Robert Šardzík

Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing

Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides. Identification of glycosylation patterns is complicated by the lack of sensitive analytical techniques that can distinguish between epimeric carbohydrates. It has now been shown that ion-mobility tandem mass spectrometry of ions derived from glycopeptides and oligosaccharides enables glycan stereochemistry to be determined, highlighting the potential of this technique for sequencing complex carbohydrates on cell surfaces.

Preparation of aminoethyl glycosides for glycoconjugation

Summary The synthesis of a number of aminoethyl glycosides of cell-surface carbohydrates, which are important intermediates for glycoarray synthesis, is described. A set of protocols was developed which provide these intermediates, in a short number of steps, from commercially available starting materials.

Chemoenzymatic synthesis of sialooligosaccharides on arrays for studies of cell surface adhesion

Sialooligosaccharides were generated by direct enzymatic glycosylation on arrays and the resulting surfaces were suitable for the study of carbohydrate-specific cell adhesion.

Enzymatic Glycosylations on Arrays

The enzymatic glycosylation of microarrays is a relatively young field in glycoscience. Platforms developed from other array technologies (e.g., proteins and nucleic acids) were successfully adopted in several proof-of-principle studies as a high-throughput tool for the generation of more complex carbohydrate structures using carbohydrate-processing enzymes. These arrays and the developed on-chip enzymatic glycosylation methodologies are reviewed in this article.

Dual purpose S-trityl-linkers for glycoarray fabrication on both polystyrene and gold

There is a wide range of immobilisation reactions to tether substrates to a variety of surfaces for array-based analysis. Most of these immobilisation strategies are specific for a particular surface and require an additional linker to be attached to the substrate or the surface. Furthermore, the analysis of functionalised surfaces is often restricted to certain analytical techniques and therefore, different immobilisation strategies for different surfaces are desirable. Here we have tested an S-tritylated linker for non-covalent or covalent immobilisation of mannosides to polystyrene or gold surfaces. S-Tritylated mannosides with varying linkers were readily synthesised and used to add to biorepulsive maleimide-terminated preformed SAMs after in situ deprotection of the S-trityl group. In addition, S-tritylated mannosides themselves formed stable glycoarrays on polystyrene microtiter plates. The glycoarrays were successfully analysed by MALDI-ToF mass spectrometry, SPR spectroscopy, and interrogated with GFP-transfected Escherichia coli cells. This work has shown that a dual purpose linker can be used on multiple surfaces to form arrays allowing for different testing as well as analytical approaches.

Formation of carbohydrate-functionalised polystyrene and glass slides and their analysis by MALDI-TOF MS.

Summary Glycans functionalised with hydrophobic trityl groups were synthesised and adsorbed onto polystyrene and glass slides in an array format. The adsorbed glycans could be analysed directly on these minimally conducting surfaces by MALDI-TOF mass spectrometry analysis after aluminium tape was attached to the underside of the slides. Furthermore, the trityl group appeared to act as an internal matrix and no additional matrix was necessary for the MS analysis. Thus, trityl groups can be used as simple hydrophobic, noncovalently linked anchors for ligands on surfaces and at the same time facilitate the in situ mass spectrometric analysis of such ligands.

Label-Free Discovery Array Platform for the Characterization of Glycan Binding Proteins and Glycoproteins

The identification of carbohydrate-protein interactions is central to our understanding of the roles of cell-surface carbohydrates (the glycocalyx), fundamental for cell-recognition events. Therefore, there is a need for fast high-throughput biochemical tools to capture the complexity of these biological interactions. Here, we describe a rapid method for qualitative label-free detection of carbohydrate-protein interactions on arrays of simple synthetic glycans, more complex natural glycosaminoglycans (GAG), and lectins/carbohydrate binding proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The platform can unequivocally identify proteins that are captured from either purified or complex sample mixtures, including biofluids. Identification of proteins bound to the functionalized array is achieved by analyzing either the intact protein mass or, after on-chip proteolytic digestion, the peptide mass fingerprint and/or tandem mass spectrometry of selected peptides, which can yield highly diagnostic sequence information. The platform described here should be a valuable addition to the limited analytical toolbox that is currently available for glycomics.

Post-translational modifications: S-linked sugars lost and found.

The vast majority of core structures of protein and peptide glycosylation motifs belong to either O-linked or N-linked glycans. A recent publication describes the structure and biosynthesis of an unusual S-linked glycan linkage in the antibacterial glycopeptide sublancin.