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B Domon

Publications and source records attributed to B Domon.

11 recordsLinked to original sources
PubMed2001

Qualitative and quantitative analysis of the glycosylation pattern of recombinant proteins.

Over the past decade, the growing number of recombinant glycoproteins used as therapeutic agents has prompted the development of robust and rugged methodologies for characterizing the glycosylation pattern of such molecules. The present study describes an alternative to the widely used HPLC approaches for profiling the N-glycan heterogeneity of proteins. The method encompasses the enzymatic deglycosylation of the glycoprotein, the permethylation of the released oligosaccharides, and the subsequent analysis of these derivatives by either matrix-assisted laser desorption/ionization or electrospray mass spectrometry. This methodology showed excellent correlation when compared with results obtained by an orthogonal technique such as the HPLC of 2-aminobenzamide-labeled glycans. In addition, it gives a more detailed insight into the glycosylation pattern by unambiguously identifying and quantifying the various glycoforms present in the mixture. Despite a somewhat complex sample preparation, reproducibility and robustness of the method were excellent. In the case of very heterogeneous glycan pools, simplification of the glycosylation pattern was achieved by performing enzymatic desialylation prior to deglycosylation and derivatization, leading to a more direct determination of the antennary distribution as well as the identification of minor components.

Amidohydrolases↗
PubMed1999

Post-source decay mass spectrometry: optimized calibration procedure and structural characterization of permethylated oligosaccharides.

Permethylated oligosaccharides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) using a reflectron time-of-flight instrument in the post-source decay (PSD) mode. Under these ionization conditions, such derivatives yield intense signals corresponding to sodium or potassium cationized molecular species. Fragments observed in the PSD spectra result exclusively from cleavage of glycosidic bonds, preferentially at N-acetylhexosamine residues. A systematic study was carried out on a series of permethylated oligosaccharides to allow rationalization of the fragmentation processes. Fragments originating from both the reducing and the non-reducing ends of the oligosaccharide yield information on sequence and branching. Moreover, glycosyl residues linked in position 3 of HexNAc units give rise to a highly specific elimination process, which allows unambiguous assignment of (1-3) interglycosidic linkages. Special attention was paid to the structural analysis of oligosaccharides carrying the commonly encountered fucosyl and sialyl end-caps. In the case of sialylated residues, a targeted methodology involving desialylation and specific CD3-labeling of the nascent free hydroxyl groups was developed to mark the initial location of sialic acid residues along the oligosaccharide backbone. As accurate mass determination of fragment ions is essential for their assignment, a simplified protocol for the calibration in the PSD mode is described. This procedure allows the determination of the correction function parameters required to process the data for an instrument that employs post-acceleration detection. MALDI/PSD-MS of permethylated oligosaccharides, by providing structural information at the low picomole level, appears to be a valuable complement, or an alternative, to the techniques currently in use for carbohydrate structural analysis.

Mass Spectrometry↗
PubMed1998

Structural assignment of permethylated oligosaccharide subunits using sequential tandem mass spectrometry.

The sequential tandem mass spectrometry (MSn) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of permethylated oligosaccharides. Under collision-induced dissociation, protonated molecular species generated in the electrospray ionization mode yield simple and predictable mass spectra. Information on sequence, branching, and, to some extent, interglycosidic linkages can be deduced from fragments resulting from the cleavage of glycosidic bonds. Simple rules for the structural assignment of carbohydrates have been established for the fragmentation of protonated species and subunits thereof and corroborated by 18O-labeling experiments. Moreover, sequential tandem mass spectrometry was demonstrated to allow the straightforward structural characterization of unknown carbohydrate moieties by comparing their CID spectra with those of a set of references. As the collision-induced dissociation patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by match of the spectra. These findings establish the basis of MSn performed on a quadrupole ion trap instrument for elucidating structures of large carbohydrates, which can be virtually degraded in the mass spectrometer into smaller entities in one or several steps. This powerful technique has been applied, used in conjunction with specific CD3 labeling, to the characterization of series of subunits generated from fucosylated and sialylated oligosaccharides, which are among the most important structures as far as biological activities are concerned.

Carbohydrate Sequence↗
PubMed1997

Structural analysis of permethylated oligosaccharides by electrospray tandem mass spectrometry.

Electrospray tandem mass spectrometry used in conjunction with reversed-phase liquid chromatography was applied to characterize permethylated oligosaccharides. N-Acetylhexosamine-containing carbohydrates yielded under these conditions promoted molecular ions which underwent extensive fragmentation, even under low-energy collision-induced dissociation. MS/MS spectra of [M + H]+ ions are characterized by simple fragmentation patterns which result from cleavage of the glycosidic bonds and thus allow a straightforward interpretation. A systematic study of various oligosaccharides showed that information on sugar sequence and branching could be obtained. The nature of the substituent linked in position 3 of HexNAc-containing fragments could easily be assigned as the result of a specific secondary fragmentation process. The nature of some internal fragments was established on the basis of MS/MS spectra of derivatives 18O-labeled at the reducing end. Furthermore, MS/MS experiments carried out on fragment ions were proven to be useful for the structural characterization of oligosaccharide subunits. Thus, this approach constitutes a powerful tool for the structural assignment of moieties derived from larger glycans.

Carbohydrate Conformation↗
PubMed1996

Structure determination of a novel uronic acid residue isolated from the exopolysaccharide produced by a bacterium originating from deep sea hydrothermal vents.

The exopolysaccharide produced by the bacterium Alteromonas sp. strain 1644 originating from deep sea hydrothermal vents was shown to contain a novel glucuronic acid derivatives: 3-O-[(R)-1-carboxyethyl]-D-glucuronic acid. The structure of this compound was established on the basis of mass spectrometric data, methylation analysis, preparation of derivatives, and chemical synthesis of references compounds.

Archaea↗
PubMed1996

Structural analysis of derivatized oligosaccharides using post-source decay matrix-assisted laser desorption/ionization mass spectrometry.

Structural information on oligosaccharides was obtained by post-source decay matrix-assisted laser desorption/ionization mass spectrometry. A systematic study of derivatized oligosaccharides showed that products relatively aminated with benzylamine allow the formation of [M+H]+ molecular species. Metastable protonated species decompose in the field-free region (post-source decay) to yield predictable and reproducible fragmentation patterns. Information on sugar sequence and branching can be derived from such mass spectra.

Acetylation↗
PubMed1994

Characterization and direct glycoform profiling of a hybrid plasminogen activator by matrix-assisted laser desorption and electrospray mass spectrometry: correlation with high-performance liquid chromatographic and nuclear magnetic resonance analyses of the released glycans.

Mass spectrometric analysis of the recombinant hybrid plasminogen activator K2tu-PA at the native glycoprotein and corresponding protein level (high mass) is described. For gross structural characterization of the major glycotypes present, a combination of enzymatic degradation and matrix-assisted laser desorption mass spectrometric analysis of the products proved convenient. In this way, mono- and di-N-glycosylated one- and two-chain molecules, unresolved in the spectra of native material, were identified, with the one-chain type monoglycosylated at Asn247 representing the major component. Actual detection of resolved original glycoforms, or unresolved groups thereof when composed of isobaric species, and their assignments regarding antennicity and degree of sialylation were possible by electrospray mass spectrometry for the major monoglycosylated one-chain species. Desialation also allowed detection of the diglycosylated one-chain species as a minor constituent. The electrospray mass spectrometric results were correlated with structural and quantitative data available from a parallel high-performance liquid chromatographic and 1H nuclear magnetic resonance study performed at the glycan (low mass) level on liberated individual carbohydrate components. The results of the two studies showed full consistency in all respects amenable to evaluation, i.e. excellent agreement for the assignments of antennicity and degree of sialylation of the major glycan components, and good agreement for the determination of proportions in which these were present in the major monoglycosylated glycoprotein. This provided a sound basis for direct glycoform profiling by electrospray mass spectrometry. Typical applications to batch quality control and to structural characterization of non-standard material are shown.

Amino Acid Sequence↗
PubMed1990

Analysis of derivatized ceramides and neutral glycosphingolipids by high-performance tandem mass spectrometry.

Microscale reduction of ceramides and neutral glycosphingolipids has been evaluated as a means of improving their analysis by fast atom bombardment mass spectrometry, alone and in combination with tandem mass spectrometry. Reduction (conversion of the amide to an amine) of native ceramides and glycosphingolipids containing one to three sugars yields derivatives that show significant signal enhancement. This sensitivity increase allows the acquisition of normal and tandem fast atom bombardment mass spectra from a submicrogram amount of sample. Concomitant permethylation is required for glycosphingolipids that contain more than three sugars. Collision induced dissociation mass spectra of protonated molecular ions, recorded on a four sector instrument, show improved fragmentation allowing the simultaneous characterization of both the ceramide and carbohydrate portions of glycosphingolipids. The reductions are carried out at the nanogram to microgram level with borane, reacting the solid sample with condensed reagent vapor. The borane reduction method has been adapted for this class of substances by adding an oxidation step in order to convert unsaturated lipids to hydroxylated derivatives by oxidation of the resulting organoborane. This approach, used in conjunction with tandem mass spectrometry, allows the determination of olefinic bond location. Labeled derivatives have been prepared by reacting the substrates with trideuterioborane and were used to ascertain the fragmentations and localize olefinic bonds. The collision induced fragmentation of reduced ceramides and neutral glycosphingolipids is only weakly affected by the presence of additional functionalities, such as methoxyl (after permethylation) and hydroxyl groups (resulting from hydroboration and oxidation), a characteristic which facilitates interpretation of the spectra of unknown compounds.

Animals↗
PubMed1988

Structure elucidation of glycosphingolipids and gangliosides using high-performance tandem mass spectrometry.

Glycosphingolipids and gangliosides have been investigated by using fast atom bombardment high-performance tandem mass spectrometry (FABMS/MS). Homologous compounds have been investigated in order to ascertain the fragmentation. Collision-induced dissociation spectra of the molecular species in the positive ion mode mainly afford information on the ceramide constitution (aglycon as a whole, N-acyl residue, and long-chain base), whereas negative ion spectra show fragments informative of the sugar sequence and the degree of branching of the carbohydrate. In the case of gangliosides carrying a complex oligosaccharide moiety, collision spectra of fragment ions have been performed in order to gain additional structural data. The advantage of tandem mass spectrometry over conventional fast atom bombardment mass spectrometry (FABMS) consists in the fact that collision spectra of the individual components from mixtures, as usually encountered with these kinds of samples, can be recorded. Furthermore, the exclusion of most of the interfering signals from the matrix allows the identification of pertinent fragments at low mass.

Gangliosides↗