Urs Peter Schlunegger

Studies on the Selection of New Matrices for Ultraviolet Matrix‐assisted Laser Desorption/Ionization Time‐of‐flight Mass Spectrometry

A new group of compounds has been successfully tested as matrices for ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (UV-MALDI MS). Several new matrices for UV-MALDI MS have been found by choosing, as potential matrices, compounds that perform an intramolecular proton transfer along an intramolecular H-bond under UV irradiation. Compounds of this type are, for example, salicylamide, salicylanilide, several ortho-hydroxyacetophenones and ortho-hydroxybenzophenones. The matrix activity of these compounds is compared to the corresponding meta- and para-isomers and to the matrix activity of such well known matrices as 2,5-dihydroxybenzoic acid and 2,4,6-trihydroxyacetophenone. It was found that meta- and para-substituted hydroxycarbonyl compounds show either a significantly lower or no matrix activity compared with the ortho isomers.

Incorporation of atmospheric 15NO2-nitrogen into free amino acids by Norway spruce Picea abies (L.) Karst.

During spring and autumn 1991, potted 6-yearold spruce trees (Picea abies (L.) Karst.) were fumigated with 60 nl·1−115NO2 for 4 days under controlled conditions in constant light. Current and previous flush needles, the bark and the fine roots were analysed for total 15N content and incorporation of 15N into the α-amino nitrogen of free amino acids. In addition, in vitro nitrate reductase activity and stomatal conductance of the needles were measured. Nitrate reductase activity was significantly higher in the needles of fumigated trees compared to control trees exposed to filtered air. With an average of 9.1% 15N, free glutamate was the pool with the most label. Taking into account the time-course of the labelling of this pool, this figure can be taken as an estimate of the minimum contribution of NO2 to the N nutrition of the needles. 15N-labelled amino acids were also detected in the bark and the roots, indicating export from the needles.

Sequestration ofVeratrum alkaloids by specialistRhadinoceraea nodicornis konow (Hymenoptera, Tenthredinidae) and its ecoethological implications

The larvae of the specialist sawflyRhadinoceraea nodicornis Konow (Hymenoptera, Tenthredinidae) store in their hemolymph ceveratrum alkaloids originating from the host plantVeratrum album L. (Liliales, Melanthiaceae). The major alkaloid found in the hemolymph is 3-acetyl-zygadenine. Qualitative and quantitative data showed that the plant alkaloid 3-angeloylzygadenine is most probably metabolized in the larval gut to zygadenine and then acetylated. A still unidentified alkaloid with a molecular weight of 591 Da was detected in plant leaves as well as in the gut, hemolymph, and excrement of larvae. Protoveratrine A and B, on the other hand, seem to be degraded by the larvae. These findings indicate that the pathway of ceveratrum alkaloids inR. nodicornis larvae is fourfold: direct sequestration, metabolism followed by sequestration, excretion of intact alkaloids, and degradation. In contrast, no ceveratrum alkaloids were detected in the hemolymph and excrement of larvae of the generalist sawflyAglaostigma sp. fed withV. album leaves. Bioassays with the antMyrmica rubra L. proved that the hemolymph ofR. nodicornis larvae is highly deterrent and toxic. In bioassays evaluating defensive efficiency against predators (ants, spiders, and bushcrickets), no larvae were eaten. Ceveratrum alkaloids were also detected in the hibernating prepupae ofR. nodicornis. In feeding bioassays, the shrewCrocidura russula Hermann rarely fed upon prepupae, suggesting that this stage is also protected from predation to some degree. In field surveys, the only parasitoids recorded were two ichneumonid species that are believed to be specialized onR. nodicornis. Bioassays and field observations enable us to suppose thatR. nodicornis and its enemies produce a food web of ion connectance.

Analysis of Hydrophobic Proteins and Peptides by Electrospray Ionization Mass Spectrometry

Bacterioopsin (BO) from Halobacterium halobium, as well as its V8-protease and CNBr fragments from the C-terminal region, were used to establish appropriate conditions for the mass determination of membrane proteins and peptides by electrospray mass spectrometry (ESI-MS). Of the tested solvents neat formic acid gave the best results for BO (26 781.8 ± 5.1 Da) and chloroform + methanol + water (2:5:2, v/v/v/) containing 2% acetic acid was best for the BO fragments (2461.5 ± 0.2 Da, 7147.1 ± 0.5 Da, 7748.0 ± 0.4 Da) with an overall mass accuracy of about 0.01% The three subunits IIABMan, IICMan and IIDMan of the mannose transporter complex in E. coli were used to establish conditions for liquid chromatography/electrospray mass spectrometry measurements (LC/ESI-MS). The subunits were separated by reversed-phase high-performance liquid chromatography using a water/formic acid gradient. Of the detergents used to solubilize the sample, the uncharged dodecylmaltoside and the positively charged dodecyltrimethylammoniumchloride had the most favorable influence on the separation and mass measurement by ESI-MS. For the first time the rather hydrophilic IIABMan (34 920.8 ± 6.7 Da) and the transmembrane IIDMan (31 909.4 ± 18.2 Da) subunits could be determined with a mass accuracy in the range of 0.01 to 0.05%. On the other hand no appropriate conditions could be found to determine the precise mass of IICMan experimentally.

The Secretion of the Ventral Glands in Hoplocampa Sawfly Larvae

The defensive secretion of the ventral glands of both species, Hoplocampa testudinea and H. flava is mainly composed of acetogenins. Major compounds are (E)-2-octenal, (E,Z)- and (E,E)-2,4-decadienal and (E,Z,Z)-2,4,7-decatrienal that was identified for the first time in an insect. Benzaldehyde is abundant in H. flava. A comparison of Hoplocampa with taxonomically related genera suggests that, for those acetogenins present as major compounds in the secretion, the chain length decreased during evolution of nematine sawflies. Only the last larval instar of Hoplocampa species produces large amounts of secretions. For H. testudinea, the last instar as compared with previous ones was more efficiently protected against ants in a bioassay, and possessed allometrically larger glands. Hoplocampa larvae live concealed within the fruit that they infest until dropping off the plant for pupation in the soil. The relative importance during the ontogeny of the mechanical protection offered by the fruit and the chemical defence provided by the ventral glands is discussed, also with respect to field observations on the interactions between ants and sawfly larvae. Our hypothesis is that nematine sawfly larvae with a concealed life style will show a relative reduction in glandular size and defensive significance of the glandular secretion.

Distribution patterns of n-alkanes in human liver, urine and sweat.

Abstract Paraffins in human liver, urine and sweat were isolated by extraction, molecular sieving, and gas chromatography-mass spectrometry, and the distribution patterns of n-alkanes in these organic materials compared. n-Alkanes in liver (about 10 mg/kg wet wt.) were found to contain 12–33 C-atoms. n-Alkanes with more than 20 C-atoms contained predominately an odd number of C-atoms, whereas in the smaller molecules even-numbered homologues predominated. An excessive concentration of n-nonacosane (C29H60) indicates a vegetable diet. In urine n-alkanes (about 1.3 mg/1) containing 13–30 C-atoms were isolated, with a striking predominance of the even-numbered homologues, showing a possible connection between the hydrocarbons in liver and those in urine. In sweat a series of n-alkanes (about 27 mg/g lipid extract from sweat) in the range from 12 to 35 C-atoms was found with a highest concentration of pentacosane (C25H52), indicating a similarity to the alkane pattern of human blood serum.

Combined Analyzer Technologies

There is no one single answer to the question of why different analyzer combinations are being used because the driving force to construct and use such instruments changes with time. Certainly one of the driving forces is the need for further technological development; for example, the need of better sensitivity and higher mass resolution for atomic [1] and molecular [2] mass determination and structure elucidation of natural compounds [3]. This was subsequently achieved by the introduction of double focusing sector instruments (combination of magnetic and electrostatic analyzers) [4,5]. The study of natural compounds, for instance, alkaloids and steroids [6], pushed mass spectrometry to a higher mass range (up to 1 kDa and more). The possibility to analyze larger molecules induced again the development of new ionization methods, thus adding “soft” ionization techniques to the commonly used electron impact ionization (EI). Methods like chemical ionization (CI), field desorption (FD), and fast atom bombardment (FAB) opened a wide research field for mass spectrometry [7–10].

Uptake and Distribution of NO2 in Norway Spruce (Picea Abies [L.] Karst.)

Potted spruce trees (Picea abies [L.] Karst.) were fumigated with NO2 or 15NO2 under controlled conditions in constant light. Current and previous flush needles, the bark and the fine roots were analysed for total 15N content and incorporation of 15N into the α-amino nitrogen of free amino acids. In addition, in vitro nitrate reductase activity and stomatal conductance of the needles were measured. In fumigated trees, nitrate reductase activity increased after a lag period of about four hours to levels which were five times higher than in controls exposed to filtered air. After a fumigation period of 24 hours nitrate reductase activity was linearly dependant on the NO2 concentration used for fumigation both in previous and current flush. With an average of 9.1% 15N, free glutamate was the pool with the most label. Taking into account the time-course of the labelling of this pool, this figure can be taken as an estimate of the minimum contribution of NO2 to the N nutrition of the needles. 15N labelled amino acids were also detected in the bark and the roots, indicating export from the needles. There were linear correlations between the stomatal conductance and the 15N uptake and between the 15N uptake and nitrate reductase activity.