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.

Sialylation of lactosyl lipids in membrane microdomains by T. cruzi trans-sialidase.

A synthetic perfluoroalkyl-tagged lactosyl glycolipid has been shown to form lipid microdomains in fluid phospholipid bilayers. When embedded in the membranes of phospholipid vesicles, this glycolipid was trans-sialylated by soluble T. cruzi trans-sialidase (TcTS) to give a perfluoroalkyl-tagged glycolipid that displayed the ganglioside GM3 epitope, with up to 35% trans-sialylation from fetuin after 18 h. Following sialylation, vesicles bearing this Neu5Ac(α2-3)Gal(ß1-4)Glc sequence in their "glycocalyx" were recognised and agglomerated by the lectin M. amurensis leukoagglutinin. Monitoring TcTS-mediated trans-sialylation by HPLC over the first 6 h revealed that enzymatic transformation of bilayer-embedded substrate was much slower than that of a soluble lactosyl substrate. Furthermore, clustering of the lactose-capped glycolipid into "acceptor" microdomains did not increase the rate of sialic acid transfer from fetuin by soluble TcTS, instead producing slight inhibition.

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.

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.

Accelerated enzymatic galactosylation of N-acetylglucosaminolipids in lipid microdomains.

A fluoro-tagged N-acetylglucosamine-capped glycolipid that can form lipid microdomains in fluid phospholipid bilayers has been shown to be enzymatically galactosylated by bovine ß(1,4)-galactosyltransferase. MALDI MS, HPLC, and LC-MS revealed that the rate of enzymatic transformation was significantly enhanced by lipid clustering; at a 1% mol/mol loading, clustered glycolipids were galactosylated 9-fold faster than glycolipids dispersed across the bilayer surface. The transformation of the GlcNAc "glycocalyx" into a Gal(ß1-4)GlcNAc "glycocalyx" relabeled these vesicles, making them susceptible to agglutination by Erythrina cristagalli lectin (ECL). The kinetic parameters for this transformation revealed a lower apparent Km when the substrate lipids were clustered, which is attributed to multivalent binding to an extended substrate cleft around the active site. These observations may have important implications where soluble enzymes act on substrates embedded within cellular lipid rafts.

Chemoenzymatic synthesis of O-mannosylpeptides in solution and on solid phase.

O-mannosyl glycans are known to play an important role in regulating the function of α-dystroglycan (α-DG), as defective glycosylation is associated with various phenotypes of congenital muscular dystrophy. Despite the well-established biological significance of these glycans, questions regarding their precise molecular function remain unanswered. Further biological investigation will require synthetic methods for the generation of pure samples of homogeneous glycopeptides with diverse sequences. Here we describe the first total syntheses of glycopeptides containing the tetrasaccharide NeuNAcα2-3Galß1-4GlcNAcß1-2Manα, which is reported to be the most abundant O-mannosyl glycan on α-DG. Our approach is based on biomimetic stepwise assembly from the reducing end and also gives access to the naturally occurring mono-, di-, and trisaccharide substructures. In addition to the total synthesis, we have developed a "one-pot" enzymatic cascade leading to the rapid synthesis of the target tetrasaccharide. Finally, solid-phase synthesis of the desired glycopeptides directly on a gold microarray platform is described.

Oxo-ester mediated native chemical ligation on microarrays: an efficient and chemoselective coupling methodology.

We report a highly efficient and selective method for the coupling of peptides and glycoconjugates bearing N-terminal cysteines to activated surfaces. This chemoselective method generates stable amide linkages without using any thiol additives.

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.

Preparation of aminoethyl glycosides for glycoconjugation.

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.

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.