Glycosylation as means of reducing sample complexity to enable quantitative proteomics

Abstract

Quantitative proteomics is a rapidly expanding field, in particular, the application to clinical biomarker studies for diagnosis or prognosis of diseases, and the systematic analysis of protein functions in biological systems. Isolation of a class of peptides or a subproteome enables reduction of sample complexity, which is essential to perform sensitive, quantitative analyses over a wider dynamic range of protein concentrations. Glycosylation is one of the most frequent PTMs, and glycans have unique chemical properties that can be leveraged to selectively enrich for a subset of peptides, and thus facilitate the downstream analysis. The isolation of glycopeptides and its benefits for mass spectrometric measurements is discussed. Keywords:Affinity chromatography / Glycoproteins / Mass spectrometry / Quantitative analysis 1488 Proteomics 2009Proteomics , 9, 1488Proteomics -1491 Glycosylation is one of the most frequent post/cotranslational modifications of cell surface and cytoplasmic proteins. Two main forms are commonly encountered: N-linked glycans attached to asparagine residues part of the consensus sequence N-X-[S/T], and O-linked oligosaccharides bound to serine or threonine residues. The structures of the oligosaccharides are complex and very diverse [1], and the fact that several different glycan structures (glycoforms) are attached to each individual glycosylation site results in a tremendous heterogeneity. Thus the full characterization of glycoproteins remains a formidable task. N-linked oligosaccharides are typically constituted of eight to twenty monosaccharides (most commonly N-acetyl-glucosamine, mannose, galactose, fucose, and N-acetyl-neuraminic acid) and their structures are strictly controlled by well-established biosynthesis rules. In contrast, O-glycans have much more diverse structures as their biosynthesis is not template driven.A broad structural diversity originates from isomeric monosaccharide constituents, branching, isomeric interglycosidic linkages, and anomeric configuration, which represents a true analytical challenge. An extensive characterization of glycoproteins requires determination of: (i) the level of glycosylation (i.e., site occupancy), (ii) the distribution of the different glycoforms present on each site, and (iii) the structure of each oligosaccharide moiety.Changes in the glycosylation profiles and alteration of the glycan structures have been reported to be associated with several diseases, including inflammation [2] and cancer [3]. However, to date the methodology to characterize systematically glycoproteins in the proteomics context remains in its infancy. Most studies have focused on either on the characterization of the released glycans as pools (typically Nglycan and O-glycan pools), or the glycopeptides obtained from purified individual glycoproteins.Nonetheless, the glycan portions represent ideal targets for a selective isolation of a subset of peptides required for proteomics analyses. This is achieved by leveraging the unique structural characteristics of...

Sialic Acid-focused Quantitative Mouse Serum Glycoproteomics by Multiple Reaction Monitoring Assay

“…However, it has been shown that in silico calculation indicates that on average 1/20 of the tryptic peptides have the N-X-S/T sequence known as the N-glycosylation consensus sequence (49). Considering the microheterogeneity of glycoforms in the redundant N-glycosylation sites, we believe that the glycoform-focused reverse glycoblotting technique greatly contributes to reducing the complexity of biological samples and accelerates discovery research of potential biomarkers on the basis of multiplex MRM MS (50,51).…”

Using lectins to harvest the plasma/serum glycoproteome

“…There is, therefore, general agreement that to detect glycoproteins, some with potential as disease biomarkers, effective sample fractionation or enrichment strategies are required. This can be achieved by first subdividing the proteome into smaller, less complex fractions, or selective enrichment of the glycoproteome subset by leveraging the unique properties of their glycans [], followed by separate analysis of individual fractions to achieve a more in‐depth coverage of the sample proteome. The suitability of the chosen technology or combination of technologies depends primarily on the biological questions to be answered and the need to enhance coverage of the targeted glycoproteome to ensure maximum interpretation and downstream utility of the data.…”

Perspectives of targeted mass spectrometry for protein biomarker verification

“…Another strategy, which has been applied in combination with SRM for the quantification of low abundance proteins in plasma, focuses on the N -glycosite subproteome [31,32]. This approach is based on the fact that most proteins present at the cell surface or secreted from tissue are glycosylated and are therefore likely to be detected in plasma.…”