Showing papers in "Journal of Mass Spectrometry in 1997"

Ion Mobility Measurements and their Applications to Clusters and Biomolecules

Abstract: Ion mobility measurements can be used to obtain structural information for large polyatomic ions in the gas phase. The methods are flexible and can be applied to a wide range of chemical systems. This article reviews the development of these methods and discusses recent applications to complex ions such as atomic clusters and large biomolecules. © John Wiley & Sons, Ltd.

An Introduction to Quadrupole Ion Trap Mass Spectrometry

Abstract: A concise introduction is presented to the theory and application of quadrupole ion trap mass spectrometry. The presentation of the theoretical treatment is based on a demonstration of the equivalence of the force acting on an ion in a quadrupole field and the force derived from the Mathieu equation; this equivalence permits the application of the solutions of Mathieu’s equation to the confinement of gaseous ions. Resonant excitation, collision-induced dissociation, mass spectrometry, tandem mass spectrometry and chemical ionization are discussed in the context of analytical applications. Sample calculations of the trapping parametersqzand βz, the axial secular frequency, mass range, mass range extension and the magnitude of the potential well depth are given. © 1997 by John Wiley & Sons, Ltd.

Electrospray: Principles and Practice

Abstract: The basic principles underlying the electrospray process are reviewed without recourse to detailed discussion of mechanisms. The essential features of the practical implementation of electrospray (at various solution flow rates) are described and the nature of the resultant gas-phase ion population is discussed. The generation by electrospray of multiply charged ions creates complications in that spectral complexity is increased and the determination of charge number must precede the measurement of mass. Multiple charging is beneficial, however, in extending the mass range and improving fragmentation yield in tandem mass spectrometry. The current breadth of application of the technique (including the analysis of non-covalently bound species) and future developments are discussed. © 1997 by John Wiley & Sons, Ltd.

Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Sample Preparation Techniques Designed for Various Peptide and Protein Analytes

Abstract: This study encompasses a collection of experiences with regard to numerous matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) sample preparation techniques in terms of their suitability for different peptide and protein analytes. Variants of both established and new sample preparation techniques for the MALDI-MS analysis of peptides and proteins are described. The importance of matrix selection, matrix and analyte concentration, pH adjustment, crystallization conditions and the use of additives is evaluated. The tolerance of the different sample preparations towards salts, buffers, synthetic polymers, detergents, denaturants and other contaminants, and also the influence of the preparation methods on undesired amino acid side-chain oxidation, are investigated. Moreover, the performance of on-target tryptic digestion and on-target disulfide reduction is shown and a microscale purification procedure is described. According to this study, there is no universally applicable sample preparation for a broad variety of analytes. Rather, it is necessary to specifically adapt the sample preparation to the analyte properties. © 1997 John Wiley & Sons, Ltd.

Probing the Non‐covalent Structure of Proteins by Amide Hydrogen Exchange and Mass Spectrometry

Abstract: The rates at which hydrogens located at peptide amide linkages in proteins undergo isotopic exchange when a protein is exposed to D2O depend on whether these amide hydrogens are hydrogen bonded and whether they are accessible to the aqueous solvent. Hence, amide hydrogen exchange rates are a sensitive probe for detecting changes in protein conformation and dynamics. Hydrogen exchange rates in proteins are most often measured by NMR or Fourier transform IR spectroscopy. After a brief introduction to model kinetics used to relate amide hydrogen exchange rates to protein structure and dynamics, information required to understand and implement a new method that uses acid proteases and mass spectrometry to determine amide hydrogen exchange rates in proteins is presented. Structural and dynamic features affecting isotopic exchange rates can be detected and localized from the deuterium levels detected by mass spectrometry in proteolytic fragments of the protein. Procedures used to adjust for isotopic exchange occurring during the analysis, to extract isotope exchange rate constants from mass spectra and to link bimodal isotope patterns to protein unfolding and structural heterogeneity are also discussed. In addition, the relative merits of using mass spectrometry or NMR combined with amide hydrogen exchange to study protein structure and dynamics are discussed. The spatial resolution of hydrogen exchange results obtained by this method is typically in the range of 1-10 residues, which is substantially less than that obtained by high-resolution NMR, but sufficient to detect many functionally significant structural changes. Advantages in the areas of sensitivity, protein solubility, detection of correlated exchange and high molecular mass proteins make this approach particularly attractive for a wide range of studies.

SPECIAL FEATURE:TUTORIAL Slow Heating Methods in Tandem Mass Spectrometry

Abstract: Several approaches to ion activation in tandem mass spectrometry have been developed in recent years for use in ion trapping instruments that allow for conditions to be reached wherein rates of ion activation and deactivation are comparable. These approaches are defined as slow heating methods and include continuous-wave laser infrared multiphoton dissociation, dissociation driven by blackbody radiation, quadrupole ion trap collisional activation and sustained off-resonance irradiation in ion cyclotron resonance mass spectrometry. In the limiting case in which ion activation and deactivation rates are equal, a steady-state parent ion internal energy distribution is achieved and the kinetics of dissociation can be interpreted in analogy with thermal dissociation. This discussion describes the thermal analogy and the limiting conditions of rapid energy exchange and slow energy exchange along with the possible ramifications for dissociation rates and product ion spectra. The figures of merit that the various slow heating methods share as a class of activation methods are also discussed. The purpose of this perspective is to provide a frame-of-reference from which slow heating methods can be considered. Such methods are seeing increasing use as the number of ion trapping instruments grows and have shown remarkable success with dissociation of high-mass ions. © 1997 by John Wiley & Sons, Ltd.

Post-source decay analysis in matrix-assisted laser desorption/ionization mass spectrometry of biomolecules

Abstract: An introduction to primary structure analysis of biomolecules by matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) is given, sketching the principles and applications of the method for scientists in molecular biology, biochemistry and biomedicine. The fundamentals of PSD are described in order to explain the potential and limitations of its applicability. Two examples of peptide sequencing of a completely unknown peptide and of a database-listed peptide are presented and the procedure of (non-automated) amino acid sequence elucidation is described. 1997 by John Wiley & Sons, Ltd.

High-resolution Mass Spectrometry and Accurate Mass Measurements with Emphasis on the Characterization of Peptides and Proteins by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Abstract: Fundamental concepts of high-resolution mass spectrometry as it relates to the issue of accurate mass measurements are presented. Important issues specific to magnetic sector instruments, Fourier transform ion cyclotron resonance and time-of-flight (TOF) methods are discussed. Recent high-resolution TOF measurements on peptides and proteins, ionized by matrix-assisted laser desorption/ionization (MALDI), are presented and discussed in terms of basic concepts important to accurate mass measurements. © 1997 by John Wiley & Sons, Ltd.

High‐energy Collision‐induced Fragmentation of Complex Oligosaccharides Ionized by Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry

Abstract: The high-energy CID spectra of the MNa+ ions from 17 underivatized oligosaccharides of the type found attached to asparagine in glycoproteins were examined with a double-focusing mass spectrometer fitted with a tandem orthogonal time-of-flight analyser Fragment ions were observed throughout the mass range from all compounds and provided considerable structural information in the low-picomole range The three types of fragmentation that were observed were glycosidic cleavages, cross-ring cleavages and the formation of internal cleavage ions The major glycosidic fragmentations were B- and Y-type cleavages (Domon and Costello nomenclature) B-cleavages were particularly abundant at GlcNAc residues Z-ions were absent when glycosidic linkages occurred at the 6-position Cross-ring cleavages were predominantly of the 1,5X-type, which provided much sequence and branching information 3,5A cleavages of the core branching mannose residue were often prominent and provided information on the composition of each of the main antennae Antenna composition was also reflected by a major internal fragment ion formed by elimination of the two GlcNAc residues of the chitobiose core together with the entire antenna at the 3-position of the core branching mannose residue A further loss of GlcNAc as 221 mass units from this ion in the spectra of the hybrid and complex carbohydrates was indicative of the presence of a "bisecting' (4-linked) GlcNAc substituent Another prominent internal fragment ion containing a mannose residue and only one GlcNAc, with its substituents, was present in the spectra of the complex sugars when branching of the 3-antenna occurred

Measuring Lipogenesis and Cholesterol Synthesis in Humans with Deuterated Water: Use of Simple Gas Chromatographic/Mass Spectrometric Techniques

Abstract: Lipogenesis and cholesterol synthesis can be studied by measuring the incorporation into fatty acids and cholesterol of deuterium from deuterated water. This has been previously achieved in human subjects using low levels of deuterium enrichment in plasma water, and thus in fatty acids and cholesterol. For the measurement of enrichment in lipids, this required the use of isotope ratio mass spectrometry, a tedious and time-consuming technique. It is shown that these measurements can be performed using the much simpler gas chromatography/mass spectrometry if higher, but always safe, deuterium enrichments in plasma water are obtained. Normal subjects ingested deuterated water in order to obtain stable enrichment in plasma water of 0.3% during a 60 h period. Enrichment in palmitate of plasma triglycerides (TG) plateaued (0.6-0.76%) whereas plasma cholesterol enrichment increased progressively [0.32 +/- 0.08% (12 h) to 0.78 +/- 0.18% (60 h)]. Endogenous synthesis was estimated to contribute, in post-absorptive subjects, 8-10% of the plasma TG pool and 3-5% of plasma free cholesterol pool. These data agree with results obtained previously using isotope ratio mass spectrometry. The present method will be useful for studies of normal and abnormal lipid metabolism in humans.

Pathways to Immonium Ions in the Fragmentation of Protonated Peptides

Abstract: A 1 formed directly from protonated di- and tripeptides most likely by concerted elimination of CO and an amino acid or smaller peptide. ions can be formed directly from protonated dipeptides in part through the sequential loss of A 2 although kinetic energy release measurements suggest direct elimination of HCOOH also may be H 2 O + CO, occurring. Internal immonium ions are shown to originate by further fragmentation of ions and by further A n fragmentation of ions. 1997 by John Wiley & Sons, Ltd. Y n / (

Analysis of epoxyeicosatrienoic and monohydroxyeicosatetraenoic acids esterified to phospholipids in human red blood cells by electrospray tandem mass spectrometry.

Abstract: Electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) were used to analyze epoxyeicosatrienoic acids (EETs) and monohydroxyeicosatetraenoic acids (HETEs) isolated from human red blood cell membranes following base hydrolysis. ESI results in the formation of an abundant isobaric carboxylate anion atm/z319 for both of these oxidized metabolites of arachidonic acid. The product ion spectra from the collision-induced dissociation of this carboxylate anion could be used to identify each of the isomeric eicosanoids from the unique fragment ions of each eicosanoid. The observed product ion spectra were identical with those previously obtained by fast atom bombardment ionization; however, ESI required less EET and HETE for analysis. Both EET and HETE phospholipids were present in human red blood cells (RBCs) and their abundance could be substantially increased by treatment under conditions that would induce free radical oxidation of membrane phospholipids. Following incubation of human RBCs withtert-butyl hydroperoxide (tBuOOH), phospholipids were extracted and purified by normal-phase high-performance liquid chromatography (HPLC) as to glycerophospholipid class containing ethanolamine (GPE), serine (GPS) and choline (GPC) as the polar head group. Each class of phospholipid was hydrolyzed to yield the free carboxylic acid prior to on-line HPLC/ESI-MS/MS analysis. The formation of oxidized arachidonic acid esterified to phospholipids in treated RBCs was found to increase significantly for both esterified EETs in GPE, GPS and GPC which increased 49-, 34- and 59-fold, respectively, and also for esterified HETEs in GPE, GPS and GPC which increased 3-, 4- and 11-fold, respectively, compared with untreated RBCs. These results provide the first characterization of EETs formed non-enzymatically as intact phospholipids in a lipid peroxidation model system. © 1997 by John Wiley & Sons, Ltd.

β-Carboline Alkaloids as Matrices for Matrix-assisted Ultraviolet Laser Desorption Time-of-flight Mass Spectrometry of Proteins and Sulfated Oligosaccharides: a Comparative Study Using Phenylcarbonyl Compounds, Carbazoles and Classical Matrices

Abstract: The successful use of six β-carbolines (i.e.nor-harmane (9H-pyrido[3,4-b]indole), harmane (1-methyl-9H-pyrido[3,4-b]indole), harmine (7-methoxy-1-methyl-9H-pyrido[3,4-b]indole), harmol (1-methyl-9H-pyrido[3,4-b]indol-7-ol), harmaline (3,4-dihydro-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole) and harmalol (3,4-dihydro-1-methyl-9H-pyrido[3,4-b]indol-7-ol)) is reported as matrices in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) (λexc=337 nm) for proteins (proteins checked: gramicidin S, bovine insulin, aprotinin, horse heart cytochromec, ribonuclease A, lysozyme, myoglobin, trypsin, protease and bovine serum albumin) and sulfated oligosaccharides (λ-carrageenans ofMr549, 712, 1570 and 1733), using stainless-steel probes and membranes (poly(vinylidene difluoride) (PVDF) polymers) to prepare the samples. The possible use of some phenylcarbonyl compounds (hydroxyphenyl ketones, amino- and hydroxybenzoic acids) and a few carbazole derivatives as matrices is also discussed briefly. In addition, the usefulness of the new matrices in MALDI/TOF-MS (λexc=337 nm) of proteins and sulfated oligosaccharides is compared with those of classical MALDI matrices such as α-cyano-4-hydroxycinnamic acid, gentisic acid, sinapinic acid, 6-aza-2-thiothymine and 3-indole-trans-β-acrylic acid. Laser desorption/TOF-MS (λexc=337 nm) of the new matrices is also described. © 1997 by John Wiley & Sons, Ltd.

Isotope Ratio Mass Spectrometric Method for the On-line Determination of Oxygen-18 in Organic Matter

Abstract: A method for the on-line determination of oxygen-18, at a naturally occurring level, in organic material is presented. After pyrolysis of the samples to form carbon monoxide, which is performed at 1300°C in a vitreous carbon tube, the pyrolysis products are transported by a stream of helium gas. Using an open split, a small part of the effluent is transferred to the ion source of an isotope ratio mass spectrometer. The ratio is obtained from a measurement of the ion current intensities atm/z30 and 28 (12C18O and 12C16O). The method was tested with the secondary water standard GISP (Greenland Ice Sheet Precipitation) and the carbonate standard NBS 19. The values obtained were -24.8‰ and 27.3‰ vs. VSMOW (Vienna Standard Mean Ocean Water) (IAEA reference values are -24.8‰ and 28.7‰ vs. VSMOW). The potential of the method was demonstrated by measuring the 18O content of samples of beet and cane sucrose and also samples of vanillin extracted from vanilla pods or of synthetic origin. © 1997 by John Wiley & Sons, Ltd.

Characterization of oligonucleotide metabolism in vivo via liquid chromatography/electrospray tandem mass spectrometry with a quadrupole ion trap mass spectrometer.

Abstract: The pattern of nuclease degradation observed for an antisense phosphorothioate oligonucleotide in pig kidney was determined using liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) and LC/ESI-MS/MS with a quadrupole ion trap mass spectrometer. Metabolites were separated by length using reversed-phase high-performance liquid chromatography with aqueous hexafluoropropan-2-ol-triethylamine and a methanol gradient. The individual masses of metabolites in each LC peak were determined via deconvolution and converted into potential nucleotide compositions. The nucleotide composition was used to locate metabolites within the known oligomer sequence. The identity of metabolites was confirmed using on-line LC/MS/MS to generate fragment ions suitable for sequence verification. A limited number of shorter oligonucleotide fragments were observed, suggesting that metabolism in vivo may be sequence dependent.

Matrix-assisted Laser Desorption/Ionization Mass Spectrometric Peptide Mapping of the Neural Cell Adhesion Protein Neurolin Purified by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis or Acidic Precipitation

Abstract: Neurolin is a cell surface protein involved in the neural regeneration and neogenesis of the central nervous system of goldfish. Its theoretical molecular mass, based on the amino acid sequence translated from the cDNA, is 58 kDa, but in SDS-PAGE it shows an apparent MW of 86 kDa. Neurolin is stated to be a glycoprotein and it contains five potential N- and 96 potential O-glycosylation sites. The complete characterization of the primary structure and initial investigations on the postulated glycosylation of neurolin, immunopurified from goldfish brains, are described. The protein was either digested in situ in the sodium dodecyl sulfate polyacrylamide gel matrix or digested after trichloroacetic acid precipitation. Trypsin and endoprotease Glu-C were used as proteases and matrix-assisted laser desorption/ionization mass spectrometry was applied for direct peptide mapping analysis of the proteolytic mixtures. Various sample preparation techniques were performed and the mass spectra were recorded in both positive- and negative-ion modes.

Improving the Microdialysis Procedure for Electrospray Ionization Mass Spectrometry of Biological Samples

Abstract: Significant advances in the area of microdialysis which allowed more effective handling of small volumes (microliters) of samples, more efficient desalting and enhanced mass spectrometric detection sensitivity are described. The previously reported on-line coupling of microdialysis with electrospray ionization (ESI) mass spectrometry has been found to be highly effective; however, direct coupling requires relatively high sample flow rates (∽2 μl min-1) to obtain a stable ESI current compared with the flow rates of newer ESI sources (e.g. ‘microspray,’ 10–100 nl min-1). To circumvent this major limitation imposed by the dimensions of currently available materials, the microdialysis procedure was modified to an off-line mode in order to avoid excessive sample consumption. A more than tenfold decrease in sample consumption was achieved using the off-line modevsthe on-line mode, which resulted in a similar quality spectrum. In addition, several other aspects of the microdialysis approach were altered to improve its performance further: (i) an increase in dialysis temperature was found to increase the desalting efficiency greatly and therefore improve the spectrum quality; (ii) the addition of piperidine and imidazole to the dialysis buffer solution resulted in a reduction of charge states and a further increase in detection sensitivity for DNA and (iii) use of low concentrations (0–2.5 mM NH4OAc) of dialysis buffer shifted the DNA negative ions to higher charge states and produced a nearly tenfold increase in detection sensitivity and a slightly decreased desalting efficiency. Protocols for desalting different samples using microdialysis are discussed. © 1997 by John Wiley & Sons, Ltd.

High-performance Liquid Chromatography/NMR Spectrometry/Mass Spectrometry:Further Advances in Hyphenated Technology

Abstract: The earlier use of combined liquid chromatography/NMR spectrometry/mass spectrometry (LC/NMR/MS) involved the use of a particle beam interface. This paper describes further developments of this hyphenated technology, in particular the incorporation of an electrospray interface into the LC/NMR/MS system. This improved LC/NMR/MS system was designed for the support of a combinatorial library program. The power of this technique is demonstrated in the successful structural elucidation of each compound in a mixture of commercially available peptides.

Promotion and Stabilization of b1 ions in Peptide Phenythiocarbamoyl Derivatives: Analogies with Condensed-phase Chemistry

Abstract: The preparation of theN-terminal phenylthiocarbamoyl (PTC) derivative is the first step in the condensed phase chemistry employed in the Edman method for peptide sequencing; subsequent treatment with anhydrous acid effects cleavage of theN-terminal peptide bond yielding a derivatized amino acid and a truncated peptide. Low-energy collisional activation of peptide PTC derivative [M+2H]2+ ions during electrospray tandem mass spectrometry results in highly favoured cleavage of theN-terminal peptide bond yielding complementary b1 and yn-1 fragments. The cleavage is evidently promoted by protonation of the peptide backbone. The apparently close mechanistic similarity between the gas-phase and condensed-phase processes may be readily understood in terms of current thinking concerning the mechanism of formation of b-type ions, which involves nucleophilic attack by anN-terminal carbonyl moiety on the carbonyl carbon of the first peptide bond. Collisionally activated decomposition of source-formed b1 ions from a peptide PTC derivative is consistent with ion rearrangement similar to the PTC–phenylthiohydantoin isomerization observed in the condensed phase. © 1997 by John Wiley & Sons, Ltd.

Delayed extraction time‐of‐flight MALDI mass spectrometry of proteins above 25000 Da

Abstract: Matrix-assisted laser desorption/ionization (MALDI) with delayed extraction (DE) has been optimized for mass analysis of high-mass proteins and glycoproteins with masses above 25000 Da. Under optimized experimental conditions, i.e. using a weak extraction field strength and a long delay time, a steep drop in mass resolution above 30000 Da is no longer observed and an improvement of more than a factor of 10 is obtained compared with the non-DE case, at least up to 66 kDa in a 1.2 m time-of-flight mass analyzer. On this level of resolution the factors limiting further improvements become apparent, i.e. adduct ion formation between matrix and analyte, but also cationization and further non-matrix-related adducts, as well as prompt fragmentation. Moreover, heterogeneity of the sample is often the reason for the detection of broad signals for larger proteins. Within these limitations, DHBs (a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid) gave by far the best results. © 1997 John Wiley & Sons, Ltd.

Direct identification of phenolic glucosides from olive leaf extracts by atmospheric pressure ionization tandem mass spectrometry

Abstract: Pneumatically assisted electrospray (or ionspray) coupled with liquid chromatography was applied to the identification of the phenolic glucoside content of olive leaf directly from the crude extracts. The mass spectra of the positive ions provide insights into the composition of the phenolic constituents. Oleuropein, ligstroside and a disaccharide containing the hydroxytyrosol moiety were found in olive leaf of Olea europea L. cv. Cassanese and their structures were thoroughly determined by tandem mass spectrometry. © 1977 by John Wiley & Sons, Ltd.

Glycopeptide profiling of human urinary erythropoietin by matrix-assisted laser desorption/ionization mass spectrometry

Abstract: The site-specific glycan heterogeneity of human urinary erythropoietin was investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Owing to the small amount of protein available, a strategy combining optimal sensitivity and specificity was used. Erythropoietin was reduced, S-alkylated and digested with endoproteinase Lys C. The peptides were separated by reversed-phase high-performance liquid chromatography and the molecular masses of the peptides determined by MALDI-MS. The peptides were identified by comparing the experimental masses with the masses predicted from the cDNA derived amino acid sequence. Glycopeptides were identified from the mass spectra based on the peak pattern caused by the glycan heterogeneity. They were further characterized after treatment with neuraminidase and endoproteases. All N-glycosylation sites exhibited fucose-containing complex-type glycans. The N-glycosylation sites at Asn38 and Asn83 are mainly occupied by tetraantennary glycans, whereas Asn24 is occupied by a mixture of bi-, tri- and tetraantennary glycans. A molecular mass glycoprofile for each glycosylation site was established based on the relative peak intensities observed in the MALDI mass spectra of the desialylated glycopeptides.

Microsample determination of lovastatin and its hydroxy acid metabolite in mouse and rat plasma by liquid chromatography/ionspray tandem mass spectrometry.

Abstract: A sensitive and specific method was developed and validated to quantitate lovastatin and its hydroxy acid in mouse and rat plasma. This method employs a solid-phase extraction procedure to isolate lovastatin and its hydroxy acid metabolite from the biological matrices (0.1 ml of mouse or rat plasma). The reconstituted extracts were analyzed by liquid chromatography/ionspray tandem mass spectrometry (LC/MS/MS). Simvastatin and simvastatin hydroxy acid were used as internal standards for lovastatin and lovastatin hydroxy acid, respectively. The assay has a lower limit of quantitation (LLQ) of 0.50 ng ml-1 in mouse and rat plasma for both lovastatin and its hydroxy acid based on 0.1 ml aliquots of plasma. The intra-and inter-assay precision (RSD), calculated from quality control (QC) samples, was <7% for lovastatin and <6% for lovastatin hydroxy acid in both matrices. The inter-assay accuracy as determined from QC samples was less than 6% for lovastatin and less than 8% for lovastatin hydroxy acid in both matrices. The overall recovery of lovastatin was 54% in mouse plasma and 55% in rat plasma, and the overall recovery of lovastatin hydroxy acid was 100% in mouse plasma and 67% in rat plasma. © 1997 by John Wiley & Sons, Ltd.

Using Natural Isotope Variations of Nitrogen in Plants as an Early Indicator of Air Pollution Stress

Abstract: Model experiments (container experiments) were used to investigate whether natural variations of nitrogen constitute a suitable means for the early indication of harmful effects on plants and the level at which changes in nitrogen metabolism may occur. Wheat plants were exposed under near-ambient, controlled conditions (open-top chambers) to ozone for 2 or 6 weeks. The responses of the nitrogen metabolism in these plants were compared with non-exposed organisms at various biochemical levels. In the short-term experiment (2 weeks), wheat plants exhibited altered δ15N values under stress conditions right at the molecular level in the individual amino acids, whereas the biochemical N-fractions (structural protein (SP), soluble protein (LP) and non-protein nitrogen (NPN)) remained nearly unchanged. In the long-term experiment (6 weeks), the impact of ozone treatment and the combined effects of ozone and carbon dioxide on the wheat plants were examined. An increase in δ15N values was found as a reaction to ozone treatment in both the LP amino acids detected and in all biochemical N-fractions. Combined exposure to ozone and carbon dioxide was found to have a lesser impact at the level of the N-fractions and also at the molecular level of amino acids. The results show an example of how stable isotopes can be used as sensitive ecotoxicological indicators to provide early warning of environmental damage. © 1997 by John Wiley & Sons, Ltd.

Structural characterization of sulfoquinovosyl, monogalactosyl and digalactosyl diacylglycerols by FAB‐CID‐MS/MS

Abstract: The glycolipids such as sulfoquinovosyl diacylglycerol (SQDG), isolated from wild-type cyanobacterium Synechocystis sp. PCC 6803, and monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), obtained from whole wheat Nour, have been investigated by using fast atom bombardment tandem mass spectrometry (FAB-MS/MS). Collision-induced dissociation (CID) of sodium-adduct molecular ions [M O H + 2Na ]e from SQDG and [M + Na ]e from MGDG and DGDG generates ions which are characteristic of the structure of the polar head group as well as the fatty acid composition. In particular, the charge-remote fragmentation along the fatty acid chains observed in the high-mass range was useful to locate the double-bond positions. The major component ion ( [M O H + 2Na ]e at m/z 865) of SQDG contains oleic and palmitic acids at the sn-1 and sn-2 positions, respectively. The predominant molecular ion ( [M + Na ]e at m/z 801) of MGDG contains two linoleic acids. One of the two major species of DGDG contains palmitic and linoleic acids ( [M + Na ]e at m/z 939) at the sn-1 and sn-2 positions, while the other contains two linoleic acids ( [M + Na ]e at m/z 963). The utility of FAB-MS/MS in the structural determination of glycolipids in mixtures of biological origin has been demonstrated. 1997 by

Improved quantification of 8-epi-prostaglandin F2α and F2-isoprostanes by gas chromatography/triple-stage quadrupole mass spectrometry : Partial cyclooxygenase-dependent formation of 8-epi-prostaglandin F2α in humans

Abstract: F2-isoprostanes are considered to be novel markers of lipid peroxidation. To study the in vivo formation of F2-isoprostanes, an improved method was developed for isotope dilution assays involving gas chromatography/triple-stage quadrupole mass spectrometry (GC/MS/MS) including thin-layer chromatography (TLC) (sum of all F2-isoprostanes) and high-performance liquid chromatographic (HPLC) purification (prostaglandin F2 alpha (PGF2 alpha) and 8-epi-PGF2 alpha). Following the addition of isotopically labeled prostaglandins to urine, the sample was acidified and applied to a C18 cartridge. After elution, prostaglandins were derivatized to pentafluorobenzyl esters and subjected to TLC. A broad zone was scratched off, isoprostanes were eluted and after formation of their trimethylsilyl ether derivatives the sum of F2-isoprostanes was determined by GC/MS/MS. For the determination of PGE2 alpha and 8-epi-PGF2 alpha prior to trimethylsilylation an additional HPLC step was performed and the fractions containing PGF2 alpha and 8-epi-PGF2 alpha were analyzed by GC/MS/MS. Using this technique, 8-epi-PGF2 alpha concentrations in urine samples as low as 5 pg ml-1 could be determined with high accuracy. The excretion rates of isoprostanes were studied in comparison with the classical prostaglandins in three different groups: healthy adults, healthy children and children with hyper-PGE syndrome (HPS), a pathological situation associated with a stimulated PGE2 synthesis. F2-isoprostanes represented the main arachidonic acid metabolites in these groups and 8-epi-PGF2 alpha excretion was comparable in its amount to the classical prostanoids. To delineate the cyclooxygenase-catalyzed contribution, the influence of indomethacin, an inhibitor of cyclooxygenases, on F2-isoprostane formation in healthy adults and in HPS children was analyzed. Significantly decreased excretion rates were observed 2 days after indomethacin administration for all prostanoids, including F2-isoprostanes and 8-epi-PGF2 alpha. However, the suppression of F2-isoprostanes and 8-epi-PGF2 alpha excretion rates was less pronounced in comparison with the classical prostanoids. An improved and reliable method for the determination of F2-isoprostanes and especially 8-epi-PGF2 alpha has been developed. The data obtained on human urine samples indicates a contribution of the cyclooxygenase pathway to the formation of isoprostanes.

Non-covalent complexes between DNA-binding drugs and double-stranded deoxyoligonucleotides: a study by ionspray mass spectrometry

Abstract: The non-covalent complexes between some DNA-binding drugs and duplex oligodeoxynucleotides were studied by ionspray mass spectrometry, with the aim of evaluating the suitability of this technique to screen rapidly a series of drugs exerting their activity through non-covalent binding to specific base sequences of DNA. Two classes of drugs were considered, distamycins (which show affinity for the minor groove of DNA) and anthracyclines (which interact through intercalation between bases). For the former, d(CGCGAATTCGCG)2 was chosen as the model oligodeoxynucleotide. Following optimization of sample preparation and instrumental conditions, the complexes of different distamycins were observed; depending on the ligand considered, 1:1 or 2:1 complexes were formed preferentially. A semi-quantitative evaluation of the relative affinities was made by measuring the ratio of the complexes signals to those of the duplex, and also by competitive binding with equimolar amounts of distamycin. For anthracyclines, the daunorubicin–d(CGATCG)2 complex was chosen as the model for a preliminary mass spectrometric study; however, the signals of the duplex and the complex were very low compared with the monomer signal. Since the complex was known to be stable in solution, this was ascribed to gas-phase instability, probably caused by electrostatic repulsion between negatively charged phosphate groups. © 1997 John Wiley & Sons, Ltd.

Characterization of organic pollutants in industrial effluents using liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry

Abstract: Contaminated industrial effluents often contain a variety of organic pollutants which are difficult to analyse by standard GC-MS methods since this technique often misses the more polar or non-volatile fraction of these organic compounds. In the present work a method for the characterization of complex mixtures of organic contaminants present in various industrial effluents is proposed. The protocol consists of setting-up a methodology based on solid phase extraction (SPE) using an Automated Sample Preparation with Extraction Columns system (ASPEC XL) and Lichrolut EN sorbent material for preconcentrating 300-500 ml of water volumes spiked with a variety of pollutants: phenolic compounds, benzophenone, isothiocyanate-cyclohexane, ethylbenzoate, 1 -methyl-2-pyrrolidinone, 2-methylbenzenesulphonamide, benzidines, acridine, 1,1,3,3-tetramethyl-2-thiourea, 2,2-dimethyl-1,3-propanediol, phosphates, phthalates and non-ionic detergents characterized by LC-MS using atmospheric pressure chemical ionization in the positive and negative ion modes. The developed protocol permitted unequivocal identification of target analytes such as pentachlorophenol, tributyl pbosphate, 4-nonylphenol, dibutylphthalate, dimethylphthalate, bis(2-ethylhexyl)phthalate, isothiocyanate-cydohexane, ethylbenzoate, 2-methylbenzene-sulphonamide, tetramethyl-thiourea, 2,2-dimethyl-1,3-propanediol and 1-methyl-2-pyrrolidinone at concentration levels varying from 0.16 to 54.4 μg l -1 .

Profile and flight time analysis of bovine insulin clusters as a probe of matrix-assisted laser desorption/ionization ion formation dynamics.

Abstract: Detailed ion signal profile and flight time analyses were performed on the time-of-flight signals obtained for bovine insulin cluster ions produced by matrix-assisted laser desorption/ionization (MALDI). Profile analyses of the ion signals strongly suggest that the signals are made up of a composite of two separate components and analysis of the flight times of the two components suggests that the ions are formed with different dynamics. The formation dynamics of one component of the ions is best described as prompt ionization to yield ions with essentially mass-independent total energies. The formation dynamics of the second component of the ions is best described as delayed gas-phase ionization of entrained material moving at a constant velocity. This model is supported by several ancillary experiments and suggests new insights into the mechanism of MALDI ion formation. © 1997 by John Wiley & Sons, Ltd.