.

Various fragmentation strategies applicable to mass spectrometry have been reported, including collision-induced dissociation (CID), higher-energy collision-induced dissociation (HCD), electron capture dissociation (ECD), electron transfer dissociation (ETD), and various forms of photodissociation (infrared photodissociation, IRPD; ultraviolet photodissociation, UVPD).

CID/HCD preferentially cleave glycosidic bonds (forming B^- and Y^- ions, providing information about the glycan composition), and depending on the collision energy, they may also generate cross-ring fragments (A^- and X^- ions, providing potential information about glycan structures). Electron-driven dissociation, on the other hand, primarily cleaves peptide bonds (e.g., forming c^- and z^- ions, providing sequence information of the peptide). Due to the differential sensitivity of glycan and peptide moieties to different dissociation methods, the mass spectrometry workflow for glycoproteomics involves alternating fragmentation using CID and ETD, followed by analysis of the fragment ions using LC-MS to obtain comprehensive information about glycopeptides.

In recent years, there has been a trend towards combining multiple fragmentation methods, known as hybrid fragmentation approaches. This method tends to generate more ion fragments and ion types, thereby providing more structural information about the analytes. For example, in the field of phosphoproteomics, a commonly used approach is to first use ETD and then HCD (referred to as electron transfer higher-energy collision dissociation, EThcD), which generates many simple ion fragments that not only provide peptide sequence information but also specific phosphorylation sites.

Hybrid fragmentation approaches include energy-resolved fragmentation techniques (energy-resolved CID and HCD), sequential ion triggering fragmentation techniques (e.g., CID-pd-ETD, HCD-pd-ETD), and the combination of multiple fragmentation techniques with precursor ion selection (e.g., EThcD and electron transfer/collision-induced dissociation, ETciD).

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(A) Overview of analysis by Tandem Mass Spectrometry. (B)Overview of the different modes of data collection in tandem mass spectrometry.

Application of Tandem Mass Spectrometry in Oligosaccharide Structural Characterization

Oligosaccharides play important roles in many biological processes. The structure of oligosaccharides differs from other polymers like peptides due to the abundance of linkage combinations and branching. This complexity makes the analysis of oligosaccharides uniquely challenging compared to peptides. Mass spectrometry provides a powerful method for determining the composition of oligosaccharides, and tandem mass spectrometry offers high sensitivity for obtaining structural information.

Oligosaccharides undergo two major types of fragmentation. Glycosidic bond cleavage occurs between two adjacent sugar rings and provides information about sequence and branching. Cross-ring cleavage involves the breaking of any two bonds within a sugar ring, although glycosidic bond cleavage is more common. They can provide valuable information about linkage patterns and branching.

Glycosidic bond cleavage ions are the major products obtained from low-energy methods, but cross-ring cleavage is often observed in high-energy methods. Factors such as charge carriers (e.g., H*, Na*), charge states, glycan types (O-linked vs N-linked), reaction energy, and ion lifetimes prior to detection can influence the extent of oligosaccharide fragmentation and the ratio between glycosidic and cross-ring cleavages.

Mannose and sialic acid residues are structurally distinct, yet under CID conditions, they can generate similar unstable characteristic structures. Both residues are prone to dissociation and loss of structural information, particularly in positive mode. Even during ionization, sialic acid is highly unstable, especially in MALDI methods. Loss of sialic acid structures frequently occurs unless the chemical properties of the molecule are stabilized. When acids are esterified, their dissociation behavior is not significantly different from that of neutral oligosaccharide residues. Although there is no specific mechanism study on sialic acid dissociation, its cleavage is consistent with acid proton-induced dissociation. The relative proximity of the carboxylic acid promotes proton migration towards the glycosidic bond, and converting the carboxylic acid into an ester removes the proton and stabilizes the residue. Esterification of sialic acid significantly reduces the loss of sialic acid during the ionization process and enhances the signal of sialylated oligosaccharides in positive mode.

Mannose, being a neutral oligosaccharide, exhibits even more unstable properties during dissociation. Fragments of mannose are among the first ions generated and are frequently observed even during the ionization process. The key difference between mannose and other neutral residues lies in the loss of the hydroxyl group at C6, which plays an important role in the structural identification of sugars. The presence or absence of this fragment as an internal residue can be used to speculate whether the C6 position is a stable terminus and whether there are significant interactions between the C6 hydroxyl and another hydroxyl group. Currently, there is no simple method similar to esterification for stabilizing mannose residues in oligosaccharides.

References

Schjoldager, Katrine T., et al. "Global view of human protein glycosylation pathways and functions." Nature reviews Molecular cell biology 21.12 (2020): 729-749.
Neagu, Anca-Narcisa, et al. "Applications of tandem mass spectrometry (MS/MS) in protein analysis for biomedical research." Molecules 27.8 (2022): 2411.

* For Research Use Only. Not for use in diagnostic procedures.

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