Structural differentiation of uronosyl substitution patterns in acidic heteroxylans by electrospray tandem mass spectrometry

The structures of two oligomers of acidic xylo-oligosaccharides (XOS) of the same molecular weight (634 Da), Xyl(2)MeGlcAHex and Xyl(2)GlcA(2) were differentiated by electrospray tandem mass spectrometry (ESI-MS/MS). These oligomers were present in a mixture of XOS obtained by acid hydrolysis of heteroxylans extracted from Eucalyptus globulus wood (Xyl(2)MeGlcAHex) and Olea europaea olive fruit (Xyl(2)GlcA(2)). In the ESI-MS spectra of the XOS, ions at m/z 657 and 652 were observed and assigned to [M + Na](+) and [M + NH(4)](+), respectively. The ESI-MS/MS spectrum of [M + Na](+) ion of Xyl(2)MeGlcAHex showed the loss of Hex residue from the reducing end followed by the loss of MeGlcA moiety. Simultaneously, the loss of a Xyl residue from either the reducing or the non-reducing ends was detected. On the other hand, the fragmentation of Xyl(2)GlcA(2) occurs mainly by the loss of one and two GlcA residues or by the loss of the GlcAXyl moiety, due to the glycosidic bond cleavage between the two Xyl residues. Loss of one and two CO(2) molecules was only observed for this oligomer, where the GlcA are in vicinal Xyl residues. The ESI-MS/MS spectra of [M + NH(4)](+) of both oligomers showed the loss of NH(3), resulting in the protonated molecule, where the presence of ions assigned as protonated molecules of aldobiuronic acid residues, [MeGlcA - Xyl + H](+) and [GlcA - Xyl + H](+), are diagnostic ions of the presence of MeGlcA and GlcA moieties in XOS. Since these structures occur in small amounts in complex acidic XOS mixtures and are very difficult, if possible, to isolate, tandem mass spectrometry revealed to be a powerful tool for the characterization of these types of substitution patterns present in heteroxylans.     

Electrospray tandem mass spectrometry of underivatised acetylated xylo-oligosaccharides

Acetylated neutral (Xyl(n)Ac(m)) and acidic xylo-oligosaccharides (Xyl(n)Ac(m)MeGlcA, and Xyl(n)Ac(m)MeGlcAHex) obtained by partial acid hydrolysis of Eucalyptus globulus wood glucuronoxylans and fractionated by preparative ligand exchange/size-exclusion chromatography were identified by electrospray ionisation mass spectrometry (ESI-MS). Low molecular weight acetylated xylo-oligosaccharides were studied by ESI-tandem mass spectrometry (MS/MS). All the acetylated xylo-oligosaccharides showed an abundant ion due to the neutral loss of 60 Da (CH(3)CO(2)H) in the MS/MS spectra. The presence of diacetylated xylo-oligosaccharides was confirmed by the ions formed by loss of two molecules of acetic acid. Furthermore, characteristic [Xyl(res)Ac(2)+Na](+) and [XylAc(2)+Na](+) ions, and ions due to loss of XylAc(2), indicate that both acetyl groups are located in the same Xyl residue. On the other hand, losses of Xyl(res)Ac and XylAc are also observed as well as [Xyl(res)Ac+Na](+) and [XylAc+Na](+) , indicating the location of both acetyl groups in different Xyl residues, in some cases even in adjacent xyloses. The MS/MS spectra of triacetylated xylo-oligosaccharides were complex due to the presence of different isobaric xylo-oligosaccharides containing the acetyl groups at different locations in the xylo-oligosaccharide backbone. In the MS/MS spectra of acidic xylo-oligosaccharides, the ion at m/z 387, [Xyl(res)AcMeGlcA+Na](+), indicates that the acetyl groups are preferentially linked to Xyl substituted with MeGlcA. However, acidic xylo-oligosaccharides with the acetyl and 4-O-methylglucuronic acid groups in different Xyl residues were also identified. In neutral and in acidic xylo-oligosaccharides several possible locations of the acetyl groups were identified, namely at terminal positions. In summary, ESI-MS/MS is shown to be a powerful tool for the characterisation of acetylated patterns in complex mixtures of oligosaccharides.     

Separation and detection of oxidation products of fluorodeoxyglucose and glucose by high-performance liquid chromatography-electrospray ionisation mass spectrometry

2-Deoxy-2-[18F]fluoro-D-glucose ([18F] FDG), the most popular positron emitting radiopharmaceutical, may oxidise by autoradiolysis in aqueous solution. The aim of this work was to use LC-MS for determination of the oxidation products of fluorodeoxyglucose and glucose (Glc) obtained by oxidation with Fenton's reagent. Asahipak NH2P-50 polyamide silica column and acetonitrile-0.025% aqueous ammonium formate (80:20 (v/v)) eluent were utilised with an Agilent 1100 HPLC-MS instrument. Ten major oxidation products of FDG and Glc were separated and identified by mass spectrometry: 2-fluorogluconic acid, 2-fluoroglucuronic acid, 2-oxoerythronic acid, arabinose, arabonic acid, araburonic acid, erythrose, erythrulose, gluconic acid, and glucuronic acid. The most intensive electrospray ionisation signals were found in the negative ion spectra and were due to HCOO- adducts, the other acids being in their lactone forms.     

The characterisation of coumarins from selected structural classes by electrospray ionisation quadrupole time-of-flight tandem mass spectrometry

Electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS) has been used for characterisation of a selection of naturally occurring and synthetic coumarins from different structural classes. The product ions, suggested in earlier studies by electrospray ionisation ion trap mass spectrometry (ESI-MS(n)), are unequivocally established for the representative coumarins by virtue of accurate mass measurement. Synthetic coumarins that are unsubstituted in the heterocyclic ring give rise to a major product ion by loss of CO(2), whereas those substituted in the heterocyclic ring generally undergo alternative fragmentation releasing neutral species such as ketene or methyl ketene. Naturally occurring coumarins, unsubstituted in the heterocyclic ring and substituted in the benzene ring with chains or rings of hydrocarbons and oxygen, principally fragment at the side chain releasing unsaturated hydrocarbons. The ESI-QTOF-MS/MS behaviour of some naturally occurring and synthetic quinolines which are structurally similar or fragment similarly are included where appropriate.     

Electrospray ionisation mass spectral studies on hydrolysed products of sulfur mustards

Off-site detection of the hydrolysed products of sulfur mustards in aqueous samples is an important task in the verification of Chemical Weapons Convention (CWC)-related chemicals. The hydrolysed products of sulfur mustards are studied under positive and negative electrospray ionisation (ESI) conditions using an additive with a view to detecting them at trace levels. In the presence of cations (Li(+), Na(+), K(+) and NH(4) (+)), the positive ion ESI mass spectra of all the compounds include the corresponding cationised species; however, only the [M+NH(4)](+) ions form [M+H](+) ions upon decomposition. The tandem mass (MS/MS) spectra of [M+H](+) ions from all the hydrolysed products of the sulfur mustard homologues were distinct and allowed these compounds to be characterised unambiguously. Similarly, the negative ion ESI mass spectra of all the compounds show prominent adducts with added anions (F(-), Cl(-), Br(-), and I(-)), but the [M-H](-) ion can only be generated by decomposition of an [M+F](-) ion. The MS/MS spectra of the [M-H](-) ions from all the compounds result in a common product ion at m/z 77. A precursor ion scan of m/z 77 is shown to be useful in the rapid screening of these compounds in aqueous samples at trace levels, even in the presence of complex masking agents, without the use of time-consuming sample preparation and chromatography steps. An MS/MS method developed to measure the detection limits of the hydrolysed products of sulfur mustards found these to be in the range of 10-500 ppb.     

Structural analysis of oligosaccharides by atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap mass spectrometry.

An ion source incorporating a fibre optic interface has been constructed for atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap mass spectrometry. The configuration has been applied to the study of linear and complex oligosaccharides. Multi-stage tandem mass spectrometry (MSn, n = 2-4) experiments carried out in the ion trap enable extended fragmentation pathways to be investigated that yield structural information. Collisional activation of sodiated oligosaccharides, as demonstrated on the model compound maltoheptaose, produces primarily B and Y fragments resulting from cleavage of glycosidic bonds; fragments from cross-ring cleavages are also observed following further stages of tandem mass spectrometry, providing additional linkage information. The analyses of mixtures of complex oligosaccharides are demonstrated for N-linked glycans from chicken egg glycoproteins and a ribonuclease glycan mixture. Mass spectrometric and tandem mass spectrometric data for sugars with molecular weights up to 4000 Da is shown for mixtures of linear dextrans and N-linked glycans. The use of MSn (n = 3, 4) on these complex molecules enabled structural information to be elucidated that confirms data observed in the MS/MS spectra.     

Negative ion mode matrix-assisted laser desorption/ionisation time-of-flight mass spectrometric analysis of oligosaccharides using halide adducts and 9-aminoacridine matrix

9-Aminoacridine was established as a matrix for the detection of neutral oligosaccharides in negative ion mode matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. Sodium iodide proved to be a useful additive inducing formation of stable iodide adducts of the analytes, in particular for oligosaccharides with a degree of polymerisation (DP) of three and higher. Lower oligomers (DP <3) and monosaccharides show more stable adducts with chloride ions. After optimisation of the sample preparation procedure, limits of quantitation were determined for alpha-cyclodextrin and cellopentaose at 7 and 13 pmol, respectively, with a linear detector response over two concentration orders. The iodide additive could be successfully employed on MALDI-TOF mass spectrometers with vacuum and atmospheric pressure ion sources. The value of the new method to solve biological problems has been demonstrated by the analysis of a mixture of beta-glucane elicitors isolated from the cell walls of Phytophthora sojae.     

Capillary zone electrophoresis of basic analytes in methanol as non-aqueous solvent mobility and ionisation constant

The electrophoretically relevant properties of monoacidic 21 bases (including common drugs) containing aliphatic or aromatic amino groups were determined in methanol as solvent. These properties are the actual mobilities (that of the fully ionised weak bases), and their pKa values. Actual mobilities were measured in acidic methanolic solutions containing perchloric acid. The ionisation constants of the amines were derived from the dependence of the ionic mobilities on the pH of the background electrolyte solution. The pH scale in methanol was established from acids with known conventional pK*a values in this solvent used as buffers, avoiding thus further adjustment with a pH sensitive electrode that might bias the scale. Actual mobilities in methanol were found larger than in water, and do not correlate well with the solvent's viscosity. The pK*a values of the cation acids, HB-, the corresponding form of the base, B, are higher in methanol, whereas a less pronounced shift was found than for neutral acids of type HA. The mean increase (compared to pure aqueous solution) for aliphatic ammonium type analytes is 1.8, for substituted anilinium 1.1, and for aromatic ammonium from pyridinium type 0.5 units. The interpretation of this shift was undertaken with the concept of the medium effect on the particles involved in the acid-base equilibrium: the proton, the molecular base, B, and the cation HB+.     

On-line liquid chromatography-electrospray ionisation mass spectrometry for kappa-carrageenan oligosaccharides with a porous graphitic carbon column

Enzymatically digested oligosaccharides of kappa-carrageenans were separated on a porous graphitic carbon (PGC) column and characterised on-line by electrospray ionisation mass spectrometry (ESI-MS). Two different developing ions were applied. Among them ammonium hydrogencarbonate showed more eluting power as it should on normal anion-exchange stationary phases. The oligosaccharides were detected by ESI-MS as fully deprotonated oligosaccharides.     

Oligosaccharide analysis using anion attachment in negative mode electrospray mass spectrometry

Eleven different anionic species were able to form adducts with neutral oligosaccharides at low cone voltage in negative ion mode electrospray mass spectrometry. Among them, fluoride and acetate have the ability to significantly enhance the absolute abundance of [M - H](-) for neutral oliogosaccharides, which otherwise have low tendencies to deprotonate due to the lack of a highly acidic group. Evidence shows that the source of high abundances of [M - H](-) for neutral oligosaccharides arises from the decomposition of [M + F](-) and [M + Ac](-) with neutral losses of HF and HAc, respectively. The chloride adducts have the best stability among all the adduct species investigated, and chloride adducts consistently appeared in higher abundances relative to [M - H](-). In tandem mass spectrometry (ES-MS/MS) experiments, upon collision induced dissociation (CID), F(-) and Ac(-) adducts gave purely analyte-related product ions, i.e., no detection of the attaching anion and no incorporation of these anions into decomposition products. Cl(-) adducts produced both Cl(-) and analyte-related product ions. For the above three anions, CID of adduct species may be used for structural determination of neutral oligosaccharides because, in each case, structurally-informative fragment ions were produced. In the presence of F(-) and Ac(-), simultaneous detection of acidic and neutral oligosaccharides was achieved, because the problem of the presence of an acidic group that can impede the deprotonation of a neutral oligosaccharide was minimized. The ratio of Cl(-):non-Cl-containing product ions obtained in CID spectra of chloride adducts of disaccharides was used to differentiate anomeric configurations of disaccharides. Density functional theory (DFT) was employed to evaluate the optimized structures of chloride adducts of disaccharides, and it was found that chloride anions favor close contact with the hydrogen from the anomeric hydroxyl group. Multiple hydrogen bonding further stabilizes the chloride adduct.     

Electron detachment dissociation of fluorescently labeled sialylated oligosaccharides

We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared with infrared multiphoton dissociation. Neutral losses and satellite ions such as C-2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA-labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared with 2-AA-labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag.     

Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis

Negative ion nano-liquid chromatography/mass spectrometry (nano-LC/MS) and tandem mass spectrometry (nano-LC/MS(2)), using graphitised carbon as separating medium, were explored for analysing neutral and acidic O-linked and N-linked oligosaccharide alditols. Compared to the sensitivity of capillary LC/MS (flow rate of 6 microL/min) coupled with a conventional electrospray ionisation source, the nano-LC/MS (flow rate of 0.6 microL/min) with a nanoflow ion source was shown to increase the sensitivity ten-fold with a detection limit in the low-femtomole range. The absolute signals for the [M-nH](n-) ions of the oligosaccharides were increased 100-fold, enabling accumulation of high-quality fragmentation data in MS(2) mode, in which detection of low abundant sequence ions is necessary for characterisation of highly sialylated N-linked oligosaccharides. Oligosaccharides with high numbers of sialic acid residues gave dominant fragments arising from the loss of sialic acid, and less abundant fragments from cleavage of other glycosidic bonds. Enzymatic off-line desialylation of oligosaccharides in the low-femtomole range prior to MS(2) analysis was shown to increase the quality of the spectra. Automated glycofragment mass fingerprinting using the GlycosidIQ software confirmed the oligosaccharide sequence for both neutral desialylated as well as sialylated structures. Furthermore, the use of graphitised carbon nano-LC/MS enabled the detection of four sialylated O-linked oligosaccharides on membrane proteins from ovarian tissue (5 microg of total amount of protein).     

Recent advances in the research of bacterial glucuronosyltransferases

Many bacterial extracellular polysaccharides, which play an important role in various biological processes, contain glucuronic acid (GlcA) as a major component. Glucuronosyltransferase (GlcA-T) is the enzyme responsible for GlcA attachment in the biosynthesis of these polysaccharides. GlcA-T has recently attracted significant attention because of its biological activities as well as its potential application to chemoenzymatic synthesis of GlcA-containing oligosaccharides and polysaccharides that are difficult to achieve chemically due to the problems related to glycosylation reactions with GlcA. At present, nine GlcA-Ts derived from Xanthomonas campestris, Pasteurella multocida, Escherichia coli, Sphingomonas paucimobilis, and Streptococci have been characterized. This article reviews the recent progresses made in understanding the structures, functions, physical, and chemical properties of reported GlcA-Ts.     

Neutral and acidic products derived from hydroxyl radical‐induced oxidation of arabinotriose assessed by electrospray ionisation mass spectrometry

The oxidation of α‐(1 → 5)‐l ‐arabinotriose (Ara₃), an oligosaccharide structurally related to side chains of coffee arabinogalactans, was studied in reaction with hydroxyl radicals generated under conditions of Fenton reaction (Fe²⁺/H₂O₂). The acidic and neutral oxidation products were separated by ligand exchange/size‐exclusion chromatography, subsequently identified by electrospray ionisation mass spectrometry (ESI–MS) and structurally characterised by tandem MS (ESI–MS/MS). In acidic fraction were identified several oxidation products containing an acidic residue at the corresponding reducing end of Ara₃, namely arabinonic acid, and erythronic, glyceric and glycolic acids formed by oxidative scission of the furanose ring. In neutral fractions were identified derivatives containing keto, hydroxy and hydroperoxy moieties, as well as derivatives resulting from the ring scission at the reducing end of Ara₃. In both acidic and neutral fractions, beyond the trisaccharide derivatives, the corresponding di‐ and monosaccharide derivatives were identified indicating the occurrence of oxidative depolymerisation. The structural characterisation of these oxidation products by ESI–MS/MS allowed the differentiation of isobaric and isomeric species of acidic and neutral character. The species identified in this study may help in detection of roasting products associated with the free radical‐mediated oxidation of coffee arabinogalactans. Copyright © 2014 John Wiley & Sons, Ltd.     

Investigation of the ionisation and fragmentation behaviour of different nitroaromatic compounds occurring as polar metabolites of explosives using electrospray ionisation tandem mass spectrometry

In order to develop a liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) method for identification and quantification of polar metabolites of explosives using a triple quadrupole system, the mass spectrometric ionisation and fragmentation behaviour of different nitrophenols, nitro- and aminonitrobenzoic acids, nitrotoluenesulfonic acids, and aminonitrotoluenes was investigated. Due to their different molecular structures, the substances concerned showed a very different ionisation efficiency in the ESI process. Interestingly, 2,4-dinitrobenzoic acid yielded no mass signals in the Q1 scan suggesting a thermal decarboxylation in the ion source, whereas the corresponding 3,5-isomer showed a high ionisation yield. Using negative ionisation polarity, carboxylic, phenolic, and sulfonic acid groups were deprotonated resulting in molecular anions, which could be fragmented in a collision cell. A pronounced dependency of the produced fragment ion series on the kind and position of substituents at the nitrobenzene ring (ortho effects) was observed and exploited for the development of substance-specific detection methods in the multiple reaction monitoring mode. In case of benzoic and sulfonic acids, decarboxylation and desulfonation, respectively, were observed as the most frequent fragmentation reactions. Furthermore, besides loss of NO(2), NO fragmentation occurred and preceded a decarbonylation of the benzene ring. The expulsion of the open-shell molecules NO and NO(2) led to a variety of distonic radical anions.     

Exploiting the carboxylate chemical shift to resolve degenerate resonances in spectra of 13C-labelled glycosaminoglycans

Glycosaminoglycans (GAG) are important vertebrate extracellular matrix polysaccharides that comprise repeated units of an acidic and an N-acetylated sugar. The constituent acidic sugars are central to their biological functions, but have been largely inaccessible to NMR because the (1)H resonances overlap with those from other residues. Here, pulse sequences that address this failure are developed using (13)C-enriched oligosaccharides of the glycosaminoglycan, hyaluronan, as model systems. Two pulse sequences are presented that exploit the unique chemical shifts and scalar couplings present at the carboxylate moiety to filter out coherences from the N-acetylated sugars and produce simple spectra containing only resonances from the acidic sugars. The first sequence uses one-bond couplings to correlate the carboxylate carbon with the adjacent carbon and its directly attached proton, while the second sequence exploits a long-range coupling to correlate the carboxylate carbon with the anomeric proton and carbon of the same residue. In addition, inclusion of an isotropic mixing block into these sequences allows resonances from the otherwise degenerate ring protons to be resolved. Spectra from the hyaluronan tetra- and hexasaccharides show that all glucuronic acid (GlcA) residues can be resolved from one another, allowing nuclei to be assigned in a sequence-specific manner. However, in some spectra, resonances are observed at positions not predicted by spin-operator analysis, and simulations reveal that these additional magnetisation transfers result from strong-coupling. These experiments represent a foundation from which new structural and biochemical information can be obtained in a sequence-specific manner for the acidic sugar residues in hyaluronan and other glycosaminoglycans.     

The signal-to-noise ratio as the measure for the quantification of lysophospholipids by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

The subject of this report is the determination of lysophospholipids; lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidic acid, by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The mean signal-to-noise ratio (S/N) was used for the first time as a measure of lysophospholipid concentration. Two different sample preparation procedures were applied, the 'standard' procedure and the 'premix' in order to check to what extent these methods influence the results of the lysophospholipid quantification. Results can be summarised as follows: (a) All classes of lysophospholipids can be easily and sensitively analysed by MALDI-TOF MS. The smallest detectable amount of lysophospholipids was 0.09 pmol on the sample plate. That is about two orders of magnitude lower than the amount detectable by standard chromatographic methods. (b) The mean S/N of all peaks detected in the positive ion mass spectra can be used as a measure of the lysophospholipid concentration. Whereas the S/N for neutral lysophospholipids correlated with the applied concentrations only when the samples were analysed as 'premix', the sample preparation and application procedure did not influence the quantification of acidic lysophospholipids. The standard deviations were not higher than 10% of the mean value. (c) All spectra were additionally analysed in the presence of CsCl. The addition of caesium ions makes the peak identification unambiguous in phospholipid mixtures, but the Cs adducts of lysophospholipids do not properly reflect their concentration and, therefore, they were not useful for quantification. (d) The applicability of the method was demonstrated on the organic extract of human neutrophils.     

Identification of sulfoglycolipids from the alga Porphyridium purpureum by matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight mass spectrometry

Sulfoglycolipids, isolated from different phototrophic organisms, particularly plants and algae, have already been identified as bioactive compounds. In addition to their antiviral activity their influence on the immune response in mammalian cells is the focus of many studies. For the first time it has been possible to investigate purified sulfoquinovosyldiacylglycerols (SQDGs) from the microalga Porphyridium purpureum by matrix-assisted laser desorption/ionisation (MALDI) in the negative ion reflectron mode. Thereby, different solid and ionic liquid matrices have been tested to improve signal intensity during the laser ionisation. By using the MALDI Trap time-of-flight (ToF) multiple-stage (MS(n)) hybrid mass spectrometer the fatty acid compositions of the SQDGs were analysed by MS, and confirmed by MS(2) and MS(3) experiments. Thereby, hexadecanoic acid (C16:0), octadecadienoic acid (C18:2), eicosatetraenoic acid (C20:4), and eicosapentaenoic acid (C20:5) were detected in the purified fraction of SQDGs. The localisation of hexadecanoic acid (C16:0) at the sn-2 position, and unsaturated fatty acids at the sn-1 position of the SQDGs, determined by specific enzymatic hydrolysis, marks a procaryotic biosynthesis of SQDGs in the eucaryotic alga cells.     

Identification of dinucleoside polyphosphates by matrix-assisted laser desorption/ionisation post-source decay mass spectrometry

Dinucleoside polyphosphates are a group of intra- and extracellular mediators controlling numerous physiological functions. In this study dinucleoside polyphosphates were examined by positive ion matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MADLI-TOFMS). 3-Hydroxypicolinic acid was used as UV-absorbing matrix. For the individual dinucleoside polyphosphates Ap(n)A (n = 2-7), Ap(n)G (n = 2-6) and Gp(n)G (n = 2-6), MALDI post-source decay (PSD) mass spectra were measured. Each mass peak in the MALDI-PSD mass spectra could be assigned to individual fragments of dinucleoside polyphosphates. The comparison of the fragmentation patterns of the dinucleoside polyphosphates presented here demonstrates that dinucleoside polyphosphates preferably cleave to fragment ions consisting of the corresponding mononucleoside polyphosphates as well as the corresponding nucleosides and bases during flight in the field-free drift path of the MALDI mass spectrometer. Therefore, the MALDI-PSD approach described here is suitable for identification of other dinucleoside polyphosphates. The present MALDI-PSD mass spectra may be used as MALDI-PSD mass reference spectra for future identification of dinucleoside polyphosphates and other nucleotides.     

Analysis of 8-aminonaphthalene-1,3,6-trisulfonic acid labelled N-glycans by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a fast and efficient analytical method which is now widely used in glycobiology for the separation and quantification of free or glycoprotein-released oligosaccharides. However, since identification by FACE of N-glycan structures is only based on their electrophoretic mobility after labelling with 8-aminonaphthalene-1,3, 6-trisulfonic acid (ANTS), co-migration of derived glycans on gel could occur which may result in erroneous structural assignments. As a consequence, a protocol was developed for the fast and efficient matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometric analysis of ANTS-labelled N-glycans. N-Glycans were isolated from plant and mammalian glycoproteins, reductively aminated with the charged fluorophore 8-aminonaphthalene-1, 3, 6-trisulfonic acid (ANTS) and separated using high resolution polyacrylamide gel electrophoresis. The ANTS-labelled glycans were eluted from FACE gel slices and then analysed by MALDI-TOF mass spectrometry in negative ion mode. Using 3-aminoquinoline containing 2.5 mM citrate NH(4)(+) as matrix, neutral N-linked N-glycans, as well as labelled sialylated oligosaccharides, were found to be easily detected in the 2-10 picomole range giving rise to ?M - H(-) ions.