Mass spectral characterization of lipooligosaccharides from Haemophilus influenzae 2019.

Lipooligosaccharide (LOS) glycoforms from Haemophilus influenzae 2019 were profiled using the high-resolution and accurate mass capabilities of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Sequence and linkage for two previously unknown LOS glycoforms were subsequently obtained through MSn analyses on FT-ICR and quadrupole ion trap (qIT) instruments. MSn analysis of negative ion precursors confirmed structural details within the lipid moiety, while CID spectra of sodiated precursor ions provided monosaccharide sequence and linkage for the oligosaccharide portion of the molecule. Results obtained in this study indicate that extensive heterogeneity exists within the oligosaccharide moieties in LOS from H. influenzae 2019. More importantly, the data suggest that additional hexose moieties, which are added onto the LOS, are not simple extensions of one particular core structure but rather that structural isomers with different connectivities are present within the heterogeneous mixture.

Mass spectrometry and immobilized enzymes for the screening of inhibitor libraries.

A technique has been developed to rapidly screen enzyme inhibitor candidates from complex mixtures, such as those created by combinatorial synthesis. Inhibitor libraries are screened by using immobilized enzyme technologies and electrospray ionization ion cyclotron resonance mass spectrometry. The library mixture is first sprayed into the mass spectrometer, and compounds are identified. The library is subsequently incubated with the immobilized enzyme of interest under the correct conditions (buffer, pH, temperature) by using an excess of enzyme to ensure a surplus of sites for ligand binding. The immobilized enzyme/inhibitor mixture is centrifuged, and an aliquot of supernatant is again analyzed by electrospray ionization mass spectrometry. Potential inhibitors are quickly identified by comparison of the spectra before and after incubation with the immobilized enzyme. Non-inhibitors show no change in ion intensity after incubation, whereas weak inhibitors exhibit a visible decrease in ion abundance. Once inhibitor candidates have been identified, the library is reinjected into the mass spectrometer, and tandem mass spectrometry is used to determine the structure of the inhibitor candidates as needed. This method has been successfully demonstrated by identifying inhibitors of the enzymes pepsin and glutathione S-transferase from a 19- and 17-component library, respectively. It is further shown that the immobilized enzyme can be recycled and reused for continuous screening of additional new libraries without adding additional enzyme.

Combined partial acid hydrolysis and electrospray ionization-mass spectrometry for the structural determination of oligosaccharides.

A general oligosaccharide acid hydrolysis method, amenable to electrospray ionization mass spectrometry (ESI-MS), is described that allows for hydrolysis of glycosidic bonds for both hexose- and N-acetylhexosamine-containing oligosaccharides. The partial acid hydrolysis of oligosaccharides is obtained by using an acid-exchange resin as the acid catalyst. A ladder sequence of the glycan is produced in solution that is directly analyzed by ESI tandem mass spectrometry, employing both ion trap and Fourier transform ion cyclotron resonance mass spectrometers, to provide sequence and linkage information. Unlike traditional acid hydrolysis procedures, there is minimal degradation of monosaccharide residues or deacetylation of N-acetylhexosamines by employing this technique. It is further demonstrated that the stereochemistry of the released monosaccharides and the anomeric configuration within disaccharides is determined by direct derivatization of the hydrolysate with Zn(dien)-Cl2 followed by ESI-MS/MS.

Fragmentation reactions in the mass spectrometry analysis of neutral oligosaccharides.

A method is described to obtain multicollision dissociation threshold (MCDT) values. These values provide relative reaction thresholds for dissociation in the three major gas-phase fragmentation reactions of oligosaccharides complexed to alkali metal ions. The quasimolecular ions are produced using matrix-assisted laser desorption/ionization Fourier transform mass spectrometry. The MCDTs for alkali metal ion dissociation and glycosidic bond and cross-ring cleavages were resolved from the kinetic energy dependence of collision-induced dissociation (CID) products. The relative strengths of alkali metal ion binding to N,N'-diacetylchitobiose (chitobiose) and N,N',N"-triacetylchitotriose (chitotriose) were probed using sustained off-resonance irradiation (SORI) CID. Experiments to evaluate MCDT values and the method for obtaining them were performed by studying alkali metal ion coordinated crown ethers. Molecular dynamic simulations were also performed to provide insight into the alkali metal ion binding of chitin-based oligosaccharides. The relative dissociation thresholds of glycosidic bond cleavages and cross-ring cleavages were determined for various alkali metal ion coordinated oligosaccharides. The activation barriers of glycosidic bond cleavages were found to depend on the size of the alkali metal ion. Cross-ring cleavages were found to be independent of the alkali metal ion but dependent on linkage type. The results suggest that glycosidic bond cleavages are charge-induced while cross-ring cleavages are charge-remote processes.

Anion dopant for oligosaccharides in matrix-assisted laser desorption/ionization mass spectrometry.

A new anion dopant for oligosaccharides is developed for use in matrix-assisted laser desorption/ionization mass spectrometry. Two types of sulfate-attached quasimolecular ions are formed in the negative ion mode when neutral oligosaccharides are doped with dilute H2SO4 solutions. Under mild conditions, i.e., low H2SO4 concentration (approximately 10(-3) M) and threshold laser fluence, a sulfate adduct [M + HSO4]- is formed. With more concentrated H2SO4 solutions (approximately 10(-2) M) and higher laser fluence, in situ derivatization of the oligosaccharides occurs to produce an ion whose m/z corresponds to a sulfate derivative [M + HSO4 - H2O]-. Hydrogen sulfate appears to be a general anion dopant because it forms complexes with a wide variety of neutral oligosaccharides. Conversely, anionic oligosaccharides form neither the adduct nor the derivative. The combination of complex formation (with neutral oligosaccharides) and the deprotonation of acidic oligosaccharides allows simultaneous detection of the respective mixture.

Oceanapiside, an antifungal bis-alpha,omega-amino alcohol glycoside from the marine sponge Oceanapia phillipensis.

The structure of oceanapiside, an antifungal alpha, omega-bis-aminohydroxylipid glycoside from the temperate marine sponge Oceanapia sp., was elucidated by a combination of 2D NMR, chemical degradation/correlation, and MALDI MS-MS spectrometry. Oceanapiside exhibits antifungal activity against Candida glabrata at 10 micrograms/mL (MIC).

Alkaline degradation of oligosaccharides coupled with matrix-assisted laser desorption/ionization Fourier transform mass spectrometry: a method for sequencing oligosaccharides.

A new technique for determining sequence and linkage information of underivatized oligosaccharides is developed using alkaline degradation and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS). Alkaline degradation (also known as the "peeling" reaction) is a chemical degradation technique that only cleaves the glycosidic bond at the reducing end by beta-elimination to yield a new reducing end. The reaction products are sampled directly with minimal cleanup and monitored by MALDI-FTMS to elucidate the oligosaccharide sequence. Linkage information is provided by cross-ring cleavage fragmentation of the new reducing ends, created by either MALDI source fragmentation or sustained off-resonance irradiation collision-induced dissociation. This method is illustrated by the successful sequence and linkage determination of neutral, branched, fucosylated, and sialylated oligosaccharides. Experiments on differently linked disaccharides are also performed to determine the specificity of the cross-ring cleavage reactions. The power of this technique is enhanced by the Fourier transform mass analyzer, which provides high-resolution, exact mass, and facile tandem mass spectrometry experiments of MALDI-produced ions.

Collision-induced dissociation of branched oligosaccharide ions with analysis and calculation of relative dissociation thresholds.

Collision-induced dissociation (CID) is used in an external source Fourier transform mass spectrometer (FTMS) equipped with matrix-assisted laser desorption/ionization (MALDI) to study a number of complex, branched oligosaccharides. The relative dissociation thresholds for various oligosaccharide fragmentation pathways have been calculated in terms of kinetic and center-of-mass frame energy. For two isomers of difucosyllacto-N-hexaose, the loss of the fucose sugar is always the lowest energy fragment observed and occurs at the same energy for both isomers when the oligosaccharide is coordinated to a sodium ion. When the oligosaccharide is complexed to cesium, the threshold for the removal of the fucose moiety increases, indicating that the cesium is involved in a coordination complex that stabilizes the sugar. MS/ MS/MS is performed on a sugar, mannose core, which does not readily fragment during MALDI. In all the sugars examined, CID produces additional structural information relative to MALDI/FTMS.

A dual vacuum chamber fourier transform mass spectrometer with rapidly interchangeable LSIMS, MALDI, and ESI sources: initial results with LSIMS and MALDI.

A design is presented involving two separate vacuum chambers to provide nearly simultaneous capabilities of liquid secondary ion mass spectrometry (LSIMS), matrix-assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI) in an external source Fourier transform mass spectrometer. The instrument consists of two vacuum chambers, one with five stages of differential pumping for a combined LSIMS/MALDI source. The chamber dedicated to ESI was formerly a three-stage chamber with LSIMS and electron ionization. Two additional stages were added with the ESI source. LSIMS and MALDI have similar vacuum requirements and were moved to a newly built chamber with two stages of pumping. We present our first results obtained on the new vacuum chamber. Data presented for the MALDI source show that, with only two stages of pumping, and with shorter radio frequency-only quadrupole rods for ion injection, spectra comparable to those obtained on the formerly three-stage instrument can be obtained. Characterization of the MALDI source and data on linear, cyclic, and branched oligosaccharides are given. Finally, the design of the secon chamber is proposed as a low-cost prototype for an external source FTMS instrument.