Stationary phases for the enrichment of glycoproteins and glycopeptides

Abstract

The analysis of protein glycosylation is important for biomedical and biopharmaceutical research. Recent advances in LC-MS analysis have enabled the identification of glycosylation sites, the characterisation of glycan structures and the identification and quantification of glycoproteins and glycopeptides. However, this type of analysis remains challenging due to the low abundance of glycopeptides in complex protein digests, the microheterogeneity at glycosylation sites, ion suppression effects and the competition for ionisation by co-eluting peptides. Specific sample preparation is necessary for comprehensive and site-specific glycosylation analyses using MS. Therefore, researchers continue to pursue new columns to broaden their applications. The current manuscript covers recent literature published from 2008 to 2013. The stationary phases containing various chemical bonding methods or ligands immobilisation strategies on solid supports that selectively enrich N-linked or sialylated N-glycopeptides are categorised with either physical or chemical modes of binding. These categories include lectin affinity, hydrophilic interactions, boronate affinity, titanium dioxide affinity, hydrazide chemistry and other separation techniques. This review should aid in better understanding the syntheses and physicochemical properties of each type of stationary phases for enriching glycoproteins and glycopeptides.

Monitoring vaccine protein glycosylation: analytics and recent developments

“…A number of methods can be used for this purpose including size exclusion chromatography, lectin affinity, hydrophilic interaction chromatography (HILIC) and porous graphite chromatography. These have been reviewed elsewhere [58,59]. HILIC enrichment is one of the most successful forms of glycopeptide enrichment and has been shown to enrich glycopeptide signal up to 240-fold with little glycopeptide loss or bias [60] as shown in Figure 3.…”

Systems biology—opportunities and challenges: the application of proteomics to study the cardiovascular extracellular matrix

“…Glycopeptides are usually enriched using lectins, hydrophilic interaction LC, hydrazide or graphite. The method of choice will determine the type of glycopeptides that will ultimately be enriched 90 , 91 . After enrichment, PNGase-F is used to enzymatically remove the glycan moiety from asparagine residues, serving two purposes: Firstly, the core peptide can be analysed without interference from sugars during MS/MS and secondly, PNGase-F via a deamidation reaction converts the asparagine to aspartic acid.…”

“…For such analysis, proteins in the sample are first digested into peptides, followed by glycopeptide enrichment using zwitterionic hydrophilic interaction LC (ZIC-HILIC) 93 or alternative approaches 91 , 94 . Recently, the combination of higher energy collision dissociation (HCD) and electron transfer dissociation (ETD) have facilitated direct MS analysis of glycopeptides.…”

Synthesis and evaluation of carboxymethyl chitosan for glycopeptide enrichment

“…Alternatively, hydrophilic interaction liquid chromatography (HILIC) approaches have demonstrated simple, fast and efficient procedures over reversed phase approaches for glycopeptide enrichment [16,17]. While these methods are based on solid phase extraction (SPE), it is worthwhile noting their compatibility towards on-line analysis with techniques such as electrospray, where mobile phases used in HILIC are often advantageous due to the higher percentage of organic solvents which are desolvated efficiently [18]. In light of this, much emphasis has been placed around designing niche 'glyco-modified' columns which can be used under HILIC conditions including 'click saccharides' onto polymer beads [19], silica beads [20,21], and magnetic nanoparticles [22].…”