Josef Voglmeir

Glycoarrays—tools for determining protein–carbohydrate interactions and glycoenzyme specificity

Carbohydrate arrays (glycoarrays) have recently emerged as a high-throughput tool for studying carbohydrate-binding proteins and carbohydrate-processing enzymes. A number of sophisticated array platforms that allow for qualitative and quantitative analysis of carbohydrate binding and modification on the array surface have been developed, including analysis by fluorescence spectroscopy, mass spectrometry and surface plasmon resonance spectroscopy. These platforms, together with examples of biologically-relevant applications are reviewed in this Feature Article.

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.

A versatile gold surface approach for fabrication and interrogation of glycoarrays

Glycoarrays on gold: A designer gold surface incorporating a self-assembled monolayer with weak protein absorption properties has been optimised for rapid display and interrogation of both native and derivatised glycans in array formats. This rapid, facile approach has diverse applications in glycomics, through exploitation of fluorescence, SPR and MALDI-ToF MS detection methods.

SPOT synthesis of peptide arrays on self-assembled monolayers and their evaluation as enzyme substrates.

amino acid to the other, and therefore the amount and purity of peptides can also vary. Monitoring reactions on the support relies on the use of a coloured dye, and quality control of the peptide requires its cleavage from the support after completion of synthesis for subsequent solution-phase analysis by HPLC or MALDI-ToFMS. [19] Consequently, assay results obtained from an array prepared by SPOT synthesis sometimes need to be confirmed by using purified samples of peptides in a solution-phase assay. [15] By combining the robustness and versatility of SAM-coated gold platforms and the easy-to-handle SPOT peptide synthesis methodology, an efficient method for preparing peptide arrays on gold surfaces is described herein. The method allows onchip monitoring of the synthesis and subsequent evaluation of enzymatic modifications by MALDI-ToFmass spectrometry (Figure 1).

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.

Green and efficient extraction of rutin from tartary buckwheat hull by using natural deep eutectic solvents

In this study, an efficient extraction technique using a combination of ultrasound and natural deep eutectic solvents (NADESs) was developed. Some basic physical properties, including viscosity, polarity, and solubility, of thirteen NADESs prepared from natural components were investigated systematically. Results show that the solubility of rutin increased in choline chloride- and glycerol-based NADESs by 660-1577times compared to water. NADESs with high rutin extractability can be designed by combining NADESs components. A maximum of 9.5mg/g rutin was extracted from tartary buckwheat hull with extraction efficiencies of 95%. NADESs can be recovered and recycled. In addition, the biocompatibility and biodegradability of the tested NADESs were also evaluated. The results demonstrated that these NADESs were excellent solvents with extremely low toxicities and favorable biodegradabilities. Our findings suggest that NADESs can be used as green solvents for the extraction of bioactive ingredients.

Comparative characterisation of recombinant invertebrate and vertebrate peptide O-Xylosyltransferases

Chondroitin and heparan sulphates have key functions in animal development and their synthesis is initiated by the action of UDP-α-D-xylose:proteoglycan core protein β-D-xylosyltransferase (EC 2.4.2.26). cDNAs encoding this enzyme have been previously cloned from mammalian species; this in turn facilitated identification of corresponding Caenorhabditiselegans (sqv-6) and Drosophilamelanogaster (oxt) genes. In the present study, we report the expression in Pichia pastoris and subsequent assay using either MALDI-TOF MS or RP-HPLC of recombinant forms of the Caenorhabditis xylosyltransferase SQV-6 and the human xylosyltransferase I, in addition to extending our previous studies on the xylosyltransferase from Drosophila. The enzyme activities were tested with a number of peptide substrates based on portions of the human bikunin, human perlecan and Drosophila syndecan core peptides. Whereas a variant of the latter, containing two Ser-Gly motifs was only modified on one of these motifs, the perlecan peptide with three Ser-Gly motifs could be multiply modified in vitro. Using this substrate, we could for the first time follow, by mass spectrometry, the xylosylation of a peptide with multiple xylosyltransferase acceptor motifs.

Discrimination of in vitro and in vivo digestion products of meat proteins from pork, beef, chicken, and fish.

In vitro digestion products of proteins were compared among beef, pork, chicken, and fish. Gastric and jejunal contents from the rats fed these meat proteins were also compared. Cooked pork, beef, chicken, and fish were homogenized and incubated with pepsin alone or followed by trypsin. The digestion products with molecular weights of less than 3000 Da were identified with MALDI‐TOF‐MS and nano‐LC‐MS/MS. Gastric and jejunal contents obtained from the rats fed the four meat proteins for 7 days were also analyzed. After pepsin digestion, pork, and beef samples had a greater number of fragments in similarity than chicken and fish samples, but the in vitro digestibility was the greatest (p < 0.05) for pork and the smallest for beef samples. After trypsin digestion, the species differences were less pronounced (p > 0.05). A total of 822 and 659 peptides were identified from the in vitro and in vivo digestion products, respectively. Our results could interpret for the differences in physiological functions after the ingestion of different species of meat.

Biochemical characterisation of the neuraminidase pool of the human gut symbiont Akkermansia muciniphila

Since the isolation and identification of Akkermansia muciniphila one decade ago, much attention has been drawn to this gut bacterium due to its role in obesity and type 2 diabetes. This report describes the discovery and biochemical characterisation of all four putative neuraminidases annotated in the A. muciniphila genome. Recombinantly expressed candidate genes, which were designated Am0705, Am0707, Am1757 and Am2085, were shown to cover complementary pH ranges between 4.0 and 9.5. Temperature optima of the enzymes lay between 37 and 42 °C. All four enzymes were strongly inhibited by Cu(2+) and Zn(2+), and loss of activity after the addition of EDTA suggests that all neuraminidases, with the exception of Am0707, require divalent metal ions for their catalytic function. Chemoenzymatically synthesised α2,3- and α2,6-linked indoyl-sialosides were utilised to determine the regioselectivity and substrate promiscuity of the neuraminidases towards C5-modifications of sialic acids with N-acetyl-, N-glycolyl-, N-propionyl-, or hydroxyl-groups. The combination of simple purification procedures and good activities of some of the characterised neuraminidases makes them potentially interesting as tools in bioanalytical or industrial applications.

2-Pyridylfuran: A New Fluorescent Tag for the Analysis of Carbohydrates

We herein report the use of 1,3-di(2-pyridyl)-1,3-propanedione (DPPD) as a fluorogenic labeling reagent for sugars. Reaction of DPPD with the anomeric carbon affords a fluorescent 2-pyridylfuran (2-PF) moiety that permits the sensitive HPLC-based detection of monosaccharides. 2-PF-labeled monosaccharides can be easily separated and analyzed from mixtures thereof, and the reported protocol compares favorably with established labeling reagents such as 2-aminobenzoic acid (2-AA) and 1-phenyl-3-methyl-5-pyrazolone (PMP), ultimately allowing subfemtomole detection of the galactose-derived product. Furthermore, we demonstrate the application of DPPD in the labeling of monosaccharides in complex biological matrices such as blood and milk samples. We envisage that DPPD will prove to be an excellent choice of labeling reagent in monosaccharide and carbohydrate analysis.