Assignment of the stereochemistry and anomeric configuration of structurally informative product ions derived from disaccharides: infrared photodissociation of glycosyl-glycolaldehydes in the negative ion mode

Using mass spectrometry in the negative ion mode, m/z 221 ions are frequently observed as product ion substructures derived from reducing disaccharides having 2, 4, or 6 linkages. The ions have been shown to be glycosyl-glycolaldehydes. All 16 stereochemical variants of their pyranosides were prepared and evaluated by infrared photodissociation, in addition to HexNAc-glycolaldehyde variants (m/z 262) of 2-acetamido-2-deoxy-d-glucose and 2-acetamido-2-deoxy-d-galactose. The stereochemistry and anomeric configuration of these ions were differentiated in the gas phase using a Fourier transform ion cyclotron resonance spectrometer with infrared multiphoton dissociation at 10.6 μm. Results were compared to those obtained by collision-induced dissociation. In some cases, differentiation was far preferable using infrared photodissociation; in others, collision-induced dissociation was preferred. Using an instrument that interfaced a linear trap with a Fourier transform ion cyclotron resonance spectrometer, either dissociation technique could be used to optimally discriminate between isomers. With infrared photodissociation, spectral differences were highly statistically significant, even between pairs of isomers having spectra that appeared to be visually somewhat similar (p <1 × 10⁻⁹, student’s t-test for key discriminatory ions). Comparisons among different instruments suggest that physical standards of the stereochemical variants of these ions will be required for their detailed structural assignments in unknowns, as some variation was observed among instruments, both using infrared photodissociation and collision-induced dissociation.     

Differentiation of the anomeric configuration and ring form of glucosyl-glycolaldehyde anions in the gas phase by mass spectrometry: isomeric discrimination between m/z 221 anions derived from disaccharides and chemical synthesis of m/z 221 standards

Mass spectrometry of disaccharides in the negative-ion mode frequently generates product anions of m/z 221. With glucose-containing disaccharides, dissociation of isolated m/z 221 product ions in a Paul trap yielded mass spectra that easily differentiated between both anomeric configurations and ring forms of the ions. These ions were shown to be glucosyl-glycolaldehydes through chemical synthesis of their standards. By labeling the reducing carbonyl oxygen of disaccharides with 18O to mass discriminate between monosaccharides, it was established that the m/z 221 ions are comprised solely of an intact nonreducing sugar with a two-carbon aglycon derived from the reducing sugar, regardless of the disaccharide linkage position. This enabled the anomeric configuration and ring form of the ion to be assigned and the location of the ion to the nonreducing side of a glycosidic linkage to be ascertained. Detailed studies of experimental factors necessary for reproducibility in a Paul trap demonstrated that the unique dissociation patterns that discriminate between the isomeric m/z 221 ions could be obtained from month-to-month in conjunction with an internal energy-input calibrant ion that ensures reproducible energy deposition into isolated m/z 221 ions. In addition, MS/MS fragmentation patterns of disaccharide m/z 341 anions in a Paul trap enabled linkage positions to be assigned, as has been previously reported with other types of mass spectrometers.     

Adenosine in the venoms from viperinae snakes of the genus Bitis: identification and quantitation using LC/MS and CE/MS

Snake venoms are rich sources of toxic proteins and small molecules. This study was directed at molecules of molecular mass below 1 kDa. Thirty different venoms, of either neurotoxic or haemorrhagic type, were fractionated using size-exclusion chromatography. Only venoms of the Puff adder (Bitis arietans), Gaboon viper (Bitis gabonica), and Rhinoceros viper (Bitis nasicornis) exhibited large absorbance peaks at lambda(280 nm) in the total volume range of the chromatographic column indicating the presence of abundant low molecular mass material. Analysis of fractions containing this material using both HPLC and capillary electrophoresis interfaced with electrospray ion-trap mass spectrometry unequivocally established that the bioactive nucleoside, adenosine, was the major component. The concentrations of adenosine found (Puff adder--97.7 x 10(-6) mol L(-1); Gaboon viper--28.0 x 10(-6) mol L(-1); and Rhinoceros viper-56.8 x 10(-6) mol L(-1)) were above those required to activate all known sub-types of adenosine receptors. Adenosine may thus act at the site of envenomation causing local vasodilatation and may play a role in the subsequent systemic hypotension observed.     

Direct evidence for the covalent modification of glutathione-S-transferase P1-1 by electrophilic prostaglandins: implications for enzyme inactivation and cell survival

Glutathione-S-transferases (GST) catalyze the conjugation of electrophilic compounds to glutathione, thus playing a key role in cell survival and tumor chemoresistance. Cyclopentenone prostaglandins (cyPG) are electrophilic eicosanoids that display potent antiproliferative properties, through multiple mechanisms not completely elucidated. Here we show that the cyPG 15-deoxy-Delta(12,14)-PGJ2 (15d-PGJ2) binds to GSTP1-1 covalently, as demonstrated by mass spectrometry and by the use of biotinylated 15d-PGJ2. Moreover, cyPG inactivate GSTP1-1 irreversibly. The presence of the cyclopentenone moiety is important for these effects. Covalent interactions also occur in cells, in which 15d-PGJ2 binds to endogenous GSTP1-1, irreversibly reduces GST free-thiol content and inhibits GST activity. Protein delivery of GSTP1-1 improves cell survival upon serum deprivation whereas 15d-PGJ2-treated GSTP1-1 displays a reduced protective effect. These results show the first evidence for the formation of stable adducts between cyPG and GSTP1-1 and may offer new perspectives for the development of irreversible GST inhibitors as anticancer agents.     

Evidence in the formation of conjugated linoleic acids from thermally induced 9t12t linoleic acid: a study by gas chromatography and infrared spectroscopy

Thermally induced isomerisation leading to the formation of conjugated linoleic acids (CLAs) has been observed for the first time during the thermal treatment of 9t12t fatty acid triacylglycerol, and methyl ester. Fifteen microlitre portions of the triacylglycerol sample containing 9t12t fatty acid (trilinoelaidin) were placed in micro glass ampoules and sealed under nitrogen, then subjected to thermal treatment at 250 °C. The glass ampoules were removed at regular time intervals, cut open, and the contents were analysed by infrared spectroscopy using a single reflectance attenuated total internal reflectance crystal accessory. The samples were then subjected to derivatisation into their methyl esters. The methyl esters of the isomerised fatty acids were analysed by gas chromatography. The same procedure was repeated with methyl ester samples containing 9t12t fatty acid (methyl linoelaidate). Each sample was subjected to infrared measurements and gas chromatographic analysis after appropriate dilution in heptane.The results show that the thermally induced isomerisation of 9t12t fatty acids from both triacylglycerol molecules and methyl esters give identical CLA profiles as those found for the thermally induced isomerisation of 9c12c fatty acids. The infrared spectrometry provides additional evidence confirming the formation of CLA acids during thermal treatment. A mechanism for the formation of the CLAs from 9t12t fatty acid molecules is also formulated for the first time. This mechanism complements the pathways of formation of CLAs from 9c12c fatty acids during thermal treatment.     

A microtechnique for the analysis of free and conjugated indole-3-acetic acid in milligram amounts of plant tissue using a benchtop gas chromatograph-mass spectrometer

A microtechnique was developed for the quantification of indole-3-acetic acid (IAA) in plant samples of one milligram fresh weight or less. The method permitted quantification of both free and conjugated IAA using a benchtop gas chromatograph-mass spectrometer. New methods for sample purification with high recovery at microscale levels, together with simple changes that result in enhanced sensitivity of the instrumentation, allowed for a significant reduction in the amount of plant material required for analysis. Single oat (Avena sativa L.) coleoptile tips could be studied with this method and were found to contain free and total IAA levels of 137 and 399 pg · mg⁻¹ fresh weight, respectively. A single 5-d-old Arabidopsis thaliana (L.) Heynh. seedling was shown to contain 61 pg · mg⁻¹ fresh weight free IAA and 7850 pg · mg⁻¹ fresh weight of total IAA following basic hydrolysis. This microtechnique provides a way to accurately measure IAA levels in very small structures and individual seedlings, thus making it a valuable research tool for elucidating the role and distribution of auxin in relation to growth and development. Received: 1 May 1994 / Accepted: 25 June 1997