C Isotopomer Analysis of Glutamate by Tandem Mass Spectrometry

Tandem mass spectrometry allows a compound to be isolated from the rest of the sample and dissociated into smaller fragments. We show here that fragmentation of glutamate mass isotopomers yields additional mass spectral data that significantly improve the analysis of metabolic fluxes compared to full-scan mass spectrometry. In order to validate the technique, tandem and full-scan mass spectrometry were used along with (13)C NMR to analyze glutamate from rat hearts perfused with three substrate mixtures (5 mM glucose plus 5 mM [2-(13)C]acetate, 5 mM [1-(13)C]glucose plus 5 U/L insulin, and 5 mM glucose plus 1 mM [3-(13)C]pyruvate). Analysis by tandem mass spectrometry showed that the enriched substrate contributed 98 +/- 2, 53 +/- 2, and 84 +/- 7%, respectively, of acetyl-coenzyme A while the rate of anaplerotic substrate entry was 7 +/- 3, 25 +/- 8, and 16 +/- 8%. Similar results were obtained with (13)C NMR data, while values from full-scan data had higher error. We believe that this is the first use of tandem mass spectrometry to determine pathway flux using (13)C-enriched substrates. Although analysis of the citric acid cycle by NMR is simpler (and more intuitive), tandem mass spectrometry has the potential to combine high sensitivity with the high information yield previously available only by NMR.     

Enzymatic synthesis of 4-methylumbelliferyl (1-->3)-beta-D-glucooligosaccharides-new substrates for beta-1,3-1,4-D-glucanase

The transglycosylation reactions catalyzed by beta-1,3-D-glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1-->3)-beta-D-gluco-oligosaccharides having the common structure [beta-D-Glcp-(1-->3)](n)-beta-D-Glcp-MeUmb, where n=1-5. The beta-1,3-D-glucanases used were purified from the culture liquid of Oerskovia sp. and from a homogenate of the marine mollusc Spisula sachalinensis. Laminaran and curdlan were used as (1-->3)-beta-D-glucan donor substrates, while MeUmb-beta-D-glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [beta-D-Glcp-(1-->3)](2)-beta-D-Glcp-MeUmb (MeUmbG(3)) gives 4,6-O-benzylidene-D-glucopyranosyl or 4,6-O-ethylidene-D-glucopyranosyl groups at the non-reducing end of artificial oligosaccharides. The structures of all oligosaccharides obtained were solved by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. The synthetic oligosaccharides were shown to be substrates for a beta-1,3-1,4-D-glucanase from Rhodothermus marinus, which releases MeUmb from beta-di- and beta-triglucosides and from acetal-protected beta-triglucosides. When acting upon substrates with d.p.>3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUmbGlcp and MeUmbG(2).     

Characterisation by mass spectrometry and 500-MHz proton nuclear magnetic resonance spectroscopy of penta- and hexasaccharide chains of human foetal gastrointestinal mucins (meconium glycoproteins)

Structural studies using liquid secondary ion mass spectrometry, gas liquid chromatography/mass spectrometry and 500-MHz 1H NMR are described of the major penta- and hexasaccharides of a fraction of human foetal gastrointestinal mucins. Glycoproteins from a blood group H active meconium pool were studied after depletion of Ii antigenic activities by immunoaffinity chromatography and treatment with mild acid hydrolysis to reduce the chain heterogeneity. Oligosaccharides were released by mild alkali/borohydride degradation and purified by Bio-Gel P4 chromatography and HPLC. Eleven penta- and hexasaccharides have been fully characterised as a result of this study and one previous report [Hounsell et al. (1988) Biochem. J. 256, 397-401] and information obtained on additional oligosaccharides present in small amounts. These oligosaccharides show the following features: (table; see text) Sequences in these oligosaccharides not commonly found in mucins so far studied are chain-terminating GlcNAc alpha 1-4Gal, repeating-type-I (Gal beta 1-3GlcNAc) backbones, the backbone branch GlcNAc beta 1-6(GlcNAc beta 1-3)Gal and the backbone sequence GlcNAc beta 1-6Gal beta 1- in the absence of a substituent at C3 of galactose.     

Structural studies utilizing 13C-enrichment of the O-antigen polysaccharide from the enterotoxigenic Escherichia coli O159 cross-reacting with Shigella dysenteriae type 4.

The structure of the O-antigen polysaccharide from Escherichia coli O159 has been determined using primarily NMR spectroscopy of the 13C-enriched polysaccharide. The sequence of the sugar residues could be determined by heteronuclear multiple bond connectivity NMR experiments. The polysaccharide is composed of a pentasaccharide repeating unit with the following structure: [sequence: see text] Matrix assisted laser desorption ionization mass spectrometry was performed on intact lipopolysaccharide and from the resulting molecular mass the O-antigen part was estimated to contain approximately 23 repeating units. Cross-reactivity of this O-antigen to that of Shigella dysenteriae type 4 was confirmed using enzyme-linked immunoabsorbant assay.     

Structural analysis of the lipopolysaccharide derived core oligosaccharides of Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and the genome strain 5b

The structures of the core oligosaccharides of the lipopolysaccharides (LPS) from Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and 5b were elucidated. The LPS's were subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structures for the core oligosaccharides were determined on the basis of the combined data from these experiments. [carbohydrate formula see text] For serotype 1: R is (1S)-GalaNAc-(1-->4,6)-alpha-Gal II-(1-->3)-beta-Gal I-(1-->, and R' is H For serotype 2: R is beta-Glc III-(1-->, and R' is D-alpha-D-Hep V-(1--> For serotypes 5a and 5b: R is H and R' is D-alpha-D-Hep V-(1--> All oligosaccharides elaborated a conserved inner core structure, as illustrated. All sugars were in the pyranose ring form apart from the open-chain N-acetylgalactosamine, the identification of which in the serotype 1 LPS was of interest.     

Structural studies on kappa-carrageenan derived oligosaccharides

Oligosaccharides were prepared through mild hydrochloric acid hydrolysis of kappa-carrageenan from Kappaphycus striatum carrageenan. Three oligosaccharides were purified by strong-anion exchange high-performance chromatography. Their structure was elucidated using mass spectral and NMR data. Negative-ion electrospray ionization (ESI) mass spectra at different fragmentor voltages provided the molecular weight of the compounds and unraveled the fragmentation pattern of the kappa-carrageenan oligosaccharides. 2D NMR techniques, including 1H-(1)H COSY, 1H-(1)H TOCSY and 13C-(1)H HMQC, were performed to determine the structure of a trisulfated pentasaccharide. 1D NMR and ESIMS were used to determine the structures of a kappa-carrageenan-derived pentasaccharide, heptasaccharide, and an undecasaccharide. All the oligosaccharides characterized have a 4-O-sulfo-D-galactopyranose residue at both the reducing and nonreducing ends.     

Structure of the exopolysaccharide produced by Streptococcus thermophilus S3

The exopolysaccharide of Streptococcus thermophilus S3, produced in skimmed milk, is composed of D-galactose and L-rhamnose in a molar ratio of 2:1. The polysaccharide contains 0.4 equiv of O-acetyl groups per repeating unit. Linkage analysis and 1D/2D NMR (1H and 13C) studies on native and O-deacetylated EPS together with nanoES-CID tandem mass spectrometry studies on oligosaccharides generated by a periodate oxidation protocol, show the polysaccharide to have the following structure: [structure: see text].     

Overexpression of a homogeneous oligosaccharide with <Superscript>13</Superscript>C labeling by genetically engineered yeast strain

This report describes a novel method for overexpression of ¹³C-labeled oligosaccharides using genetically engineered Saccharomyces cerevisiae cells, in which a homogeneous high-mannose-type oligosaccharide accumulates because of deletions of genes encoding three enzymes involved in the processing pathway of asparagine-linked oligosaccharides in the Golgi complex. Using uniformly ¹³C-labeled glucose as the sole carbon source in the culture medium of these engineered yeast cells, high yields of the isotopically labeled Man₈GlcNAc₂ oligosaccharide could be successfully harvested from glycoprotein extracts of the cells. Furthermore, ¹³C labeling at selected positions of the sugar residues in the oligosaccharide could be achieved using a site-specific ¹³C-enriched glucose as the metabolic precursor, facilitating NMR spectral assignments. The ¹³C-labeling method presented provides the technical basis for NMR analyses of structures, dynamics, and interactions of larger, branched oligosaccharides.     

Synthesis of four novel trisaccharides by induction of loose acceptor specificity in Gal beta 1----4 transferase (EC 2.4.1.22): Galp(beta 1----4)Glcp(X)Glc where X = beta 1----3: beta 1----4: beta 1----6: alpha 1----4.

Development of tandem mass spectral methods for direct linkage determination in oligosaccharides requires sets of trisaccharides differing only in one structural parameter. In this case, we chose the position of linkage to the reducing-end hexose. These sets of compounds would also be useful for the development of high-resolution separation techniques geared to resolve linkage types. Conventional organic synthesis of such a set could take as long as 2-5 months for each member of the set. Each trisaccharide would require 10-20 steps of synthesis. Instead, we utilized low pH to induce a loose acceptor specificity for bovine milk galactosyltransferase (lactose synthase: EC 2.4.1.22) and by this method, within 2 weeks, generated four novel oligosaccharides for NMR and mass spectral studies. The disaccharides cellobiose (beta 1----4), laminaribiose (beta 1----3), gentiobiose (beta 1----6) and maltose (alpha 1----4) acted as acceptors for EC 2.4.1.22 under these conditions. The beta 1----2-linked disaccharide, sophorose, was not commercially available and is not included in this study. The alpha-linked disaccharides were also examined, but except for the alpha 1----4 disaccharide maltose, were very poor acceptors under a variety of conditions. From these four acceptors, the following four novel trisaccharides were synthesized in micromole amounts, suitable for studies of linkage position using low-energy collision-induced-dissociation tandem mass spectrometry (FAB-MS-CID-MS), and for NMR: Galp(beta 1----4)Glcp(beta 1----3)-Glc, Galp(beta 1----4)Glcp(beta 1----4)Glc, Galp(beta 1----4)Glcp(beta 1----6)-Glc and Galp(beta 1----4)Glcp(alpha 1----4)Glc.     

Characterization of N-linked oligosaccharides in chorion peroxidase of Aedes aegypti mosquito

A peroxidase is present in the chorion of Aedes aegypti eggs and catalyzes chorion protein cross-linking during chorion hardening, which is critical for egg survival in the environment. The unique chorion peroxidase (CPO) is a glycoprotein. This study deals with the N-glycosylation site, structures, and profile of CPO-associated oligosaccharides using mass spectrometric techniques and enzymatic digestion. CPO was isolated from chorion by solubilization and several chromatographic methods. Mono-saccharide composition was analyzed by HPLC with fluorescent detection. Our data revealed that carbohydrate (D-mannose, N-acetyl D-glucosamine, D-arabinose, N-acetyl D-galactosamine, and L-fucose) accounted for 2.24% of the CPO molecular weight. A single N-glycosylation site (Asn328-Cys- Thr) was identified by tryptic peptide mapping and de novo sequencing of native and PNGase A-deglycosylated CPO using matrix-assisted laser/desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The Asn328 was proven to be a major fully glycosylated site. Potential tryptic glycopeptides and profile were first assessed by MALDI/TOF/MS and then by precursor ion scanning during LC/MS/MS. The structures of N-linked oligosaccharides were elucidated from the MS/MS spectra of glycopeptides and exoglycosidase sequencing of PNGase A-released oligosaccharides. These CPO-associated oligosaccharides had dominant Man3GlcNAc2 and Man3 (Fuc) GlcNAc2 and high mannose-type structures (Man(4-8)GlcNAc2). The truncated structures, Man2GlcNAc2 and Man2 (Fuc) GlcNAc2, were also identified. Comparison of CPO activity and Stokes radius between native and deglycosylated CPO suggests that the N-linked oligosaccharides influence the enzyme activity by stabilizing its folded state.     

Structural features of the arabinan component of the lipoarabinomannan of Mycobacterium tuberculosis

The recent availability of pure lipoarabinomannan (LAM) from Mycobacterium spp. has resulted in its implication in host-parasite interaction, which events may be mediated by the presence of a phosphatidylinositol unit at the reducing end of LAM. Herein we address the structure of the antigenic, nonreducing end of the molecule. Through the process of 13C NMR analysis of the whole molecule and gas chromatography/mass spectrometry of alditol acetates derived from the differential per-O-alkylated lipopolysaccharide, the majority of the arabinosyl residues were recognized as furanosides. Second, through analysis of per-O-alkylated oligoarabinosyl arabinitol fragments of partially hydrolyzed LAM, it was established that the internal segments of the arabinan component consists of branched 3,5-linked alpha-D-arabinofuranosyl (Araf) units with stretches of linear 5-linked alpha-D-Araf residues attached at both branch positions, whereas the nonreducing terminal segments of LAM consist of either of the two arrangements, beta-D-Araf-(1----2)-alpha-D-Araf-(1----5)- alpha-D-Araf---- or [beta-D-Araf-(1----2)-alpha-D-Araf-(1----]2---- (3 and 5)-alpha-D-Araf----. Since this latter arrangement also characterizes the terminal segments of the peptidoglycan-bound arabinogalactan of Mycobacterium spp., we propose that mycobacteria elaborate unique terminal arabinan motifs in two distinct settings. In the case of the bound arabinogalactan, these motifs provide the nucleus for the esterified mycolic acids, entities which dominate the physicochemical features of mycobacteria and their peculiar pathogenesis. In the case of LAM, these motifs, non-mycolylated, are the dominant B-cell antigens responsible for the majority of the copious antibody response evident in most mycobacterial infections.     

Identification of lysine 346 as a functionally important residue for pyridoxal 5'-phosphate binding and catalysis in lysine 2, 3-aminomutase from Bacillus subtilis

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of L-lysine and L-beta-lysine. The enzyme contains pyridoxal 5'-phosphate (PLP) and a [4Fe-4S] center and requires S-adenosylmethionine (SAM) for activity. The hydrogen transfer is mediated by the 5'-deoxyadenosyl radical generated in a reaction of the iron-sulfur cluster with SAM. PLP facilitates the radical rearrangement by forming a lysine-PLP aldimine, in which the imine group participates in the isomerization mechanism. We here report the identification of lysine 346 as important for PLP binding and catalysis. Reduction of LAM with NaBH(4) rapidly inactivated the enzyme with concomitant UV/visible spectrum changes characteristic of reduction of an aldimine formed between PLP and lysine. Following reduction with NaBH(4) and proteolysis with trypsin, a single phosphopyridoxyl peptide of 36 amino acid residues was identified by reverse-phase liquid chromatography/mass spectrometry (LC/MS). The purified phosphopyridoxyl peptide exhibited an absorption band at 325 nm, and its identity was further confirmed by tandem mass spectrometry (MS/MS) sequencing. The bound PLP is linked to lysine 346 in a PGGGGK (PLP) structure. The sequence of this binding motif is conserved in LAMs from Bacillus and Clostridium and other homologous proteins but is distinct from the PLP-binding motifs found in other PLP enzymes. The function of lysine 346 was further studied by site-directed mutagenesis. The purified K346Q mutant was inactive, and its content of PLP was only approximately 15% of that of the wild-type enzyme. The data indicate that the formation of the aldimine linkage between lysine 346 and PLP is important for LAM catalysis. Sequences similar to the PLP-binding motifs in other enzymes were also present in LAM. However, lysine residues within these motifs neither are the PLP-binding sites in LAM nor are directly involved in LAM catalysis. This study represents the first comprehensive investigation of PLP binding in a SAM-dependent iron-sulfur enzyme.     

Structure of the complex group-specific polysaccharide of group B Streptococcus

The group-specific antigen was isolated from a type Ia group B streptococcal strain and is a complex polysaccharide composed of alpha-L-rhamnopyranosyl, alpha-D-galactopyranosyl, 2-acetamido-2-deoxy-beta-D-glucopyranosyl, D-glucitol, and phosphate residues. The complexity of the group B polysaccharide antigen is evident from the fact that when depolymerized by basic hydrolysis it yielded three structurally related, but nevertheless significantly different, oligosaccharides. These oligosaccharides were obtained in different molar quantities as their monophosphate esters. This evidence strongly suggests that they are linked by phosphodiester bonds in the original group B antigen. If these oligosaccharides are in fact randomly situated throughout the linear polysaccharide, then this type of heterogeneous repeating unit is unusual for a polysaccharide of bacterial origin. However, this structural arrangement of the oligosaccharides has yet to be unambiguously established because the alternate explanation of there being three different polysaccharides in the group B antigen cannot be discounted in the evidence presented here. The oligosaccharides were enzymatically dephosphorylated, and the structures of two of the three oligosaccharides are (formula: see text) Despite their structural differences, the two oligosaccharides are related by the smaller being an integral part of the larger. In the structural analysis of the group B antigen, methylation analysis, periodate oxidation, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, fast atom bombardment mass spectrometry, and various specific chemical and enzymatic degradations were the principal methods used. Of particular interest was the use of an alpha-rhamnosidase to selectively degrade the larger oligosaccharide. This facilitated the assignment of signals in its 1H and 13C NMR spectra.     

A Comparative Study on Structural Elucidation of Sialyl Oligosaccharides by Mass Spectrometry with Fast Atom Bombardment,Electrospray Ionization,and Matrix Assisted Laser Desorption/Ionization

To establish a new protocol for sensitive detection and structural characterization of sialyl oligosaccharides, their sensitivities and structural information from mass spectrometry and tandem mass spectrometry with FAB-, ESI-, and MALDI were evaluated in detail. Among these ionization methods, FAB-MS and FAB-MS/MS gave reproducible and predictable spectra carrying information on sequence and branching of sialyl oligosaccharides after derivatization with 2-aminopyridine (PA). With both positive and negative ion modes, their structural elucidation promises to be straightforward, MS/MS specta being measurable at as low as 200 pmol. Thus, this method consitutes a powerful tool for sensitive detection and structural characterization of limited quantities of sialyl oligosaccharides by FAB-MS and FAB-MS/MS.     

Profiling the morphological distribution of O-linked oligosaccharides

The morphological distribution of oligosaccharides is determined in the egg jelly surrounding Xenopus laevis eggs. This biological system is used to illustrate a method for readily identifying and quantifying oligosaccharides in specific tissues. The extracellular matrix surrounding X. laevis eggs consists of a vitelline envelope and a jelly coat. The jelly coat contains three morphologically distinct layers designated J1, J2, and J3 from the innermost to the outermost and is composed of 9-11 distinct glycoproteins. Each jelly layer is known to have specific functions in the fertilization of the egg. We developed a rapid method to separate and identify the oligosaccharides from X. laevis egg jelly layers. Identification was based on the retention times in high-performance liquid chromatography (porous graphitized carbon column), exact masses, and tandem mass spectrometry. Over 40 neutral and 30 sulfated oligosaccharides were observed in the three jelly layers. Neutral oligosaccharide structures from different jelly layers were both unique and overlapping, while sulfated oligosaccharides were detected only in layers J1 and J2. Neutral oligosaccharides unique to jelly layer J3 and the combined layers J1+J2 had similar core structures and similar residues. However, differences between these two sets of unique oligosaccharides were also observed and were primarily due to the branching carbohydrate moieties rather than the core structures.     

A comparative study on structural elucidation of sialyl oligosaccharides by mass spectrometry with fast atom bombardment, electrospray ionization, and matrix assisted laser desorption/ionization.

To establish a new protocol for sensitive detection and structural characterization of sialyl oligosaccharides, their sensitivities and structural information from mass spectrometry and tandem mass spectrometry with FAB-, ESI-, and MALDI were evaluated in detail. Among these ionization methods, FAB-MS and FAB-MS/MS gave reproducible and predictable spectra carrying information on sequence and branching of sialyl oligosaccharides after derivatization with 2-aminopyridine (PA). With both positive and negative ion modes, their structural elucidation promises to be straightforward, MS/MS spectra being measurable at as low as 200 pmol. Thus, this method constitutes a powerful tool for sensitive detection and structural characterization of limited quantities of sialyl oligosaccharides by FAB-MS and FAB-MS/MS.     

3-(3-hydroxy-2,3-dimethyl-5-oxoprolyl)amino-3,6-dideoxy-D-glucose: a new amino sugar of the antigenic polysaccharide from Pseudomonas fluorescens

O-specific polysaccharide chain of the Pseudomonas fluorescens lipopolysaccharide is composed of 6-deoxy-L-talose, N-acetyl-D-fucosamine and N-acyl-3,6-dideoxy-D-glucose. Analysis of the latter sugar, obtained from the polysaccharide hydrolysate, by 1H NMR (including NOE), 13C NMR, and FAB mass spectrometry proved the unusual N-acyl substituent to be a 3-hydroxy-2,3-dimethyl-5-oxoproline residue.     

Structural elucidation of the core-lipid A backbone from the lipopolysaccharide of Acinetobacter radioresistens S13, an organic solvent tolerant Gram-negative bacterium

The structure of the core oligosaccharide of the lipopolysaccharide from an organic solvent tolerant Gram-negative bacterium, Acinetobacter radioresistens S13, was investigated by chemical analysis, NMR spectroscopy and MALDI-TOF mass spectrometry. All the experiments were performed on the oligosaccharides obtained either by alkaline degradation or mild acid hydrolysis. The data showed the presence of two novel oligosaccharide molecules containing a trisaccharide of 3-deoxy-D-manno-octulopyranosonic acid in the inner core region and a glucose rich outer core whose structure is the following: [structure: see text] R=H in the main oligosaccharide and beta-Glc in the minor product. The bacterium was grown on aromatic (phenol and benzoic acid) and nonaromatic carbon sources and the core oligosaccharide resulted to occur always with this novel structure.     

Structural determination of novel lacto-N-decaose and its monofucosylated analogue from human milk by electrospray tandem mass spectrometry and 1H NMR spectroscopy

We have isolated and characterised two neutral oligosaccharides, one nonfucosylated and the other monofucosylated, from human milk that are based on the doubly branched lacto-N-decaose core. Their structures have been determined by a combined use of electrospray tandem mass spectrometry (ES-MS/MS) and NMR spectroscopy. The sequences of the three branches resulted from the double-branching, including the identity and location of the blood-group-related Lewis determinant and partial linkages, were elucidated by the unique method of high sensitivity negative-ion ES-MS/MS analysis. Their full structure assignment was completed by methylation analysis and 1H NMR. The monofucosylated lacto-N-decaose, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc is a novel sequence, whereas the nonfucosylated lacto-N-decaose, Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc, has not been isolated and identified as an individual oligosaccharide.