Matthias C. Letzel

SIRIUS: decomposing isotope patterns for metabolite identification

Motivation: High-resolution mass spectrometry (MS) is among the most widely used technologies in metabolomics. Metabolites participate in almost all cellular processes, but most metabolites still remain uncharacterized. Determination of the sum formula is a crucial step in the identification of an unknown metabolite, as it reduces its possible structures to a hopefully manageable set. Results: We present a method for determining the sum formula of a metabolite solely from its mass and the natural distribution of its isotopes. Our input is a measured isotope pattern from a high resolution mass spectrometer, and we want to find those molecules that best match this pattern. Our method is computationally efficient, and results on experimental data are very promising: for orthogonal time-of-flight mass spectrometry, we correctly identify sum formulas for >90% of the molecules, ranging in mass up to 1000 Da. Availability: SIRIUS is available under the LGPL license at http://bio.informatik.uni-jena.de/sirius/ Contact: anton.pervukhin@minet.uni-jena.de Supplementary information: Supplementary data are available at Bioinformatics online.

Glial Glycolysis Is Essential for Neuronal Survival in Drosophila

Neuronal information processing requires a large amount of energy, indicating that sugars and other metabolites must be efficiently delivered. However, reliable neuronal function also depends on the maintenance of a constant microenvironment in the brain. Therefore, neurons are efficiently separated from circulation by the blood-brain barrier, and their long axons are insulated by glial processes. At the example of the Drosophila brain, we addressed how sugar is shuttled across the barrier to nurture neurons. We show that glial cells of the blood-brain barrier specifically take up sugars and that their metabolism relies on glycolysis, which, surprisingly, is dispensable in neurons. Glial cells secrete alanine and lactate to fuel neuronal mitochondria, and lack of glial glycolysis specifically in the adult brain causes neurodegeneration. Our work implies that a global metabolic compartmentalization and coupling of neurons and glial cells is a conserved, fundamental feature of bilaterian nervous systems independent of their size.

Inhibition of furin by serpin Spn4A from Drosophila melanogaster

The serpin gene Spn4 from Drosophila melanogaster encodes multiple isoforms with alternative reactive site loops (RSL). Here, we show that isoform Spn4A inhibits human furin with an apparent k assoc of 5.5 × 106 M−1 s−1. The serpin forms SDS‐stable complexes with the enzyme and the RSL of Spn4A is cleaved C‐terminally to the sequence –Arg–Arg–Lys–Arg↓ in accord with the recognition/cleavage site of furin. Immunofluorescence studies show that Spn4A is localized in the endoplasmic reticulum (ER), suggesting that the inhibitor is an interesting tool for investigating the cellular mechanisms regulating furin and for the design of agents controlling prohormone convertases.

Decomposing metabolomic isotope patterns

We present a method for determining the sum formula of metabolites solely from their mass and isotope pattern. Metabolites, such as sugars or lipids, participate in almost all cellular processes, but the majority still remains uncharacterized. Our input is a measured isotope pattern from a high resolution mass spectrometer, and we want to find those molecules that best match this pattern. Determination of the sum formula is a crucial step in the identification of an unknown metabolite, as it reduces its possible structures to a hopefully manageable set. Our method is computationally efficient, and first results on experimental data indicate good identification rates for chemical compounds up to 700 Dalton. Above 1000 Dalton, the number of molecules with a certain mass increases rapidly. To efficiently analyze mass spectra of such molecules, we define several additive invariants extracted from the input and then propose to solve a joint decomposition problem.

Alternating copolymerization by nitroxide-mediated polymerization and subsequent orthogonal functionalization.

A novel method for the preparation of functionalized alternating copolymers is presented. Nitroxide-mediated polymerization of hexafluoroisopropyl acrylate with 7-octenyl vinyl ether provides the corresponding alternating polymer, which can be chemically modified using two orthogonal polymer-analogous reactions. A thiol-ene click reaction followed by amidation provides dual-functionalized alternating copolymers. The potential of this method is illustrated by the preparation of a small library (15 examples) of functionalized alternating copolymers.

A novel assay for determination of diadenosine polyphosphates in human platelets: studies in normotensive subjects and in patients with essential hypertension

Objective Diadenosine polyphosphates (APnAs, n = 3–6) are a family of endogenous vasoactive purine dinucleotides which have been isolated from thrombocytes. Diadenosine pentaphosphate (AP5A) and diadenosine hexaphosphate (AP6A) are more potent than diadenosine tetraphosphate (AP4A) and diadenosine triphosphate (AP3A) and cause skeletal muscle vasoconstriction in rats. Little is known about their physiological and pathophysiological significance in humans. The aims of the present study were to compare thrombocyte APnA concentrations in patients with essential hypertension (HYP) and in healthy normotensive humans (CON) using a novel quantitative assay and to assess a possible relationship between thrombocyte APnA concentrations and skeletal muscle vascular resistance. Design and methods We describe a novel assay for quantification of APnAs in human platelets, involving platelet isolation from human blood, a solid-phase extracting procedure with a derivatized resin, desalting and quantitative determination of the substances with an ion-pair reversed-phase high-performance liquid chromatography (HPLC) system. The structural integrity of the isolated APnAs was confirmed by mixed assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) measurements and co-elution with added standards. The detection threshold for all four APnAs was 1 pmol/l and the inter-assay coefficients of variation were < 11% (n = 12). After venous blood sampling, mean arterial blood pressure (MAP) and forearm blood flow (FBF, using venous occlusion plethysmography) were measured in HYP and CON. Forearm vascular resistance (FVR) was calculated as MAP/FBF. Results HYP (n = 15) and CON (n = 15) did not significantly differ in platelet AP3A and AP4A content, but HYP had significantly higher thrombocyte concentrations of AP5A (56 ± 7 versus 32 ± 3 ng/μg β-thromboglobulin, P = 0.003) and AP6A (10 ± 1 versus 6 ± 1 ng/μg β-thromboglobulin, P = 0.015) than CON. HYP had significantly elevated FVR (50 ± 6 versus 33 ± 2 arbitrary units, P = 0.01) compared to CON. Significant correlations were found between AP5A and FVR (ρ = 0.38, P = 0.04) as well as between AP6A and FVR (ρ = 0.42, P = 0.02). In contrast, there were no significant correlations between APnAs and MAP. Conclusions The study shows that thrombocyte concentrations of AP5A and AP6A are elevated in patients with essential hypertension. Vasoconstriction caused by release of AP5A and AP6A from thrombocytes may contribute to the increase of vascular resistance in hypertensive patients.

Analysis of Free Fatty Acids by Ultraviolet Laser Desorption Ionization Mass Spectrometry Using Insect Wings as Hydrophobic Sample Substrates

Physiologically relevant free fatty acids (FFAs) were analyzed by UV-laser desorption/ionization orthogonal extracting time-of-flight mass spectrometry (LDI-oTOF-MS). Dissected wings from Drosophila melanogaster fruit flies were used as the hydrophobic, laser energy strongly absorbing sample substrates. Using untreated substrates produces predominantly molecular [M + K](+) ions of the FFAs, whereas other alkali metal adducts can be generated by treating the wings with the corresponding alkali hydroxide before spotting of analyte. Limits of detection for the positive ion mode were determined for mixtures of isolated FFAs to values in the low 10 pmol range. Specific values depend on chain length and degree of unsaturation. R(2) coefficients for the analysis of saturated FFAs were found to be generally close to 0.98 over about 3 orders of magnitude if an internal standard (15:0 FFA) was added. Semiquantitative analyses of mixtures containing unsaturated FFAs are also possible but require more effort on the calibration strategy. Notably, both saturated and (poly-)unsaturated FFAs are detected sensitively in the presence of relatively high concentrations of other physiologically abundant lipids (phospholipids and triacyclglycerols). This simplifies screening of the FFA composition in crude tissue extracts. This feature is demonstrated by the analysis of a crude liver extract and that of fingermarks.

On the way to supramolecular photochemistry at the single-molecule level

Two examples of artificial supramolecular host-guest systems derived from resorc[4]arenes (calix[n]arenes based on resorcinol) and ammonium ions as guests have been studied by atomic force microscopy (AFM). For the first time, real single-molecule events have been determined for this type of supramolecular complexes and off-rates as well as molecular parameters of single-molecule aggregates such as the depths of the binding pocket (molecular length parameter) could be measured by applying the methods of dynamic force spectroscopy. In addition, this technique was also applied to differentiate between the two states (open and closed) of a photoswitchable resorc[4]arene-anthracene tweezer. An investigation of the exchange rates of various complexes in the gas phase by means of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry confirmed the results of the AFM study.

Cyclochiral resorcin[4]arenes as effective enantioselectors in the gas phase.

The effect of cyclochirality of rccc-2,8,14,20-tetra-n-decyl-4,10,16,22-tetra-O-methylresorcin[4]arene (C) on the enantiodiscrimination of a number of chiral bidentate and tridentate aromatic and aliphatic biomolecules (G) has been investigated by nano-electrospray ionization (nano-ESI)-Fourier transform ion cyclotron resonance mass spectrometry. The experimental approach is based on the formation of diastereomeric proton-bound [C·H·G](+) complexes by nano-ESI of solutions containing an equimolar amount of quasi-enantiomers (C) together with the chiral guest (G) and the subsequent measurement of the rate of the G substitution by the attack of several achiral and chiral amines. In general, the heterochiral complexes react faster than the homochiral ones, except when G is an aminoalcoholic neurotransmitter whose complexes, beyond that, exhibit the highest enantioselectivity. The kinetic results were further supported by both collision-induced dissociation experiments on some of the relevant [C(2) ·H·G](+) three-body species and Density functional theory (DFT) calculations performed on the most selective systems.

Chiral discrimination on the host-guest-complexation of resorc[4]arenes with quarternary amines.

The interaction of inherently chiral resorc[4]arenes with different chiral ammonium ions was measured by electrospray ionization mass spectrometry. For this purpose, one enantiomer of the ammonium guests was labeled with deuterium to distinguish the enantiomers by their mass. We synthesized the ammonium salts by the reaction of chiral primary amines with either CH3I or CD3I and analyzed the resulting ammonium iodides by nuclear magnetic resonance and optical rotation. The complexation experiments were performed by mixing the chiral host with various ratios of the unlabeled guest and its labeled enantiomer. By analyzing the integrals of the host–guest complexes, we observed a chiral discrimination effect and a secondary isotope effect as well.