Hydrophilic Affinity Isolation and MALDI Multiple-Stage Tandem Mass Spectrometry of Glycopeptides for Glycoproteomics

Abstract

In glycoproteomics, key structural issues, protein identification, locations of glycosylation sites, and evaluation of the glycosylation site microheterogeneity should be easily evaluated in a large number of glycoproteins, while mass spectrometry (MS) provides substantial information about individual purified glycoproteins. Considering that structural issues are elucidated by studying glycopeptides and that the tandem MS of a tryptic peptide composed of several amino acid residues is enough for protein identification, construction of an MS-based method handling tryptic glycopeptides would be of considerable benefit in research. To this end, a simple and efficient method, utilizing hydrophilic binding of carbohydrate matrixes such as cellulose and Sepharose to oligosaccharides, was successfully applied to the isolation of tryptic glycopeptides. Both peptide and oligosaccharide structures were elucidated by multiple-stage tandem MS (MS(n)) of the ions generated by matrix-assisted laser desorption/ionization (MALDI), as follows. The MALDI ion trap mass spectrum of a tryptic glycopeptide mixture from N-linked glycoproteins was composed of the [M + H]+ ions of component glycopeptides. Collision-induced dissociation (CID) of the glycopeptide [M + H]+ ion generated saccharide-spaced peaks, with an interval of, for example, 146, 162, and 203 Da, and their fragment ions corresponding to the peptide and peptide + N-acetylglucosamine (GlcNAc) species in the MS2 spectrum. The saccharide-spaced ladder served to outline oligosaccharide structures, which were then selected as precursors for subsequent MS(n) analyses. The peptide or peptide + GlcNAc ions in the MS2 spectrum or the corresponding ions abundant in the MS1 spectrum were subjected to CID for determination of peptide sequences, to identify proteins and their glycosylation sites. The strategy, isolation of glycopeptides followed by MS(n) analysis, efficiently characterized the structures of beta2-glycoprotein I with four N-glycosylation sites and was applied to an analysis of total serum glycoproteins.

Recent advances in blood‐related proteomics

“…The identification of proteins per se is not sufficient to understand biological function because most proteins are post-translationally modified. The promise of phosphoproteomics [181], or of glycoproteomics [182], which enables the study of important physiological PTMs of proteins will revolutionize our understanding of the function of proteins. …”

Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry

“…Glycopeptide enrichment under normal-phase LC (NPLC) conditions has been demonstrated using various hydrophilic media and different capture and elution conditions (23)(24)(25)(26)(27)(28). NPLC allows either direct enrichment of peptides modified by various N-linked glycan structures using a ZICா-HILIC column (23)(24)(25)(26)(27) or targeting sialylated glycopeptides using a titanium dioxide micro-column (28).…”

“…NPLC allows either direct enrichment of peptides modified by various N-linked glycan structures using a ZICா-HILIC column (23)(24)(25)(26)(27) or targeting sialylated glycopeptides using a titanium dioxide micro-column (28). However, NPLC is neither effective for enriching less hydrophilic glycopeptides, e.g.…”

A glycomics platform for the analysis of permethylated oligosaccharide alditols

“…Some examples of these include normal phase (NPC) [22], reversedphase (RPC) [23,24], porous graphitized carbon (PGC) [25], hydrophilic interaction (HILIC) [26], ion-exchange (IEC) [27], and high pH anion exchange (HPAEC) [27] chromatographies. PGC, HILIC and HPAEC provide superior resolution of oligosaccharides and allow separation of some isomers.…”