Anne Dell

F.A.B.-Mass Spectrometry of Carbohydrates

Publisher Summary This chapter provides an overview of fast atom bombardment-mass spectrometry (FAB-MS) of carbohydrates. It focuses on those carbohydrate chemists who are not specialists in mass spectrometry and discusses the experimental aspects of FAB-MS. It also gives an indication of the types of problems that can now be solved by using the technique. The FAB-MS technique currently plays two major roles in carbohydrate structure analysis: (1) molecular weight determination, for which it has superseded field-desorption-mass spectrometry (FD-MS) and, to a large extent, chemical ionization and (2) sequence assignment, where it has very largely replaced direct-probe, electron-impact mass spectrometry, particularly for high-molecular weight compounds. On its own, FAB-MS cannot solve the complete structure of a carbohydrate and it should always be incorporated into experimental programs that include the well established methods of methylation analysis, enzymic digestion, chemical degradation, and nuclear magnetic resonance (NMR) spectroscopy. The chapter discusses the principles of FAB-MS technique and describes the choice of supporting matrix and matrix additives. It also provides an overview of sample purity and the analysis of mixtures in FAB-MS and elaborates the FAB-MS of high-molecular weight samples.

Identification and location of l-glycerate, an unusual acyl substituent in gellan gum☆

Abstract Gellan gum, the extracellular polysaccharide produced by Pseudomonas elodea , had been shown to be composed of a repeating, partially acetylated tetrasaccharide. Through the use of selective solvolysis of the rhamnosyl linkages in the polysaccharide with anhydrous, liquid hydrogen fluoride at −40°, oligosaccharides representing the repeating unit of the polymer in various acylated forms have now been generated. By a combination of n.m.r. spectroscopy, f.a.b.-mass spectrometry, and gas-liquid chromatographic analysis, these oligomers were shown to be substituted mainly with l -glyceric esters on O-2 of what was the 3-linked d -glucose in the original polymer, and substituted to a lesser degree with an acetic ester at O-6 of the same residue. The native gum must have the following structure.

The amino acid sequence of the delta haemolysin of Staphylococcus aureus

The 6 haemolysin of Sfaphylococcus aureus is one of a number of extracellular cytolytic and cytotoxic polypeptides produced by most strains of the microorganism [l-4] and is notable for its physlco-chemical and biological properties. It is stable to boiling and soluble in chloroform-methanol (2: 1, v/v) and aqueous solutions (reviewed [4]) and is inhibited by phospholipids, fatty acids, and normal serum lipoproteins [5-71. Three different purification procedures [8-lo] yield material with broadly similar properties and an amino acid analysis which is notable for the absence of arginine, proline, histidine, tyrosine and cysteine, and the presence of a high proportion of hydrophobic amino acids. Although most earlier studies suggested that native 6 haemolysin was likely to have mol. wt >lOO 000, it was apparent from gel permeation chromatography under denaturing conditions that the minimum molecular weight for the polypeptide subunit was likely to be <6000 [lo]. This investigation of the primary structure of 6 haemolysin was prompted by the realization that the solvent-transfer method 181 provided substantial quantities of highly purified 6 haemolysin from culture filtrates of S. aureus, that it did so more rapidly and reproducibly than other methods [7,8] and that the polypeptide shared all of the characteristic amphipathic and cytolytic properties observed with preparations of 6 haemolysin previously purified by ion exchange or adsorption chromatography and by gel filtration. The most striking properties of 6 haemolysin are

Fast-atom-bombardment mass-spectrometry for carbohydrate-structure determination

Abstract The potential of fast-atom-bombardment (f.a.b.) mass-spectrometry in the carbohydrate field was assessed with the aid of unmodified, permethylated, and peracetylated oligosaccharides and glycosphingolipids. F.a.b. spectra are presented for ( d -Gal→ d -GlcNAc) 5 →( d -Man) 3 → d -GlcNAc, permethylated ( d -Gal→ d -GlcNAc) 5 →( d -Man) 3 - d -[ 2 H]GlcNAcol, permethylated gangliotetraosylceramide, and reduced and peracetylated tetra- to hepta-saccharides from human milk. From unmodified oligosaccharides, pseudomolecular ions (M + Na + ) were obtained as major ions in the high-mass range. Permethylated oligosaccharides and glycosphingolipids gave pseudomolecular ions (M + H + ) of high intensity, together with fragment ions of high diagnostic importance. At ambient temperature, f.a.b. spectra could only be obtained for the lower homologs of per- O -acetylated oligosaccharides. Reduced and peracetylated penta- to hepta-saccharides from human milk gave f.a.b. spectra only after heating of the target.

Characterization of a structurally complex heptasaccharide isolated from the pectic polysaccharide rhamnogalacturonan II

Abstract A heptasaccharide was released from the plant cell-wall, pectic polysaccharide rhamnogalacturonan II by selective acid hydrolysis of the glycosidic linkages of apiosyl residues. The heptasaccharide was purified to homogeneity by gel filtration and anion-exchange chromatography. Some of the heptasaccharide molecules were found to be mono- and some di- O -acetylated, but the location of the acetic ester groups was not determined. The heptasaccharide was found to have the following structure, where AceA = an aceryl (3- C -carboxy-5-deoxy- l -xylosyl) residue, and Api = an apiose residue.

The cyclic structure of β-d-(1→2)-linked d-glucans secreted by Rhizobia and Agrobacteria

Abstract The β- d -(1→2)-linked d -glucans secreted by Rhizobia and Agrobacteria have been characterized by using fast-atom bombardment—mass spectrometry and 13 C-nuclear magnetic resonance spectroscopy. The d -glucans are shown to be unbranched, cyclic molecules varying in size from 17 to at least 24 residues. Some of these molecules are fragmented during permethylation procedures.

The discernible, structural features of the acidic polysaccharides secreted by different Rhizobium species are the same

Abstract Octasaccharide repeating-units have been isolated from the acidic polysaccharides secreted by Rhizobium trifolii strain NA30, R. trifolii strain LPR5, R. leguminosarum strain LPR1, and R. phaseoli strain LPR49. ( R. trifolii is the symbiont of clover, R. leguminosarum , of peas, and R. phaseoli , of beans). The repeating units were formed by treating the polysaccharides with an enzyme produced by a bacteriophage. The glycosyl sequence and the structures and locations of the non-glycosyl substituents were shown to be identical for repeating units derived from all of these polysaccharides, except for that derived from the polysaccharide produced by R. trifolii NA30. Therefore, the discernible structural features of the acidic polysaccharides secreted by Rhizobium species cannot be the determinant of host specificity. In support of this conclusion is the observation that R. trifolii LPR5045, produced by curing R. trifolii LPR5 of its Sym plasmid (the Sym plasmid is required for symbiosis and host specificity), secreted a polysaccharide having the same structure (including identities and locations of nonglycosyl substituents) as that of the polysaccharide secreted by its plasmid-containing parent. Thus, the structural genes that encode for synthesis of the acidic polysaccharide secreted by R. trifolii LPR5045 are not located on the Sym plasmid, and neither are the genes that encode for synthesis and attachment of non-glycosyl substituents of the polysaccharide. The possibility remains that a quantitatively minor component of the acidic polysaccharide could be a host-specific determinant.

Structural characterization of oligosaccharides isolated from the pectic polysaccharide rhamnogalacturonan II

Abstract Rhamnogalacturonan II (RG-II) is a structurally complex pectic ( d -galactosyl-uronic acid-rich) polysaccharide that is present in the primary (growing) cell-walls of higher plants. RG-II is composed of ∼60 glycosyl residues. The isolation and structural characterization of 23 oligosaccharide fragments of the residue of RG-II that remained after removal of hepta- and di-saccharides by partial hydrolysis with acid are reported. In order to obtain the oligosaccharide fragments characterized herein, the carboxyl groups of RG-II were dideuterio-reduced, and the carboxyl-reduced polysaccharide was per-O-methylated. The per-O-methylated polysaccharide was fragmented by partial hydrolysis with acid, producing partially O-methylated oligosaccharides. These derivatized oligosaccharides were reduced, to afford a mixture of partially O-methylated oligoglycosyl-alditols, which was then per-O-methylated. The structures of the resulting per-O-methylated oligoglycosylalditols were determined by chemical-ionization mass spectrometry, electron-impact mass spectrometry, fast-atom-bombardment mass spectrometry, 1H-n.m.r. spectroscopy, and analysis of corresponding, partially O-acetylated, partially O-methylated alditols. Seventeen of the oligosaccharides isolated from RG-II were parts of a single heptasaccharide, namely.

The enterobacterial common-antigen, a cyclic polysaccharide.

Structural studies of the enterobacterial common-antigen, using chemical methods and fast-atom-bombardment mass spectrometry, indicate that it is a cyclic polysaccharide, composed of four, five, and, to a smaller extent, six trisaccharide repeating-units. In the structure of the antigen, given below, D-Fuc4NAc stands for 4-acetamido-4,6-dideoxy-D-galactose.

Structure determination of carbohydrates and glycosphingolipids by fast atom bombardment mass spectrometry

Abstract We have used fast atom bombardment mass spectrometry to aid the structural analysis of blood group glycosphingolipids, carbohydrates excreted by gangliosidosis patients, glucans from fast growing Rhizobium species and a variety of plant cell wall oligosaccharides. Samples have been examined in their native form or as acetyl, permethyl, methoxime or pentafluorobenzyloxime derivatives. Negative FAB spectra of permethylated carbohydrates have been obtained for the first time by salt dosing. High sensitivity for permethylated samples has been achieved in the positive mode by ammonium cationisation. Spectra up to m/z 6620 are reported for permethylated glucans.