Biomedical subjects

Matthias Witt

Publications and source records attributed to Matthias Witt.

29 records · Page 2Linked to original sources

PubMed2003

Accurate mass measurements by Fourier transform mass spectrometry in the study of advanced glycation end products/peptides.

The Maillard reaction occurring between sugars and amino groups is important in living systems. When amino groups belonging to protein chains are involved, the Maillard reaction has been invoked as responsible for protein cross-linking and the production of 'toxic' compounds. The reaction leads to the production of a heterogeneous group of substances, usually called advanced glycation end products (AGEs). Classical analytical approaches, such as spectroscopic (ultraviolet, fluorescence) and mass spectrometric (matrix-assisted laser desorption/ionization, liquid chromatography/electrospray ionization mass spectrometry) methods, have shown that the digestion mixture is highly complex. However, there are clear differences between the digestion mixtures of glycated and unglycated human serum albumin (HSA). In the former case, possible glycated peptides belonging to the AGE peptide class may be identified. Tandem mass spectrometric experiments on selected species seemed to be promising as regards structural information, but it was thought of interest to undertake the present investigation, based on liquid chromatography/electrospray ionization Fourier transform mass spectrometry, in order to obtain definitive results on their elemental composition. Using this approach, about 20 glycated peptides were detected and their possible structures were postulated by examining the known sequence of HSA.

Amino Acid Sequence↗

PubMed2003

Combined infrared multiphoton dissociation and electron capture dissociation with a hollow electron beam in Fourier transform ion cyclotron resonance mass spectrometry.

An electron injection system based on an indirectly heated ring-shaped dispenser cathode has been developed and installed in a 7 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This new hardware design allows high-rate electron capture dissociation (ECD) to be carried out by a hollow electron beam coaxial with the ion cyclotron resonance (ICR) trap. Infrared multiphoton dissociation (IRMPD) can also be performed with an on-axis IR-laser beam passing through a hole at the centre of the dispenser cathode. Electron and photon irradiation times of the order of 100 ms are required for efficient ECD and IRMPD, respectively. As ECD and IRMPD generate fragments of different types (mostly c, z and b, y, respectively), complementary structural information that improves the characterization of peptides and proteins by FTICR mass spectrometry can be obtained. The developed technique enables the consecutive or simultaneous use of the ECD and IRMPD methods within a single FTICR experimental sequence and on the same ensemble of trapped ions in multistage tandem (MS/MS/MS or MS(n)) mass spectrometry. Flexible changing between ECD and IRMPD should present advantages for the analysis of protein digests separated by liquid chromatography prior to FTICRMS. Furthermore, ion activation by either electron or laser irradiation prior to, as well as after, dissociation by IRMPD or ECD increases the efficiency of ion fragmentation, including the w-type fragment ion formation, and improves sequencing of peptides with multiple disulfide bridges. The developed instrumental configuration is essential for combined ECD and IRMPD on FTICR mass spectrometers with limited access into the ICR trap.

Animals↗

PubMed2003

Fourier transform ion cyclotron resonance mass spectrometry with NanoLC/microelectrospray ionization and matrix-assisted laser desorption/ionization: analytical performance in peptide mass fingerprint analysis.

Protein identifications by peptide mass fingerprint analyses with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were performed using microelectrospray ionization coupled to nano liquid chromatography (NanoLC), as well as using matrix-assisted laser desorption/ionization (MALDI). Tryptic digests of bovine serum albumin (BSA), diluted down to femtomole quantities, have been desalted by fast NanoLC under isocratic elution conditions as the high resolving power of FT-ICR MS enables peptides to be separated during the mass analysis stage of the experiment. The high mass accuracy achieved with FT-ICR MS (a few ppm with external calibration) facilitated unambiguous protein identification from protein database searches, even when only a few tryptic peptides of a protein were detected. Statistical confidence in the database search results was further improved by internal calibration due to increased mass accuracy. Matrix-assisted laser desorption/ionization and micro electrospray ionization (ESI) FT-ICR showed good mass accuracies in the low femtomole range, yet a better sensitivity was observed with MALDI. However, in higher femtomole ranges slightly lower mass accuracies were observed with MALDI FT-ICR than with microESI FT-ICR due to scan-to-scan variations of the ion population in the ICR cell. Database search results and protein sequence coverage results from NanoLC FT-ICR MS and MALDI FT-ICR MS, as well as the effect of mass accuracy on protein identification for the peptide mass fingerprint analysis are evaluated.

Animals↗

PubMed2003

Terpenoids of Salvia hydrangea: two new, rearranged 20-norabietanes and the effect of oleanolic acid on erythrocyte membranes.

Four abietane-type terpenoids, including two known royleanones and two new, rearranged 20-norabietanes, were isolated from the roots of the Iranian medicinal plant Salvia hydrangea DC. ex Bentham (Lamiaceae), which is used as an anthelmintic and antileishmanial remedy. Their structures were established using COSY, NOESY, HSQC, and HMBC spectral data. The possible identity of one of the 20-norabietanes with demethylmulticauline, previously reported from a different Salvia species, is discussed. A moderate in vitro antiplasmodial effect of the extract of S. hydrangea flowers was found to be associated with the presence of large amounts of pentacyclic triterpenes, mainly oleanolic acid. The observed antiplasmodial activity of oleanolic acid is apparently due to its incorporation into the erythrocyte membrane, which adversely affects the growth of Plasmodium falciparum parasites. Thus, oleanolic acid caused transformation of erythrocytes into stomatocytes in the concentration range where the in vitro antiplasmodial activity was observed.

Abietanes↗

PubMed2003

Effects of internal hydrogen bonds between amide groups: protonation of alicyclic diamides.

The proton affinity (PA) of cyclopentane carboxamide 1, cyclohexane carboxamide 2 and their secondary and tertiary amide derivatives S1, S2, T1 and T2, was determined by the thermokinetic method and the kinetic method [PA(1) = 888 +/- 5 kJ mol(1); PA(2) = 892 +/- 5 kJ mol(1); PA(S1) = 920 +/- 6 kJ mol(1); PA(S2) = 920 +/- 6 kJ mol(1); PA(T1) = 938 +/- 6 kJ mol(1); PA(T2) = 938 +/- 6 kJ mol(1)]. Special entropy effects are not observed. Additionally, the effects of protonation have been studied using an advanced kinetic method for all isomers 37 of cyclopentane dicarboxamides and cyclohexane dicarboxamides (with the exception of cis-cyclopentane-1,2-dicarboxamide) and their bis-tertiary derivatives T3T7 by estimating the PA and the apparent entropy of protonation Delta(DeltaS(app)). Finally, the study was extended to bicyclo[2.2.1]hepta-2,5-diene-2,3-dicarboxamide 8 and its bis-tertiary derivative T8, to all stereoisomers of bicyclo[2.2.1]heptane-2,3-dicarboxamide 9, their secondary and tertiary amide derivatives S9 and T9, and to endoendobicyclo[2.2.1]heptane-2,5-dicarboxamide 10 and the corresponding secondary and tertiary derivatives S10 and T10. Compared with 1 and 2, all alicyclic diamides exhibit a significant increase of the PA (DeltaPA) and special entropy effects on protonation. For alicyclic diamides, which can not accommodate a conformation appropriate for building a proton bridge, the values of DeltaPA and Delta(DeltaS(app)) are small to moderate. This is explained by ion / dipole interactions between the protonated and neutral amide group which stabilize the protonated species but hinder the free rotation of the amide groups. If any of the conformations of the alicyclic diamide allows formation of a proton bridge, DeltaPA and Delta(DeltaS(app)) increase considerably. A spectacular case is cis-cyclohexane-1,4-dicarboxamide 7c which is the most basic monocyclic diamide, although generation of the proton bridge requires the unfavorable boat conformation with both amide substituents at a flagpole position. A pre-orientation of the two amide groups in such a 1,4-position in 10 results in a particularly large PA of < 1000 kJ mol(1). The observation of comparable values for Delta(DeltaS(app)) for linear and monocyclic diamides indicates that a major part of the entropy effects originates from freezing the free rotation of the amide groups by formation of the proton bridge. This is corroborated by observing corresponding effects during the protonation of dicarboxamides containing the rigid bicyclo[2.2.1]heptane carbon skeleton, where the only internal movements of the molecules corresponds to rotation of the amide substituents.

Amides↗

PubMed2002

Mass spectrometric characterization and gas-phase chemistry of self-assembling supramolecular squares and triangles.

A detailed mass spectrometric characterization of self-assembling polynuclear metal complexes is described. The complexes can only be ionized as intact species under a surprisingly narrow range of conditions by electrospray ionization. Comparison with the results from NMR experiments shows that several solution-phase features of these squares and triangles (such as trends in bond energies, ligand-exchange reactions, or square-triangle equilibria) are qualitatively reflected in the gas-phase data. Consequently, mass spectrometry represents a valuable method for the characterization of these compounds. Nevertheless, the formation of unspecific aggregates during the ionization process occurs and its implications are discussed. Beyond the chemistry in solution, the fragmentation pathways of these complexes in the gas phase have been studied by infrared multiphoton dissociation (IRMPD) experiments. The results of IRMPD studies allow us to draw conclusions with respect to the structure and energetics of fragmentation products. In this tandem MS experiment, reaction pathways can be observed directly which can hardly be analyzed in solution. According to these results, the equilibration of triangles and squares involves the supramolecular analogue of a neighboring-group effect.

Journal Article↗

PubMed2002

A combined ion source for fast switching between electrospray and matrix-assisted laser desorption/ionization in Fourier transform ion cyclotron resonance mass spectrometry.

A new ion source has been developed for Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) that enables quick changes between matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) modes. When operating as an ESI source, the sample solution is sprayed through an angled nebulizer. The generated ions pass through a glass capillary followed by a skimmer and three sequential hexapole ion guides. Ions can be accumulated in the third hexapole (storage hexapole) before they are injected into the ICR trap. The second hexapole is mounted on a movable platform which also carries the MALDI sample plate. During the switch from ESI to MALDI, this platform moves the second hexapole out of the hexapole series and locates a MALDI sample plate with 384 sample positions into the area directly in front of the storage hexapole. The storage hexapole is in a medium pressure chamber (MPC) which has windows both for the incoming laser beam and for the observation optics, as well as a gas tube for pulsing collision gas into the chamber. During the MALDI operation the focused laser beam enters the MPC, passes between the hexapole rods and irradiates a MALDI sample on the target plate. The sample molecules are desorbed/ionized into the storage hexapole and simultaneously cooled by collisions with the pulsed gas. Ions desorbed from multiple laser shots can be accumulated in this hexapole before they are transferred to the ICR trap. With the combined ion source a computer-controlled switch between MALDI and ESI modes is possible in less than a minute, depending on the position of the MALDI target on the 384-spot plate. Immediate acquisition of mass spectra is possible after mode switching without the need for tuning or re-calibration.

Animals↗

PubMed2002

In-source H/D exchange and ion-molecule reactions using matrix assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry with pulsed collision and reaction gases.

Controlled in-source ion-molecule reactions are performed for the first time in an external matrix assisted laser desorption ionization (MALDI) source of a Fourier transform ion cyclotron resonance mass spectrometer. The MALDI source with a hexapole ion guide that was originally designed to incorporate pulsed gas to collisionally cool ions (Baykut, G.; Jertz, R.; Witt, M. Rapid Commun. Mass Spectrom. 2000, 14, 1238-1247) has been modified to allow the study of in-source ion-molecule reactions. Upon laser desorption, a reaction gas was introduced through a second inlet and allowed to interact with the MALDI-generated ions trapped in the hexapole ion guide. Performing ion-molecule reactions in the high pressure range of the ion source prior to analysis in the ion cyclotron resonance (ICR) cell allows to maintain the ultra high vacuum in the cell which is crucial for high mass resolution measurements. In addition, due to the reaction gas pressure in the hexapole product ion formation is much faster than would be otherwise possible in the ICR cell. H/D exchange reactions with different peptides are investigated, as are proton-bound complex formations. A typical experimental sequence would be ion accumulation in the hexapole ion guide from multiple laser shots, addition of cooling gas during ion formation, addition of reaction gas, varied time delays for the ion-molecule reactions, and transmission of the product ions into the ICR cell for mass analysis. In this MALDI source H/D exchange reactions for different protonated peptides are investigated, as well as proton-bound complex formations with the reaction gas triethylamine. Amino acid sequence, structural flexibility and folding state of the peptides can be seen to play a part in the reactivity of such ions.

Cyclotrons↗

PubMed2002

Proton bound homodimers and heterodimers of amides and amines in the gas phase. Equilibrium studies by Fourier transform ion cyclotron resonance spectrometry.

By injection of the proton bound homodimer [DMF.H+.DMF] of N,N-dimethylformamide (DMF) generated in an external ion source into a mixture of DMF and a second base within the cell of a Fourier transform ion cyclotron resonance (FT-ICR) spectrometer the equilibria between [DMF.H+.DMF] and the other possible proton bound dimers [DMF.H+.base] and [base.H+.base] have been studied for 13 different bases. Strongly polar bases like aliphatic amides and dimethyl sulfoxide (DMSO) exchange both DMF in [DMF.H+.DMF] by a two step process, while the almost non-polar amines exchange only one DMF. If the base is a primary or secondary amine, the proton bound heterodimer [DMF.H+.amine] reacts further by the addition of one DMF to create a proton bound trimer [(DMF)2.H+.amine]. The affinity deltaG(DMFH+) of the bases towards protonated DMF relative to neutral DMF depends linearly on the difference deltaGB of the gas phase basicity of DMF and the other base, but different correlation lines are obtained for polar and non-polar ligands (deltaGDMFH+ = 0.44GB(base)-375 [kJ/mol] (r = 0.97) and deltaGDMFH+ = 0.46GB(base)-397 [kJ/mol] (r = 0.99), respectively). This different behavior is explained by a different character of the proton bridge in the heterodimers containing only polar ligands and those incorporating a non-polar ligand besides DMF. The former dimers contain a more or less symmetric proton bridge while the latter can be viewed as a protonated base solvated by DMF. The available data have been used to calculate the molecular pair gas phase basicity of DMF and the 13 bases used and to estimate the dissociation energies of the bonds of the proton bridge in various proton bound heterodimers.

Algorithms↗

PubMed2002

Leishmanicidal and antiplasmodial activity of constituents of Smirnowia iranica.

Three unusual, highly oxygenated novel phenylpropanoids (1-3) and two novel isoflavans, 8-prenylmucronulatol (4) and smiranicin (6), were isolated from Smirnowia iranica together with a previously described isoflavan, glyasperin H (5). The structures were established using homo- and heteronuclear two-dimensional NMR experiments. The isoflavans significantly inhibited the growth of extracellular stages of three Leishmania species in vitro, their activity against the intracellular stages being considerably lower. 8-Prenylmucronulatol (4) showed moderate in vitro toxicity against Plasmodium falciparum, without noticeable erythrocyte membrane effects at the inhibitory concentration. Because of the structural relationship of isoflavans with chalcones and aurones, some of which are potent antiprotozoal agents, the isoflavan skeleton may be a template structure in search for new compounds with leishmanicidal and antiplasmodial activity.

Animals↗

PubMedDate not supplied

Application of accurate mass measurement to urine drug screening.

Poor availability of reference standards for designer drugs, metabolites, and new substances prevents toxicology laboratories from rapidly responding to the changing analytical challenges of drug abuse. A novel screening approach comprising determination of accurate masses of sample components and comparison of these with databases of theoretical monoisotopic masses is described. Using liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS), a routine mass search window of 20-30 ppm was applied to urine samples. The ultimate reference technique, liquid chromatography-Fourier transform mass spectrometry (LC-FTMS), was capable of confirming the findings within a 3 ppm mass accuracy. Using a target database of 7640 compounds, the number of potential elemental formulas ranged from one to three with LC-TOFMS, and it was always one with LC-FTMS. In contrast to ordinary techniques requiring primary reference standards, the formula-based databases can be updated instantly with fresh numeric data from scientific literature and authority sources.

Chromatography, High Pressure Liquid↗