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Biomedical subjects

Matthias Witt

Publications and source records attributed to Matthias Witt.

At least 19 recordsLinked to original sources

Two-fold efficiency increase by selective excitation of ions for consecutive activation by ion-electron reactions and vibrational excitation in tandem fourier transform ion cyclotron resonance mass spectrometry.

A new technique called selective excitation of ions for consecutive activation (SEICA) is proposed for obtaining complementary fragmentation mass spectra from the same precursor ion population. SEICA utilizes precursor ions remaining intact after electron capture dissociation or another ion-electron reaction for efficient MS/MS based on a vibrational excitation (VE) technique, such as infrared multiphoton dissociation. SEICA uses the ability of ion-trapping instruments to detect product ions while retaining inside the trap intact precursor ions, making the latter available for consecutive activation by a VE technique. The possibility of practical implementation of SEICA by software-only modification of a commercial instrument is demonstrated. A 2-fold increase in the efficiency is achieved for both "single-scan" and "multiple-scan" experiments. This improvement can be particularly important for high-sensitivity applications in, for example, proteomics, where limited ECD efficiency poses an obstacle for broad implementation of this technique.

Cyclotrons↗

Side-chain-anchored N(alpha)-Fmoc-Tyr-OPfp for bidirectional solid-phase synthesis.

[reaction: see text] A mild resin-immobilization strategy employing a readily prepared trityl bromide resin for anchoring building blocks via a phenol group has been developed. With N(alpha)-Fmoc-Tyr-OPfp as a starter building block, it was possible to prepare asymmetrically substituted hybrids of spider- and wasp-type polyamine toxins using solid-phase peptide synthesis conditions.

Combinatorial Chemistry Techniques↗

The effects of conformational constraints and steric bulk in the amino acid moiety of philanthotoxins on AMPAR antagonism.

Philanthotoxin-343 (PhTX-343), a synthetic analogue of wasp toxin PhTX-433, is a noncompetitive antagonist at ionotropic receptors (e.g., AChR or iGluR). To determine possible effects of variations of the amino acid side chain, a library consisting of seventeen PhTX-343 analogues was prepared. Thus, tyrosine was replaced by either apolar, conformationally constrained, or bulky amino acids, whereas the acyl unit and the polyamine moiety were kept unchanged. Analogues with tertiary amide groups were prepared for the first time. Pentafluorophenyl esters were employed for amide bond formation, establishing general protocols for philanthotoxin solution- and solid-phase synthesis (39-90% and 42-54% overall yields, respectively). The analogues were tested for their ability to antagonize kainate-induced currents of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazoyl)propanoic acid receptors (AMPAR) expressed in Xenopus oocytes from rat brain mRNA. This showed that steric bulk in the amino acid moiety is well tolerated and suggests that binding to AMPAR does not involve the alpha-NHCO group as a donor in hydrogen bonding.

Animals↗

Fast gas-phase hydrogen/deuterium exchange observed for a DNA G-quadruplex.

The gas-phase hydrogen/deuterium (H/D) exchange kinetics of DNA G-quadruplexes has been investigated using Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). The quadruplex [(TGGGGT)4 . 3NH4+] undergoes very fast H/D exchange, in both the positive and in the negative ion modes, compared to DNA duplexes and other quadruplexes tested, and compared to the corresponding single-stranded TGGGGT. Substitution of NH4+ for K+ did not alter this fast H/D exchange, indicating that the hydrogens of the ammonium ions are not those exchanged. However, stripping of the interior cations of the quadruplex by source collision-induced dissociation (CID) in the positive ion mode showed that the presence of the inner cations is essential for the fast exchange to be possible. Molecular dynamics simulations show that the G-quadruplex is very rigid in the gas phase with NH4+ ions inside the tetrads. We suggest that the fast H/D exchange is favored by this rigid quadruplex conformation. This example illustrates that the concept that compact DNA structures exchange H for D slower than unfolded ones is a misconception.

DNA↗

Solid-phase synthesis of rigid acylpolyamines using temporary N-4,4'-dimethoxytrityl protection in the presence of trityl linkers.

An N-protection protocol employing the 4,4'-dimethoxytrityl (Dmt) group in combination with borane reduction of resin-bound polyamides was shown to be an efficient methodology that enables synthesis of novel analogues of natural acylpolyamine toxins. Thus, three philanthotoxins containing polyamine chains with piperidyl and cyclohexyl structural elements, which introduce conformational rigidity, increased lipophilicity, and altered proteolytic properties, were obtained in 39-44% overall yield.

Boranes↗

Expedient protocol for solid-phase synthesis of secondary and tertiary amines.

[reaction: see text] An expedient solid-phase synthetic approach to secondary and tertiary amines was developed. The protocol employs conversion of resin-bound amino alcohols to the corresponding iodides, followed by iodide displacement with primary or secondary amines or with unprotected amino alcohols. This two-step procedure, affording products in good to excellent yields, is suitable for solid-phase synthesis of polyamines.

Journal Article↗

Peptide and protein characterization by high-rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry.

The analytical utility of the electron capture dissociation (ECD) technique, developed by McLafferty and co-workers, has substantially improved peptide and protein characterization using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The limitations of the first ECD implementations on commercial instruments were eliminated by the employment of low-energy electron-injection systems based on indirectly heated dispenser cathodes. In particular, the ECD rate and reliability were greatly increased, enabling the combination of ECD/FTICR-MS with on-line liquid separation techniques. Further technique development allowed the combination of two rapid fragmentation techniques, high-rate ECD and infrared multiphoton dissociation (IRMPD), in a single experimental configuration. Simultaneous and consecutive irradiations of trapped ions with electrons and photons extended the possibilities for ion activation/dissociation and led to improved peptide and protein characterization. The application of high-rate ECD/FTICR-MS has demonstrated its power and unique capabilities in top-down sequencing of peptides and proteins, including characterization of post-translational modifications, improved sequencing of peptides with multiple disulfide bridges and secondary fragmentation (w-ion formation). Analysis of peptide mixtures has been accomplished using high-rate ECD in bottom-up mass spectrometry based on mixture separation by liquid chromatography and capillary electrophoresis. This paper summarizes the current impact of high-rate ECD/FTICR-MS for top-down and bottom-up mass spectrometry of peptides and proteins.

Animals↗

Electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry in the electron energy range 0-50 eV.

Electron capture dissociation (ECD) of polypeptide cations was obtained with pencil and hollow electron beams for both sidekick and gas-assisted dynamic ion trapping (GADT) using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) with an electrostatic ion transfer line. Increasing the number of trapped ions by multiple ICR trap loads using GADT improved the ECD sensitivity in comparison with sidekick ion trapping and ECD efficiency in comparison with single ion trap load by GADT. Furthermore, enhanced sensitivity made it possible to observe ECD in a wide range of electron energies (0-50 eV). The degree, rate and fragmentation characteristics of ECD FTICR-MS were investigated as functions of electron energy, electron irradiation time, electron flux and ion trapping parameters for this broad energy range. The results obtained show that the rate of ECD is higher for more energetic (>1 eV) electrons. Long electron irradiation time with energetic electrons reduces average fragment ion mass and decreases efficiency of formation of c- and z-type ions. The obtained dependencies suggest that the average fragment ion mass and the ECD efficiency are functions of the total fluence of the electron beam (electron energy multiplied by irradiation time). The measured electron energy distributions in low-energy ECD and hot ECD regimes are about 1 eV at full width half maximum in employed experimental configurations.

Electrons↗

Influence of minimally invasive surgery on implant positioning and the functional outcome for medial unicompartmental knee arthroplasty.

We describe postoperative functional outcome and accuracy of implant position in 38 cases after unicompartmental knee arthroplasty with the Oxford knee prosthesis with a standard open approach compared with 30 cases with a minimally invasive approach. Patients with the minimally invasive approach had significantly better functional results, with an average Hospital for Special Surgery scores of 92 (range, 81-98) compared with 78 (range, 24-99). Range of motion 1 year postoperatively was better in the minimally invasive group (113 degrees vs 107 degrees ), but the results were not statistically significant. The number of patients with extension lag was significantly higher after the open approach. The minimal invasive approach had no negative effect on positioning of the prosthesis. In our opinion, minimally invasive implantation is the method of choice for the treatment of anteromedial osteoarthritis by unicompartmental knee arthroplasty.

Aged↗

Labdanes and isopimaranes from Platycladus orientalis and their effects on erythrocyte membrane and on Plasmodium falciparum growth in the erythrocyte host cells.

Six labdanes (1-6) and four isopimaranes (7-10), including three new natural products (7, 9, and 10), were isolated from Platycladus orientalis, and their structures determined using 1D and 2D NMR methods, ion-cyclotron resonance HRMS, and optical rotation data. Relative configurations of all chiral centers in the isopimaranes were determined using NOESY experiments at 600 and 800 MHz. Specific optical rotation data were used to correlate absolute configurations. Compounds 1-9 and aframodial (11) were tested for their in vitro antiplasmodial activity and for their ability to induce changes of erythrocyte shape in order to obtain data about possible correlation between the two effects. All compounds tested exhibited weak (IC(50) > 25 microM) in vitro antiplasmodial effects against Plasmodium falciparum strain 3D7. At the same time, the compounds caused echinocytic or stomatocytic changes of the erythrocyte membrane curvature, indicative of their incorporation into the lipid bilayer, in the concentration region where the antiplasmodial activity was observed. The antiplasmodial effect of these compounds thus appears to be an indirect effect on the erythrocyte host cell. Weak or moderate antiplasmodial activity observed with many other apolar natural products, in particular those with amphiphilic structures, is also likely to be an indirect effect.

Animals↗

Cyclopentanoid cyanohydrin glucosides and amides of Lindackeria dentata.

A mixture of cyanogenic glucosides epivolkenin and taraktophyllin, 1,4-dihydroxy-2-cyclopentenecarboxamide, and uridine were isolated from leaves of Lindackeria dentata (Flacourtiaceae). Another cyclopentanoid amide, (1 R,4 S,5 R)-1,4,5-trihydroxy-2-cyclopentenecarboxamide, was synthesized in two steps from gynocardin, and shown to have the same relative configuration as a partially identified amide previously isolated from L. dentata bark.

Amides↗

Selagoline, a new alkaloid from Huperzia selago.

A new natural product, named selagoline, and two known alkaloids, huperzine A and serratidine, were isolated from Huperzia selago (Lycopodiaceae) collected in Iceland. Their structures were determined using 600 and 800MHz one- and two-dimensional NMR methods supported by Fourier-transform mass spectrometry. Possible role of selagoline as a precursor of 5,15-oxidolycopodane, a component of the classical alkaloids L28 and L31, is discussed.

Alkaloids↗

Solid-phase polyamine synthesis using piperazine and piperidine building blocks.

[reaction: see text]. Polyamines containing piperidine and piperazine moieties have been synthesized on solid support using SN2 alkylation of resin-bound secondary amines with 2-nitrobenzenesulfonates (nosylates). The effect of solvent on this alkylation was investigated. The methodology was employed for the total synthesis of novel analogues of wasp polyamine toxins (philanthotoxins).

Journal Article↗

A new class of spermidine-derived alkaloids.

[structure: see text] Four new alkaloids, dovyalicins A-D, were isolated from Dovyalis macrocalyx. Their structures were established by two-dimensional COSY, NOESY, HSQC, and HMBC NMR experiments as well as chiroptical methods. The compounds, possessing spermidine as part of a perhydro-1,5-diazocine or a perhydro-1,4-diazepine moiety, constitute a new group of polyamine-derived alkaloids.

Journal Article↗

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↗

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↗

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↗