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William F Siems

Publications and source records attributed to William F Siems.

18 recordsLinked to original sources
PubMed2006

Gas-phase chiral separations by ion mobility spectrometry.

This article introduces the concept of chiral ion mobility spectrometry (CIMS) and presents examples demonstrating the gas-phase separation of enantiomers of a wide range of racemates including pharmaceuticals, amino acids, and carbohydrates. CIMS is similar to traditional ion mobility spectrometry, where gas-phase ions, when subjected to a potential gradient, are separated at atmospheric pressure due to differences in their shapes and sizes. In addition to size and shape, CIMS separates ions based on their stereospecific interaction with a chiral gas. In order to achieve chiral discrimination by CIMS, an asymmetric environment was provided by doping the drift gas with a volatile chiral reagent. In this study (S)-(+)-2-butanol was used as a chiral modifier to demonstrate enantiomeric separations of atenolol, serine, methionine, threonine, methyl alpha-glucopyranoside, glucose, penicillamine, valinol, phenylalanine, and tryptophan from their respective racemic mixtures.

Amino Acids↗
PubMed2006

Effect of diastolic pressure on MLC2v phosphorylation in the rat left ventricle.

The effect of passive muscle stretch on the extent of MLC2v phosphorylation was investigated. We used an isolated rat heart preparation and controlled the passive pressure of the left ventricle (LV) at 0 or 15 mmHg. The hearts were flash frozen and the LV free wall was split into epicardial and the endocardial halves. The samples were solubilized using a novel method that minimizes changes in the phosphate content of MLC2v under non-denaturing conditions. The proteins were separated by urea glycerol PAGE and identified by mass spectrometry and Western blots. At 0 mmHg passive pressure, the extent of MLC2v phosphorylation of the epicardium (34.1+/-1.7%) was the same as that of the endocardium (35.3+/-3.4%). At 15 mmHg passive pressure, we found a significant increase in MLC2v phosphorylation in the epicardium (to 41.5+/-2.0%) and a significant reduction in the endocardium (to 24.2+/-1.2%), giving rise to a gradient in the extent of MLC2v phosphorylation from epicardium (high) to endocardium (low). These changes in MLC2v phosphorylation that take place in response to increased diastolic pressure are likely to impact on the calcium sensitivity of actomyosin interaction (with an increased sensitivity towards the epicardium) and may play a role in the Frank-Starling mechanism of the heart.

Animals↗
PubMed2006

Isolation and characterization of isoinhibitors of the potato protease inhibitor I family from the latex of the rubber trees, Hevea brasiliensis.

Three isoinhibitors have been isolated to homogeneity from the C-serum of the latex of the rubber tree, Hevea brasiliensis clone RRIM 600, and named HPI-1, HPI-2a and HPI-2b. The three inhibitors share the same amino acid sequence (69 residues) but the masses of the three forms were determined to be 14,893+/-10, 7757+/-5, and 7565+/-5, respectively, indicating that post-translational modifications of the protein have occurred during latex collection. One adduct could be removed by reducing agents, and was determined to be glutathione, while the other adduct could not be removed by reducing agents and has not been identified. The N-termini of the inhibitor proteins were blocked by an acetylated Ala, but the complete amino acid sequence analysis of the deblocked inhibitors by Edman degradation of fragments from endopeptidase C digestion and mass spectrometry confirmed that the three isoinhibitors were derived from a single protein. The amino acid sequence of the protein differed at two positions from the sequence deduced from a cDNA reported in GenBank. The gene coding for the inhibitor is wound-inducible and is a member of the potato inhibitor I family of protease inhibitors. The inhibitor strongly inhibited subtilisin A, weakly inhibited trypsin, and did not inhibit chymotrypsin. The amino acid residues at the reactive site P(1) and P(1)(') were determined to be Gln45 and Asp46, respectively, residues rarely reported at the reactive site in potato inhibitor I family members. Comparison of amino acid sequences revealed that the HPI isoinhibitors shared from 33% to 55% identity (50-74% similarity) to inhibitors of the potato inhibitor I family. The properties of the isoinhibitors suggest that they may play a defensive role in the latex against pathogens and/or herbivores.

Amino Acid Sequence↗
PubMed2006

Hadamard transform ion mobility spectrometry.

Traditionally, the spectrum acquired using ion mobility spectrometry (IMS) is an average of multiple experimental cycles. Each cycle is initiated by passing a short burst of ions into a drift tube containing a homogeneous electric field. Prior to starting the subsequent cycle, all ions in the system must arrive at the detector or spectral overlap may occur. To maximize resolution, the ion pulse admitted to the drift tube is small in relation to the total scan time with the unfortunate consequence of an inherently low duty cycle (approximately 1%). Offering an improved SNR through a 50% duty cycle, the Hadamard transform (HT) applied to ion mobility spectrometry represents a fresh alternative to signal-averaged data acquisition. Initial results from measurements of amphetamine and cytochrome c samples indicate a 2-10-fold increase in SNR for the HT-IMS technique with no reduction in resolution.

Journal Article↗
PubMed2005

Effects of drugs with muscle-related side effects and affinity for calsequestrin on the calcium regulatory function of sarcoplasmic reticulum microsomes.

The tight regulation of Ca2+ release to and clearance from the cytosol is essential for normal excitation-contraction coupling in both skeletal and cardiac muscles. Calsequestrin (CSQ) is one of the major components in the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. Previously, we showed that several pharmaceutical drugs, such as phenothiazine derivatives, tricyclic antidepressants, anthracycline derivatives, and other hydrophobic compounds bind CSQ with K(d) values in the micromolar range and significantly reduce the Ca2+ binding capacity of cardiac CSQ (Mol Pharmacol 67:97-104, 2005). Because of its key role in Ca2+ regulation, this interference with CSQ function could well produce adverse physiological consequences and potentially be linked to the known muscle-related side effects of these drugs. To further understand the molecular mechanism of undesirable drug effects or adverse drug reactions among those compounds, we examined their effect on the SR microsome. The results clearly showed that these compounds affect Ca2+ release and reduce the total Ca2+ content of the purified SR microsomes, matching well with our previous results with purified recombinant CSQ. Liquid chromatography-mass spectrometry/mass spectrometry showed that the antipsychotic drug trifluoperazine penetrates well into the SR microsome as expected from the reported and calculated log S (aqueous solubility) and log P (partition coefficient) values among the phenothiazine derivatives. We therefore propose that a certain portion of the muscle-related (both cardiac and skeletal) complications of these drugs is caused by the altered Ca2+ regulation of the SR mediated by their adverse interaction with CSQ.

Animals↗
PubMed2005

A hybrid LC-Gel-MS method for proteomics research and its application to protease functional pathway mapping.

Two-dimensional (2D) gel electrophoresis is the most common protein separation method in proteomics research. It can provide high resolution and high sensitivity. However, 2D gel methods have several limitations, such as labor-intensive procedures, poor reproducibility, and limited dynamic range of detection. In fact, many investigators have returned to couple the one-dimensional (1D) SDS-PAGE with mass spectrometry for protein identification. The limitation of this approach is the increased protein complexity in each one-dimensional gel band. To overcome this problem and provide reproducible quantitative information, we describe here a 2D method for protein mixture separation using a combination of high performance liquid chromatography (HPLC) and 1D SDS-PAGE. The study shows that the step-gradient fractionation method we have applied provides excellent reproducibility. In addition, high mass accuracy of LC-FTICR-MS can allow more confident protein identifications by high resolution and ultra-high mass measurement accuracy. This approach was applied to comparative proteomics since protein abundance level changes can be easily visualized with side-by-side vertical comparison in one gel. Furthermore, separation of multi-samples in the same gel significantly reduces run-to-run variation, as is shown with differential image gel electrophoresis (DIGE). Finally, this approach readily incorporates immunological methods to normalize relative abundances of multiple samples within a single gel. This paper presents the results of our developments and our initial application of this strategy for mapping protease function of beta amyloid cleaving enzyme (BACE) in biological systems.

Amyloid Precursor Protein Secretases↗
PubMed2005

Mass spectrometry identifiable cross-linking strategy for studying protein-protein interactions.

A new mass spectrometry identifiable cross-linking strategy has been developed to study protein-protein interactions. The new cross-linker was designed to have two low-energy MS/MS-cleavable bonds in the spacer chain to provide three primary benefits: First, a reporter tag can be released from cross-link due to cleavage of the two labile bonds in the spacer chain. Second, a relatively simple MS/MS spectrum can be generated owing to favorable cleavage of labile bonds. And finally, the cross-linked peptide chains are dissociated from each other, and each then can be fragmented separately to get sequence information. Therefore, this novel type of cross-linker was named protein interaction reporter (PIR). To this end, two RINK groups were utilized to make our first-generation cross-linker using solid-phase peptide synthesis chemistry. The RINK group contains a bond more labile than peptide bonds during low-energy activation. The new cross-linker was applied to cross-link ribonuclease S (RNase S), a noncovalent complex of S-peptide and S-protein. The results demonstrated that the new cross-linker effectively reacted with RNase S to generate various types of cross-linked products. More importantly, the cross-linked peptides successfully released reporter ions during selective MS/MS conditions, and the dissociated peptide chains remained intact during MS(2), thus enabling MS(3) to be performed subsequently. In addition, dead-end, intra-, and inter-cross-linked peptides can be distinguished by analyzing MS/MS spectra.

Amino Acid Sequence↗
PubMed2005

A new maleimide-bound acid-cleavable solid-support reagent for profiling phosphorylation.

A new chemical strategy for phosphopeptide profiling is reported in this study. Phosphorylation represents one of the most important classes of posttranslational modifications of proteins. Here we report a generalized strategy that employs solid-phase capture and mass-encoding steps to selectively enrich phosphopeptides from complex mixtures. This method exploits conversion of phosphates into thiols and reactive compounds to selectively isolate products of phosphorylation. Selective isolation of phosphopeptides is achieved with a simple, novel, acid-cleavable, solid-support-bound maleimide reagent. Our chemistry efforts have focused on minimization of linker size and simplification of reagent production with incorporation of common solid-phase peptide synthesis steps. Relative quantitation was demonstrated by modifying phosphopeptides with incorporation of ethanedithiol and propanedithiol. We observed that appropriate normalization is necessary to utilize mass tag strategies for relative quantitation of posttranslational modifications. The utility of solid-phase capture was determined with model phosphopeptides, and the method was demonstrated with enriching phosphopeptides from beta-casein, alpha-casein and ovalbumin. The solid-phase capture and release methods were also demonstrated with unfractionated whole histone protein mixtures to show this compound applicability in real biological samples. The new chemical strategy will ultimately be utilized for high-throughput profiling of phosphorylation and possibly other posttranslational modifications.

Animals↗
PubMed2005

Identification of novel antigenic proteins in a complex Anaplasma marginale outer membrane immunogen by mass spectrometry and genomic mapping.

Immunization with purified Anaplasma marginale outer membranes induces complete protection against infection that is associated with CD4+ T-lymphocyte-mediated gamma interferon secretion and immunoglobulin G2 (IgG2) antibody titers. However, knowledge of the composition of the outer membrane immunogen is limited. Recent sequencing and annotation of the A. marginale genome predicts at least 62 outer membrane proteins (OMP), enabling a proteomic and genomic approach for identification of novel OMP by use of IgG serum antibody from outer membrane vaccinates. Outer membrane proteins were separated by two-dimensional electrophoresis, and proteins recognized by total IgG and IgG2 in immune sera of outer membrane-vaccinated cattle were detected by immunoblotting. Immunoreactive protein spots were excised and subjected to liquid chromatography-tandem mass spectrometry. A database search of the A. marginale genome identified 24 antigenic proteins that were predicted to be outer membrane, inner membrane, or membrane-associated proteins. These included the previously characterized surface-exposed outer membrane proteins MSP2, operon associated gene 2 (OpAG2), MSP3, and MSP5 as well as recently identified appendage-associated proteins. Among the 21 newly described antigenic proteins, 14 are annotated in the A. marginale genome and include type IV secretion system proteins, elongation factor Tu, and members of the MSP2 superfamily. The identification of these novel antigenic proteins markedly expands current understanding of the composition of the protective immunogen and provides new candidates for vaccine development.

Anaplasma marginale↗
PubMed2004

In vivo posttranslational modifications of the high mobility group A1a proteins in breast cancer cells of differing metastatic potential.

The high mobility group (HMG) proteins are important modulators of chromatin structure and gene transcription. Overexpression of HMGA1 proteins in vivo induces neoplastic transformation and promotes a highly metastatic cellular phenotype. This study focuses on characterization of HMGA1a in vivo posttranslational modification (PTM) patterns found in a nonmetastatic and two metastatic lines of MCF-7 human breast cancer cells of differing tumorigenic potential. PTM types and the amino acids on which they occur were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Mass analysis was restricted to MALDI peaks having less than +/-150 parts per million (ppm) error, thereby holding our analysis to a more stringent criterion than previously published work with the HMG proteins. Validation of MALDI-TOF MS analysis was accomplished utilizing electrospray ionization tandem mass spectrometry (ESI MS/MS) and manual analysis of ion fragmentation spectra. Patterns and sites of PTMs identified in this study suggest that HMGA1a proteins, like the histones, exhibit a biochemical modification "code" that relates to cellular function. For example, both increased levels of acetylation and a previously unidentified dimethylation of both lysine and arginine residues were found on HMGA1a proteins from metastatic cells compared to proteins found in their nonmetastatic precursors. Additionally, the types of modification present on lysine-45 (e.g., unmodified, acetylation, or dimethylation) varied, depending on the metastatic potential of cells. These findings suggest that examination of the PTM patterns on HMGA1 proteins may provide valuable information concerning the physiological and phenotypic state of mammalian cells.

Amino Acid Sequence↗
PubMed2004

Mechanisms of water oxidation catalyzed by the cis,cis-[(bpy)2Ru(OH2)]2O4+ ion.

The cis,cis-[(bpy)(2)Ru(III)(OH(2))](2)O(4+) micro-oxo dimeric coordination complex is an efficient catalyst for water oxidation by strong oxidants that proceeds via intermediary formation of cis,cis-[(bpy)(2)Ru(V)(O)](2)O(4+) (hereafter, [5,5]). Repetitive mass spectrometric measurement of the isotopic distribution of O(2) formed in reactions catalyzed by (18)O-labeled catalyst established the existence of two reaction pathways characterized by products containing either one atom each from a ruthenyl O and solvent H(2)O or both O atoms from solvent molecules. The apparent activation parameters for micro-oxo ion-catalyzed water oxidation by Ce(4+) and for [5,5] decay were nearly identical, with DeltaH(++) = 7.6 (+/-1.2) kcal/mol, DeltaS() = -43 (+/-4) cal/deg mol (23 degrees C) and DeltaH(++) = 7.9 (+/-1.1) kcal/mol, DeltaS(++) = -44 (+/-4) cal/deg mol, respectively, in 0.5 M CF(3)SO(3)H. An apparent solvent deuterium kinetic isotope effect (KIE) of 1.7 was measured for O(2) evolution at 23 degrees C; the corresponding KIE for [5,5] decay was 1.6. The (32)O(2)/(34)O(2) isotope distribution was also insensitive to solvent deuteration. On the basis of these results and previously established chemical properties of this class of compounds, mechanisms are proposed that feature as critical reaction steps H(2)O addition to the complex to form covalent hydrates. For the first pathway, the elements of H(2)O are added as OH and H to the adjacent terminal ruthenyl O atoms, and for the second pathway, OH is added to a bipyridine ring and H is added to one of the ruthenyl O atoms.

Journal Article↗
PubMed2004

Rapid screening for S-adenosylmethionine-dependent methylation products by enzyme-transferred isotope patterns analysis.

We report here an isotopic labeling and mass spectrometric method to rapidly identify S-adenosylmethionine (AdoMet)-dependent methylation products. In the presence of CH(3)- and CD(3)-labeled AdoMet, a methyl transfer product appears as a doublet separated by 3 Da in a mass spectrum, while other compounds show their normal isotopic distribution. Based on this unique isotopic pattern, methylation product(s) can be easily detected even from a mixture of cellular components. To validate our method, the product of human thiopurine methyltransferase (TPMT, EC 2.1.1.67) has been successfully identified from both an in vitro assay and a whole-cell assay. This method is generally applicable to AdoMet-dependent transmethylation and other group-transfer reactions, and constitutes the first example of a general strategy of enzyme-transferred isotope patterns (ETIPs) analysis.

Deuterium↗
PubMed2003

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipids: ionization and prompt fragmentation patterns.

Ionization and prompt fragmentation patterns of triacylglycerols, phospholipids (PLs) and galactolipids were investigated using matrix-assisted laser desorption/ionization (MALDI). Positive ions of non-nitrogen-containing lipids appeared only in the sodiated form, while nitrogen-containing lipids were detected as both sodiated and protonated adducts. Lipids containing acidic hydroxyls were detected as multiple sodium adducts or deprotonated ions in the positive and negative modes, respectively, with the exception of phosphatidylcholines. The positive MALDI spectra of triacylglycerols contained prompt fragments equivalent to the loss of RCOO(-) from the neutral molecules. Prompt fragment ions [PL-polar head](+) were observed in the positive MALDI spectra of all phospholipids except phosphatidylcholines. The phosphatidylcholines produced only a minor positive fragment corresponding to the head group itself (m/z 184). Galactolipids did not undergo prompt fragmentation. Post-source decay (PSD) was used to examine the source of prompt fragments. PSD fragment patterns indicated that the lipid prompt fragment ions did not originate from the observed molecular ions (sodiated or protonated), and suggested that the prompt fragmentation followed the formation of highly unstable, probably protonated, precursor ions. Pathways leading to the formation of prompt fragment ions are proposed.

Galactolipids↗
PubMed2003

Structural analysis of phosphatidylcholines by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The utility of post-source decay (PSD) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was investigated for the structural analysis of phosphatidylcholine (PC). PC did not produce detectable negative molecular ion from MALDI, but positive ions were observed as both [PC+H](+) and [PC+Na](+). The PSD spectra of the protonated PC species contained only one fragment corresponding to the head group (m/z 184), while the sodiated precursors produced many fragment ions, including those derived from the loss of fatty acids. The loss of fatty acid from the C-1 position (sn-1) of the glycerol backbone was favored over the loss of fatty acid from the C-2 position (sn-2). Ions emanating from the fragmentation of the head group (phosphocholine) included [PC+Na-59](+), [PC+Na-183](+) and [PC+Na-205](+), which corresponded to the loss of trimethylamine (TMA), non-sodiated choline phosphate and sodiated choline phosphate, respectively. Other fragments reflecting the structure of the head group were observed at m/z 183, 146 and 86. The difference in the fragmentation patterns for the PSD of [PC+Na](+) compared to [PC+H](+) is attributed to difference in the binding of Na(+) and H(+). While the proton binds to a negatively charged oxygen of the phosphate group, the sodium ion can be associated with several regions of the PC molecule. Hence, in the sodiated PC, intermolecular interaction of the negatively charged oxygen of the phosphate group, along with sodium association at multiple sites, can lead to a complex and characteristic ion fragmentation pattern. The preferential loss of sn-1 fatty acid group could be explained by the formation of an energetically favorable six-member ring intermediate, as apposed to the five-member ring intermediate formed prior to the loss of sn-2 fatty acid group.

Molecular Structure↗
PubMed2002

Structural model of the regulatory domain of smooth muscle heavy meromyosin.

The goal of this study was to provide structural information about the regulatory domains of double-headed smooth muscle heavy meromyosin, including the N terminus of the regulatory light chain, in both the phosphorylated and unphosphorylated states. We extended our previous photo-cross-linking studies (Wu, X., Clack, B. A., Zhi, G., Stull, J. T., and Cremo, C. R. (1999) J. Biol. Chem. 274, 20328-20335) to determine regions of the regulatory light chain that are cross-linked by a cross-linker attached to Cys(108) on the partner regulatory light chain. For this purpose, we have synthesized two new biotinylated sulfhydryl reactive photo-cross-linking reagents, benzophenone, 4-(N-iodoacetamido)-4'-(N-biotinylamido) and benzophenone, 4-(N-maleimido)-4'-(N-biotinylamido). Cross-linked peptides were purified by avidin affinity chromatography and characterized by Edman sequencing and mass spectrometry. Labeled Cys(108) from one regulatory light chain cross-linked to (71)GMMSEAPGPIN(81), a loop in the N-terminal half of the regulatory light chain, and to (4)RAKAKTTKKRPQR(16), a region for which there is no atomic resolution data. Both cross-links were to the partner regulatory light chain and occurred in unphosphorylated but not phosphorylated heavy meromyosin. Using these data, data from our previous study, and atomic coordinates from various myosin isoforms, we have constructed a structural model of the regulatory domain in an unphosphorylated double-headed molecule that predicts the general location of the N terminus. The implications for the structural basis of the phosphorylation-mediated regulatory mechanism are discussed.

Amino Acid Sequence↗
PubMed2002

Rapid screening of aqueous chemical warfare agent degradation products: ambient pressure ion mobility mass spectrometry.

The use of electrospray ionization ambient pressure ion mobility spectrometry with an orthogonal reflector time-of-flight mass spectrometer to analyze chemical warfare (CW) degradation products from aqueous environmental samples has been demonstrated. Certified reference materials of analytical standards for the detection of Schedule 1, 2, or 3 toxic chemicals or their precursors as defined by the chemical warfare convention treaty verification were used in this study. A combination of six G/V-type nerve and four S-type vesicant related CW agent degradation products were separated with baseline resolution by this instrumental technique. Analytical figures of merit for each CW degradation product were determined. In some cases, reduced mobility constants (K0) have been reported for the first time. linear response ranges for the selected CW degradation products were found to be generally approximately 2 orders of magnitude, where the overall dynamic response ranges were found to extend to 4 orders of magnitude. Limits of detection for five of the nine chemical products tested were found to be less than 1 ppm. To demonstrate the potential of this instrumental method with complex mixtures, four CW degradation products were separated and detected from a spiked Palouse River water sample in less than 1 min. Finally, a homologous series of n-alkylamines were used as baseline reference standards, producing a mobility/mass trend line to which the CW degradation products could be compared. Comparison of these products in this manner is expected to reduce the number of false positive/negative responses.

Biodegradation, Environmental↗
PubMed2002

Relationship between MALDI-TOF analysis of beta-CN f193-209 concentration and sensory evaluation of bitterness intensity of aged cheddar cheese.

An internal standard method was previously developed to measure the concentration of a synthetic bitter peptide, beta-CN f193-209, by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The objective of this study was to evaluate the relationship between beta-CN f193-209 concentration in an aqueous extract of aged Cheddar cheese and bitterness intensity of the cheese. Concentrations of beta-CN f193-209 in cheese extracts were determined by MALDI-TOF at 0, 120, 180, and 270 days. Trained panels evaluated the bitterness intensity of the cheeses at 180 and 270 days. Correlation coefficients between MALDI and sensory data at 180 and 270 days were 0.803 and 0.554, respectively. The decreased correlation may be due to the presence of other bitter peptides more responsible for bitterness at longer aging or the production of compounds that mask bitterness intensity.

Aminopeptidases↗