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W F Siems

Publications and source records attributed to W F Siems.

13 recordsLinked to original sources
PubMed2001

Expression, purification, characterization, and NMR studies of highly deuterated recombinant cytochrome c peroxidase.

Two forms of extensively deuterated S. cerevisiae cytochrome c peroxidase (CcP; EC 1.11.1.5) have been overexpressed in E. coli by growth in highly deuterated medium. One of these ferriheme enzyme forms (recDCcP) was produced using >97% deuterated growth medium and was determined to be approximately 84% deuterated. The second form [recD(His)CcP] was grown in the same highly deuterated medium that had been supplemented with excess histidine (at natural hydrogen isotope abundance) and was also approximately 84% deuterated. This resulted in direct histidine incorporation without isotope scrambling. Both of these enzymes along with the corresponding recombinant native CcP (recNATCcP), which was expressed in a standard medium with normal hydrogen isotope abundance, consisted of 294 amino acid polypeptide chains having the identical sequence to the yeast-isolated enzyme, without any N-terminal modifications. Comparative characterizations of all three enzymes have been carried out for the resting-state, high-spin forms and in the cyanide-ligated, low-spin forms. The primary physical methods employed were electrophoresis, UV-visible spectroscopy, hydrogen peroxide reaction kinetics, mass spectrometry, and (1)H NMR spectroscopy. The results indicate that high-level deuteration does not significantly alter CcP's reactivity or spectroscopy. As an example of potential NMR uses, recDCcPCN and recD(His)CcPCN have been used to achieve complete, unambiguous, stereospecific (1)H resonance assignments for the heme hyperfine-shifted protons, which also allows the heme side chain conformations to be assessed. Assigning these important active-site protons has been an elusive goal since the first NMR spectra on this enzyme were reported 18 years ago, due to a combination of the enzyme's comparatively large size, paramagnetism, and limited thermal stability.

Cytochrome-c Peroxidase↗
PubMed2001

Calcium-stimulated autophosphorylation site of plant chimeric calcium/calmodulin-dependent protein kinase.

The existence of two molecular switches regulating plant chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK), namely the C-terminal visinin-like domain acting as Ca(2+)-sensitive molecular switch and calmodulin binding domain acting as Ca(2+)-stimulated autophosphorylation-sensitive molecular switch, has been described (Sathyanarayanan, P. V., Cremo, C. R., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 30417-30422). Here we report the identification of Ca(2+)-stimulated autophosphorylation site of CCaMK by matrix-assisted laser desorption ionization time of flight-mass spectrometry. Thr(267) was confirmed as the Ca(2+)-stimulated autophosphorylation site by post-source decay experiments and by site-directed mutagenesis. The purified T267A mutant form of CCaMK did not show Ca(2+)-stimulated autophosphorylation, autophosphorylation-dependent variable calmodulin affinity, or Ca(2+)/calmodulin stimulation of kinase activity. Sequence comparison of CCaMK from monocotyledonous plant (lily) and dicotyledonous plant (tobacco) suggests that the autophosphorylation site is conserved. This is the first identification of a phosphorylation site specifically responding to activation by second messenger system (Ca(2+) messenger system) in plants. Homology modeling of the kinase and calmodulin binding domain of CCaMK with the crystal structure of calcium/calmodulin-dependent protein kinase 1 suggests that the Ca(2+)-stimulated autophosphorylation site is located on the surface of the kinase and far from the catalytic site. Analysis of Ca(2+)-stimulated autophosphorylation with increasing concentration of CCaMK indicates the possibility that the Ca(2+)-stimulated phosphorylation occurs by an intermolecular mechanism.

Amino Acid Sequence↗
PubMed2001

Sampling artifacts from the use of denuder tubes with glycerol based coatings in the measurement of atmospheric particulate matter.

Chemical speciation studies use sampling configurations that often require the deployment of denuder tubes of various types to measure certain species or control particulate sampling artifacts. Concurrent sets of measurements on Teflon membrane and quartz filters in specific sampling configurations were used to evaluate the potential influence of denuder tubes with glycerol-based coatings on particulate mass and carbon measurements on downstream filters. Positive biases were observed in the measurement of gravimetric mass and carbon on Teflon and quartz filters, respectively, downstream of sodium carbonate/ glycerol and citric acid/glycerol coated denuder tubes relative to those without upstream denuder tubes. The magnitude of the bias is dependent on the level of ambient particulate loading.

Aerosols↗
PubMed2001

Analysis of plant phosphatidylcholines by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) for the quantitative determination of phospholipid (PL) molecular species has been problematic, due primarily to the formation of multiple signals (corresponding to the molecular ion and other adducts) for some classes of PL. For example, analysis of phosphatidylcholine (PC) yielded signals that corresponded to protonated and sodiated molecules in the MALDI spectrum. The resulting spectral overlap among various molecular species (e.g. [PC(16:0/18:2) + Na] and [PC(18:2/18:3)]) made it impossible to ascertain their relative amounts using this technique. Other spectral ambiguities existed among different structural isomers, such as PC(18:1/18:1) and PC(18:0/18:2). We determined that molecular species could be resolved by MALDI-TOFMS by first removing the polar head (e.g. phosphocholine) from the phospholipid to effect production of only the sodiated molecules of the corresponding diacylglycerols (DAGs). Analysis of the resulting spectrum allowed unequivocal determination of the molecular species profile of PC from potato tuber and soybean. Estimation of fatty acid composition based on the molecular species determined by MALDI-TOFMS analysis agreed with that from GC-FID analysis. Post-source decay (PSD) was used to resolve standard isomers of PC (e.g. 18:1/18:1 vs. 18:0/18:2). Our results indicated that PSD is a useful approach for resolving structural isomers of PL molecular species.

Chromatography, Gas↗
PubMed2001

Electrospray ionization with ambient pressure ion mobility separation and mass analysis by orthogonal time-of-flight mass spectrometry.

Rapid screening and identification of drug and other mixtures are possible using a novel ambient pressure high-resolution ion mobility (APIMS) orthogonal reflector time-of-flight mass spectrometer (TOFMS). Departing ions from the APIMS drift tube traversed a pressure interface between the APIMS and TOFMS where they were subjected to numerous gas collisions that could produce selective fragmentation. By increasing the accelerating field in the pressure interface region, the ions generated using water-cooled electrospray ionization (ESI) underwent collision-induced dissociation (CID). Mixtures of ESI ions were separated by APIMS based on their respective size-to-charge (s/z) ratios while CID and analysis of mass-to-charge (m/z) ratios occurred in the pressure interface and TOFMS. Product ions that were formed in this pressure interface region could be readily assigned to precursor ions by matching the mobility drift times. This process was demonstrated by the examination of a mixture of amphetamines and the resulting fragmentation patterns of the mobility-separated precursor ion species [M + H](+).

Amphetamines↗
PubMed2000

Separation of isomeric peptides using electrospray ionization/high-resolution ion mobility spectrometry.

In this paper, the first examples of baseline separation of isomeric macromolecules by electrospray ionization/ion mobility spectrometry (ESI/IMS) at atmospheric pressure are presented. The behavior of a number of different isomeric peptides in the IMS was investigated using nitrogen as a drift gas. The IMS was coupled to a quadrupole mass spectrometer, which was used for identification and selective detection of the electrosprayed ions. The mobility data were used to determine their average collision cross sections. The gas-phase ions of isomeric peptides were found to have different collision cross sections. In all cases, doubly charged ions exhibited significantly (8-20%) larger collision cross sections than the respective singly charged species. The analysis of mixtures of the isomeric peptides clearly demonstrated the capability of IMS to separate gas-phase peptide ions due to small differences in their conformational structures, which cannot be determined by mass spectrometry. An actual resolving power of 80 was achieved for two doubly charged reversed sequenced pentapeptides. Baseline separation was provided for ions differing by only 2.5% in their measured collision cross sections; partial separation was shown for isomeric ions exhibiting differences as small as 1.1%.

Algorithms↗
PubMed2000

Secondary electrospray ionization ion mobility spectrometry/mass spectrometry of illicit drugs.

A secondary electrospray ionization (SESI) method was developed as a nonradioactive ionization source for ion mobility spectrometry (IMS). This SESI method relied on the gas-phase interaction between charged particles created by electrospray ionization (ESI) and neutral gaseous sample molecules. Mass spectrometry (MS) was used as the detection method after ion mobility separation for ion identification. Preliminary investigations focussed on understanding the ionization process of SESI. The performance of ESI-IMS and SESI-IMS for illicit drug detection was evaluated by determining the analytical figures of merit. In general, SESI had a higher ionization efficiency for small volatile molecules compared with the electrospray method. The potential of developing a universal interface for both GC- and LC-MS with an addition stage of mobility separation was demonstrated.

Electrochemistry↗
PubMed1999

Fingerprint patterns from laser-induced azido photochemistry of spin-labeled photoaffinity ATP analogs in matrix-assisted laser desorption/ionization mass spectrometry.

The photochemical reaction of azide derivatives induced by ultraviolet (UV) laser in matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is reported. A novel synthesized class of azide aromatic derivatives, spin-labeled photoaffinity non-nucleoside adenosine triphosphate (ATP) analogs which are useful probes in study of muscle contraction mechanism, is used in this investigation. In the negative ion MALDI spectra of these ATP analogs, "fingerprint" peaks corresponding to [M - 10 - 1]-, [M - 12 - 1]-, [M - 16 - 1]-, [M - 26 - 1]-, [M - 28 - 1]-, [M - 41 - 1]-, and [M - 42 - 1]- were observed with relative intensities depending on the MALDI matrix. Only the [M - 16 - 1]- is present in the similar mass spectra of the analog in which the azido group is replaced by a hydrogen. A model is suggested for the photochemical reactions of azide derivatives under UV laser irradiation. The photoreaction fingerprint information is diagnostically useful in characterization of azido compounds, especially for spin-labeled photoaffinity non-nucleoside ATP analogs.

Adenosine Triphosphate↗
PubMed1990

Ion mobility detection of polydimethylsilicone oligomers following supercritical fluid chromatographic separation.

A mixture of polydimethylsilicones (Dow Corning 200), average molecular weight 2000 a.m.u., was separated by simultaneous density and temperature-programmed supercritical fluid chromatography and detected by ion mobility detection. Ion mobility spectra were captured by Fourier transform ion mobility spectrometry. Using information from these spectra it was possible to selectively detect a single compound in the complex mixture. A detector temperature investigation demonstrated that, for the efficient transfer of high-molecular-weight compounds from the column to the detector, the interface to the detector must be heated. Using a 50 microns I.D. column, a Guthrie-type restrictor and a detection temperature of 250 degrees C, as many as 70 oligomers were separated and detected.

Chromatography, High Pressure Liquid↗
PubMed1989

Preliminary investigation of ion mobility spectrometry after capillary electrophoretic introduction.

Using standard capillary electrophoretic and ion mobility methods, several electrospray interface designs were investigated for the capillary electrophoretic introduction of samples into the ion mobility spectrometer. Of the interfaces investigated, the flow assisted interface and the direct coupled interface showed the most promise. These preliminary experiments were encouraging. The ion mobility spectrometer coupled with a capillary electrophoretic introduction system operated with excellent separation efficiency and ion mobility reproducibility. Using tetrabutylammonium iodide, the number of theoretical plates for the spectrometer was calculated to be 3.10(3) and reduced mobilities were found to be reproducible with a relative standard deviation of 1.43%. Because of the desire to hold the spectrometer as hot as possible, the solvent would often vaporize in the interface, creating an unstable spray and inhomogeneities in the electrophoretic field. More work is needed to improve the spray process which contributed to the overall noise of the system and to eliminate the phenomenon of solvent vaporization which limited the reproducibility of electrophoretic migration times.

Electrophoresis↗
PubMed1989

Evaluation of direct axial sample introduction for ion mobility detection after capillary gas chromatography.

An ion mobility detector has been designed and constructed for direct axial interfacing with capillary gas chromatography. The principle advantages of this detector were the following: (1) Direct concentric introduction of the capillary column into the ionization region, eliminating peak broadening in the transfer line and improving the efficiency with which neutral molecules were swept from the detector. (2) A variable capillary insertion distance, providing a sensitivity/resolution interplay that could be modified in response to the needs of the assay. (3) An inert gas flow external to the drift cylinder, preventing atmospheric impurities from infiltrating the ultratrace detector. Qualitative and quantitative capabilities of the detector were evaluated using standard preparations of n-hexyl ether.

Chromatography, Gas↗