Electron detachment dissociation of underivatized chloride-adducted oligosaccharides.

Chloride anion attachment has previously been shown to aid determination of saccharide anomeric configuration and generation of linkage information in negative ion post-source decay MALDI tandem mass spectrometry. Here, we employ electron detachment dissociation (EDD) and collision activated dissociation (CAD) for the structural characterization of underivatized oligosaccharides bearing a chloride ion adduct. Both neutral and sialylated oligosaccharides are examined, including maltoheptaose, an asialo biantennary glycan (NA2), disialylacto-N-tetraose (DSLNT), and two LS tetrasaccharides (LSTa and LSTb). Gas-phase chloride-adducted species are generated by negative ion mode electrospray ionization. EDD and CAD spectra of chloride-adducted oligosaccharides are compared to the corresponding spectra for doubly deprotonated species not containing a chloride anion to assess the role of chloride adduction in the stimulation of alternative fragmentation pathways and altered charge locations allowing detection of additional product ions. In all cases, EDD of singly chloridated and singly deprotonated species resulted in an increase in observed cross-ring cleavages, which are essential to providing saccharide linkage information. Glycosidic cleavages also increased in EDD of chloride-adducted oligosaccharides to reveal complementary structural information compared to traditional (non-chloride-assisted) EDD and CAD. Results indicate that chloride adduction is of interest in alternative anion activation methods such as EDD for oligosaccharide structural characterization.

Electron detachment dissociation of neutral and sialylated oligosaccharides.

Electron detachment dissociation (EDD) has recently been shown by Amster and coworkers to constitute a valuable analytical approach for structural characterization of glycosaminoglycans. Here, we extend the application of EDD to neutral and sialylated oligosaccharides. Both branched and linear structures are examined, to determine whether branching has an effect on EDD fragmentation behavior. EDD spectra are compared to collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) spectra of the doubly and singly deprotonated species. Our results demonstrate that EDD of both neutral and sialylated oligosaccharides provides structural information that is complementary to that obtained from both CAD and IRMPD. In all cases, EDD resulted in additional cross-ring cleavages. In most cases, cross-ring fragmentation obtained by EDD is more extensive than that obtained from IRMPD or CAD. Our results also indicate that branching does not affect EDD fragmentation, contrary to what has been observed for electron capture dissociation (ECD).

A new alpha-galactosyl-binding protein from the mushroom Lyophyllum decastes.

A new alpha-galactosyl binding lectin was isolated from the fruiting bodies of the mushroom Lyopyllum decastes. It is a homodimer composed of noncovalently-associated monomers of molecular mass 10,276Da. The lectin's amino acid sequence was determined by cloning from a cDNA library using partial sequences determined by automated Edman sequencing and by mass spectrometry of enzyme-derived peptides. The sequence shows no significant homology to any known protein sequence. Analysis of carbohydrate binding specificity by a variety of approaches including precipitation with glycoconjugates and microcalorimetric titration reveals specificity towards galabiose (Gal alpha1,4Gal), a relatively rare disaccharide in humans. The lectin shares carbohydrate binding preference with the Shiga-like toxin, also known as verocytoxin, present in the bacteria Shigella dysenteriae and Escherichia. coli 0157:H7, both of which are causes of outbreaks of sometimes fatal food-borne illnesses.

Electron capture dissociation of oligosaccharides ionized with alkali, alkaline Earth, and transition metals.

We extend the application of electron capture dissociation (ECD) (which requires at least two charges) to oligosaccharides without basic functionalities by utilizing alkali, alkaline earth, and transition metals (Na+, K+, Ca2+, Ba2+, Mg2+, Mn2+, Co2+, and Zn2+) as charge carriers in electrospray ionization. Both linear and branched oligosaccharides were examined, including maltoheptoase, p-lacto-N-hexaose, and an N-linked glycan from human alpha1-acid glycoprotein. For comparison, infrared multiphoton dissociation (IRMPD) was also applied to all oligosaccharide species. We show that, for certain metal-adducted oligosaccharides, particularly maltoheptaose, cross-ring cleavage, which can provide saccharide linkage information, is the dominant fragmentation pathway in ECD. By contrast, glycosidic cleavages dominate in IRMPD although cross-ring fragmentation was also observed to varying degrees depending on metal ion type. The branched N-linked glycan did not fragment as easily following ECD compared to the linear oligosaccharides, presumably due to intramolecular noncovalent interactions. However, this limitation was partially overcome with a combined ECD/IRMPD approach (activated ion ECD). For all metal-adducted oligosaccharides, complementary structural information was obtained with ECD as compared to IRMPD. Our results demonstrate that ECD of metal-adducted oligosaccharides is a valuable tool for structural characterization of oligosaccharides.

Infrared multiphoton dissociation and electron capture dissociation of high-mannose type glycopeptides.

The combination of electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) for the structural characterization of high-mannose type glycopeptides is explored in depth for the first time. Contrary to previous applications to other glycan types, our analyses reveal that IRMPD does not necessarily selectively induce glycan cleavage in high-mannose type glycopeptides; rather peptide backbone cleavage can effectively compete with glycosidic cleavage. Poor glycan cleavage with IRMPD is due to a higher gas-phase stability of mannose-linking glycosidic bonds. This reasoning also explains mannose cleavage patterns observed for a xylose type glycopeptide with IRMPD. In addition, extensive peptide backbone cleavage is observed for a >6 kDa glycopeptide with ECD, to our knowledge the largest glycopeptide examined with this technique to date.

Characterization of oligodeoxynucleotides by electron detachment dissociation fourier transform ion cyclotron resonance mass spectrometry.

Electron detachment dissociation (EDD), recently introduced by Zubarev and co-workers for the dissociation of multiply charged biomolecular anions via a radical ion intermediate, has been shown to be analogous to electron capture dissociation (ECD) in several respects, including more random peptide fragmentation and retention of labile posttranslational modifications. We have previously demonstrated unique fragmentation behavior in ECD compared to vibrational excitation for oligodeoxynucleotide cations. However, that approach is limited by the poor sensitivity for oligonucleotide ionization in positive ion mode. Here, we show implementation of EDD on a commercial Fourier transform ion cyclotron resonance mass spectrometer utilizing two different configurations: a heated filament electron source and an indirectly heated hollow dispenser cathode electron source. The dispenser cathode configuration provides higher EDD efficiency and additional fragmentation channels for hexamer oligodeoxynucleotides. As in ECD, even-electron d/w ion series dominate the spectra, but we also detect numerous a/z (both even-electron and radical species), (a/z - B), c/x, (c/x - B), and (d/w - B) ions with minimal nucleobase loss from the precursor ions. In contrast to previous high-energy collision-activated dissociation (CAD) and ion trap CAD of radical oligonucleotide anions, we only observe minimum sugar cross-ring cleavage, possibly due to the short time scale of EDD, which limits secondary fragmentation. Thus, EDD provides fragmentation similar to ECD for oligodeoxynucleotides but at enhanced sensitivity. Finally, we show that noncovalent bonding in a DNA duplex can be preserved following EDD, illustrating another analogy with ECD. We believe the latter finding implies EDD has promise for characterization of nucleic acid structure and folding.

Dextrin characterization by high-performance anion-exchange chromatography--pulsed amperometric detection and size-exclusion chromatography--multi-angle light scattering--refractive index detection.

Starch hydrolysis products, or dextrins, are widely used throughout the food industry for their functional properties. Dextrins are saccharide polymers linked primarily by alpha-(1 --> 4) D-glucose units and are prepared by partial hydrolysis of starch. Hydrolysis can be accomplished by the use of acid, enzymes, or by a combination of both. The hydrolysis products are typically characterized by the "dextrose equivalent" (DE), which refers to the total reducing power of all sugars present relative to glucose. While the DE gives the supplier and buyer a rough guide to the bulk properties of the material, the physiochemical properties of dextrins are dependent on the overall oligosaccharide profile. High-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection and size-exclusion chromatography (SEC) with multi-angle light-scattering and refractive index detection were used to characterize dextrins from commercial sources. HPAEC was used to acquire the oligosaccharide profile, and SEC to obtain an overall molar mass distribution. These methods in combination extended our understanding of the relationship between oligosaccharide profile, DE, and the hydrolysis process. Data from the two techniques enabled a method for estimating the DE that gave results in reasonable agreement with the accepted titration method.