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Stephen Naylor

Publications and source records attributed to Stephen Naylor.

At least 37 records · Page 2Linked to original sources
PubMed2003

Parallel determination of multiple protein metabolite interactions using cell extract, protein microarrays and mass spectrometric detection.

Analysis of interactive networks between proteins and other molecular constituents is of paramount importance to delineate complex cellular processes. In order to facilitate this process, new technologies that allow rapid, high-throughput parallel screening, as well as identification of constituents, are necessary. A particularly powerful combination in this regard could be the use of multiprotein microarrays coupled with mass spectrometry (MS). In the initial step of the method development we applied MS to single-protein microarrays. We demonstrated that even a simplified version of the method allows rapid parallel label-free assay of specific protein interactions with multiple metabolites derived from complex artificial and natural mixtures. The microarrays fabricated by the electrospray deposition technique and cross-linked in glutaraldehyde vapor were brought into contact with droplets of solution containing either a natural extract of baker's yeast cells or an artificial cocktail of metabolites. After washing, the microarrays were placed into 75% methanol to denature proteins and release specifically bound metabolites. The eluates were then analyzed by electrospray ionization mass spectrometry (ESI-MS) to simultaneously detect all the metabolites bound. Such a procedure applied to ten different proteins demonstrated that 50-400 ng of cross-linked protein is enough to obtain ion intensities from metabolites that are well distinguishable above noise. The compatibility of microplates and different microarray designs with MS detection is discussed.

Cell Extracts↗
PubMed2003

Protein-metal ion interactions, stoichiometries and relative affinities determined by on-line size exclusion gel filtration mass spectrometry.

The modulation of metal ions on protein function is well recognized and of paramount importance in protein biochemistry. To date, very few methods allow direct determination of protein-metal ion interactions, as well as exact stoichiometric binding ratios. In this work we demonstrate the usefulness of two on-line size exclusion gel filtration mass spectrometry approaches to directly detect protein-metal ion adducts, as well as determine exact protein-metal ion stoichiometries. We show that on-line size exclusion column chromatography (SEC) and rapid in-line desalting (RILED) coupled to microelectrospray mass spectrometry (microESI-MS) can be used for such analyses. The SEC approach can be effectively used to both separate proteins in a complex mixture and exchange buffers prior to the electrospray process. While RILED does not allow for protein separation, it provides a much faster high-throughput desalting procedure than the conventional SEC technique. Specifically, we show that SEC/microESI-MS and RILED/MS can be used to determine calcium ion binding stoichiometries to a high-affinity, metal ion binding protein, calbindin D(28K). Furthermore, the same approaches can also be used to determine metal ion binding stoichiometries of low-affinity metal-binding proteins such as Spo0F.

Animals↗
PubMed2003

An on-line assay for clinical detection of amyloidogenic transthyretin variants directly from serum.

We report here for the first time the on-line analysis of transthyretin genetic variants by mass spectral analysis. The use of mass spectrometry to analyze immunoprecipitated transthyretin has been previously described. However, the on-line analysis of TTR directly from serum reported here will allow for a fully automated high throughput analysis. Mutations in the plasma transport protein TTR are readily observed and distinguished from normal TTR. Free TTR as well as TTR-cysteine and TTR-cysteinylglycine adducts are clearly evident. The resulting assay from serum to final interpretation requires less than twenty minutes. The assay should be an effective first line discriminator of patients who are being considered to have Familial Amyloidotic Polyneuropathy (FAP) and an adjunct to definitive diagnosis by sequencing of the TTR gene or protein.

Amyloid Neuropathies, Familial↗
PubMed2003

Structural characterization of a case-implicated contaminant, "Peak X," in commercial preparations of 5-hydroxytryptophan.

OBJECTIVE: To determine the chemical structure of a contaminant, X1, previously found in eosinophilia myalgia syndrome case-implicated 5-hydroxytryptophan (5-OHTrp), and also present in over-the-counter (OTC) commercially available 5-OHTrp. METHODS: Case-implicated 5-OHTrp as well as 6 OTC samples were subjected to accurate mass HPLC-mass spectrometry and HPLC-electrochemical detection, and reacted with reduced glutathione. Peak X1 was subsequently subjected to HPLC-tandem mass spectrometry (MS/MS), as well as the resulting nucleophilic glutathione product. All these data were compared with analysis carried out under identical conditions on authentic 4,5-tryptophan-dione (Trp-4,5D). RESULTS: Based on accurate mass, tandem mass spectrometric analysis, and comparision with authentic standard compound analysis, X1 was determined to be 4,5-tryptophan-dione, a putative neurotoxin. The presence of X1 in OTC samples varied from 0.5 to 10.3% of the amount of Trp-4,5D present in case-implicated 5-OHTrp. CONCLUSION: Peak X1 was identified as the putative neurotoxin Trp-4,5D. It was found in case-implicated 5-OHTrp as well as 6 OTC samples. This gives some cause for concern in terms of the safety of such commercial preparations of 5-OHTrp.

5-Hydroxytryptophan↗
PubMed2002

Molecular basis for the loss of CD28 expression in senescent T cells.

CD28(null) T cells are the most consistent biological indicator of the aging immune system in humans and are predictors of immunoincompetence in the elderly. The loss of CD28 is the result of an inoperative transcriptional initiator (INR), which consists of two nonoverlapping alpha and beta motifs that have distinct protein binding profiles but function as a unit. In CD28(null) T cells, there is a coordinate loss of alpha-/beta-bound complexes, hence the alphabeta-INR is inactive. In the present work therefore, studies were conducted to identify the components of such complexes that may account for the trans-activation of the alphabeta-INR. By affinity chromatography and tandem mass spectrometry, two proteins, namely, nucleolin and the A isoform of heterogeneous nuclear ribonucleoprotein-D0 (hnRNP-D0A), were identified to be among the key components of the site alpha complex. In DNA binding assays, specific antibodies indicated their antigenic presence in alpha-bound complexes. Transcription assays showed that they are both required in the trans-activation of alphabeta-INR-driven DNA templates. Because CD28 is T cell-restricted, and nucleolin and hnRNP-D0A are ubiquitous proteins, these results support the notion that cell-specific functions can be regulated by commonly expressed proteins. The present data also provide evidence for INR-regulated transcription that is independent of the known components of the basal transcription complex.

Base Sequence↗
PubMed2002

Macromolecular assembly of the transition state regulator AbrB in its unbound and complexed states probed by microelectrospray ionization mass spectrometry.

The Bacillus subtilis global transition-state regulator AbrB specifically recognizes over 60 different DNA regulatory regions of genes expressed during cellular response to suboptimal environments. Most interestingly the DNA regions recognized by AbrB share no obvious consensus base sequence. To more clearly understand the functional aspects of AbrB activity, microelectrospray ionization mass spectrometry has been employed to resolve the macromolecular assembly of unbound and DNA-bound AbrB. Analysis of the N-terminal DNA binding domain of AbrB (AbrBN53, residues 1-53) demonstrates that AbrBN53 is a stable dimer, showing no apparent exchange with a monomeric form as a function of pH, ionic strength, solvent, or protein concentration. AbrBN53 demonstrates a capacity for DNA binding, underscoring the role of the N-terminal domain in both DNA recognition and dimerization. Full-length AbrB is shown to exist as a homotetramer. An investigation of the binding of AbrBN53 and AbrB to the natural DNA target element sinIR shows that AbrBN53 binds as a dimer and AbrB binds as a tetramer. This study represents the first detailed characterization of the stoichiometry of a transition-state regulator binding to one of its target promoters.

Bacillus subtilis↗
PubMed2002

Binding stoichiometry of an RNA aptamer and its transcription factor target.

RNA molecules serve informational, structural, and catalytic roles in cells. RNA also offers an interesting raw material for the design or genetic selection of modifiers of gene expression. We have been interested in the possibility that natural and/or artificial RNA ligands might be identified for DNA-binding proteins. With these concepts in mind, our laboratory previously isolated a 31-nucleotide RNA aptamer that specifically binds to human transcription factor NF-kappaB. This RNA aptamer (alpha-p50) competitively inhibits DNA binding by NF-kappaB in vitro. The aptamer may target the DNA-binding groove formed by the junction of the two monomers of NF-kappaB, perhaps mimicking kappaB duplex DNA. This model predicts a binding stoichiometry of one RNA aptamer per NF-kappaB dimer. To test this hypothesis, two complementary biophysical methods were utilized. Both analytical ultracentrifugation and microelectrospray mass spectrometry suggest that 1 mol of alpha-p50 RNA binds per mole of NF-kappaB p50 homodimer. Such a result is consistent with the observed ability of the RNA aptamer to block the access of transcription factor NF-kappaB to its binding site on DNA and highlights the question of how an RNA stem-loop structurally mimics a DNA duplex. This work also demonstrates the successful application of mass spectrometry to characterize noncovalent RNA/protein interactions.

Protein Binding↗
PubMed2002

Stoichiometries of protein-protein/DNA binding and conformational changes for the transition-state regulator AbrB measured by pseudo cell-size exclusion chromatography-mass spectrometry.

We have developed on-line pseudo cell-size exclusion chromatography-mass spectrometry (PsC-SEC-MS) for the rapid, real time analyses of noncovalently bound protein complexes. The methodology can be used to determine constituent components of such complexes, as well as exact stoichiometries. Furthermore, it enables the efficient determination of gross conformational changes upon complexation. The power of the new approach is demonstrated in the analysis of the global transition-state regulator AbrB and its complex with a target DNA sequence from the promoter sinIR. Using PsC-SEC-MS, we confirm that AbrB is assembled as a homotetramer and not as a homohexamer as previously suggested. Additionally, we show that AbrB binds to the sinIR DNA target element as a homotetramer, affording a 4:1 protein:DNA stoichiometry. Finally, we demonstrate that when the complex binds to sinIR, the hydrodynamic volume (size) of the complex is notably reduced compared to that of the apoprotein, indicating a protein conformational change.

Bacillus subtilis↗
PubMed2002

The metal-binding properties of DREAM: evidence for calcium-mediated changes in DREAM structure.

DREAM, an EF-hand protein, associates with and modulates the activity of presenilins and Kv4 potassium channels in neural and cardiac tissues and represses prodynorphin and c-fos gene expression by binding to DNA response elements in these genes. Information concerning the metal-binding properties of DREAM and the consequences of metal binding on protein structure are important in understanding how this protein functions in cells. We now show that DREAM binds 1 mol of calcium/mol of protein with relatively high affinity and another 3 mol of calcium with lower affinity. DREAM binds 1 mol of magnesium/mol of protein. DREAM, pre-loaded with 1 mol of calcium, binds 1 mol of magnesium, thus demonstrating that the magnesium-binding site is distinct from the high affinity calcium-binding site. Analysis of metal binding to mutant DREAM protein constructs localizes the high affinity calcium-binding site and the magnesium-binding site to EF-hands 3 or 4. Binding of calcium but not magnesium changes the conformation, stability, and alpha-helical content of DREAM. Calcium, but not magnesium, reduces the affinity of apo-DREAM for specific DNA response elements in the prodynorphin and c-fos genes. We conclude that DREAM binds calcium and magnesium and that calcium, but not magnesium, modulates DREAM structure and function.

Amino Acid Sequence↗
PubMed2002

Simultaneous analysis of oxidized and reduced glutathione in cell extracts by capillary zone electrophoresis.

Glutathione (GSH) and glutathione disulfide (GSSG) levels in cells constitute a thiol redox system. They can be used as an indicator of oxidative stress of the cell. In this study, a capillary zone electrophoresis (CZE) method is described that enables quantitation of GSH and GSSG from cellular extracts. The CZE buffer used was 20 mM ammonium acetate containing 5% (v/v) acetic acid at pH 3.1 in conjunction with a polybrene coated capillary operated in reverse polarity mode. Effects of different acids used to prepare cell samples were investigated on CZE performance. The acids include meta phosphoric acid (MPA), trichloroacetic acid (TCA), phosphoric acid (PA) and sulfosalicylic acid (SSA) and are used to stabilize GSH and GSSG before performing CZE analysis. The method features a limit of detection of 4 microM and a limit of quantitation of 12 microM for both GSSG and GSH and recoveries of 94% for GSH and 100% for GSSG. Quantitative analysis of GSSG and GSH in HaCaT cell extracts (5% SSA, w/v) was performed with this method and changes in the ratio of GSH to GSSG in N-ethylmaleimide treated cell sample was observed by comparing with control cell samples.

Cell Extracts↗
PubMed2002

Capillary isoelectric focusing-mass spectrometry: analysis of protein mixtures from human body fluids.

Isoelectric focusing within a fused silica capillary (cIEF) has proved to be a powerful and practical method for high-resolution separation of analytes from complex biological mixtures. This technique overcomes many of the problems of isoelectric focusing within slab gel media. However current cIEF systems commonly utilize UV detection which limits the detail of analyte structural information that is obtained during analysis. The use of mass spectrometry (MS) as the detection system provides much greater structural information about the detected analytes allowing accurate relative molecular mass (M(r)) determination for proteins and polypeptides. We have constructed a cIEF-MS interface and compared the separation of standard proteins analyzed by cIEF-UV with cIEF-MS. This allowed rapid optimization of the cIEF-MS system performance. Further we have demonstrated the use of MS as a detection system provides accurate M(r) information and can provide analyte modification details. These factors increase the likelihood of absolute identification for physiological proteins within complex in vivo-derived mixtures. To demonstrate the value of cIEF-MS in such analyses we have undertaken an examination of cerebrospinal fluid (CSF), and tentatively identified a number of constituent proteins. We have also analyzed whole blood from control and diabetic patients. We show that glycated alpha- and beta- chains of hemoglobin are found in almost equal abundance in diabetic patient blood. From these results we suggest cIEF-MS is an efficient and useful tool for the separation and examination of in vivo-derived analytes within physiological fluids.

Adult↗
PubMed2002

On-Line HPLC-UV-mass spectrometry and tandem mass spectrometry for the rapid delineation and characterization of differences in complex mixtures: a case study using toxic oil variants.

An integrated differential approach to the characterization of complex mixtures is presented which includes the targeting of liquid chromatography (LC) peaks for identification using characteristic UV adsorption of the LC peak, subsequent molecular weight and formula determination using accurate mass LC mass spectrometry (MS), and structure characterization using accurate mass LC-tandem mass spectrometry. The use of differential UV adsorption aids in narrowing the scope of the study to only specific peaks of interest. Accurate mass measurement of the molecular ion species provides molecular weight information as well as atomic composition information. The tandem MS (MS/MS) spectra provide fragmentation information which allows for structural characterization of each component. Accurate mass assignment of each of the fragment ions in the MS/MS spectrum provides atomic composition for each of the fragment ions and thus further aids in the structural characterization. These experiments are facilitated through the use of on-line LC-MS and LC-MS/MS with in-line UV detection. A synthetic toxic oil (STO) related to Toxic Oil Syndrome is studied with a focus on possible contaminants resulting from the interaction of aniline, used as a denaturant, with the normal components of the oil. A differential analysis between the STO and a control oil is performed. LC peaks were targeted using UV absorbance to indicate the possible presence of the aniline moiety. Further differential analysis was performed through the determination of the MS signals associated with each component separated on the LC. Finally, the MS/MS data was also used to determine if the fragmentation of the targeted components indicated the presence of aniline. The MS/MS and accurate mass data were used to assign the structures for the targeted components.

Chromatography, High Pressure Liquid↗
PubMed2002

India ink staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis and in conjunction with Western blots for peptide mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We present an approach that allows matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) peptide mapping of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose (NC). After blocking the nitrocellulose membrane with polyvinylpyrrolidone-40 the immobilized proteins are visualized using India Ink staining which allows the detection of low nanogram amounts of protein. The utilization of a low concentration of Tween 20 (0.05%) in the India Ink staining solution does not negatively impair the quality of the mass spectra. Due to the virtual nondestructive nature of the stain proteolytic peptides could be recovered from the NC membrane. Taking into account minor precautions during the sample manipulation and concentration and by loading the sample onto a pre-crystallized matrix layer, high quality mass spectral data were obtained on <100 femtomoles of protein loaded onto the gel. Finally, the use of India Ink in conjunction with Western blot analysis is also demonstrated. A rat plasma protein, characterized by Western blot as a covalently modified protein-drug compound, was subjected to peptide mapping and post source decay (PSD) sequencing of peptides. The zomepirac-modified protein was identified as the alpha-subunit of fibrinogen.

Amino Acid Sequence↗
PubMed2002

Rare, structurally homologous self-peptides promote thymocyte positive selection.

Although it is clear that positive selection of T cells involves recognition of specific self-peptide/MHC complexes, the nature of these self-ligands and their relationship to the cognate antigen are controversial. Here we used two complementary strategies to identify naturally occurring self-peptides able to induce positive selection of T cells bearing a specific T cell receptor, OT-I. Both the bioassay- and bioinformatics-based strategies identified the same self-peptides, derived from F-actin capping protein and beta-catenin. These peptides displayed charge conservation at two key TCR contact residues. The biological activity of 43 other self-peptides and of complex peptide libraries directly correlated to the extent of conservation at TCR contact residues. These results demonstrate that selecting self-peptides are rare and can be identified by homology-based search strategies.

Actin Depolymerizing Factors↗
PubMed2002

Cefazolin administration and 2-methyl-1,3,4-thiadiazole-5-thiol in human tissue: possible relationship to hypoprothrombinemia.

Cephalosporin antibiotics with structures that include the heterocyclic leaving group 1-methyltetrazole-5-thiol (MTT) can cause hypoprothrombinemia and hemorrhage as a result of MTT-dependent inhibition of the gamma-carboxylation of glutamate. The structure of cefazolin also includes a heterocyclic thiol, 2-methyl-1,3,4-thiadiazole-5-thiol (MTD), and this compound can also inhibit the gamma-carboxylation of glutamate. However, unlike MTT, which is known to be present in vivo after the administration of drugs that include this structure, there have been no reports that MTD is present in vivo after cefazolin administration. We set out to determine whether MTD might be present in the tissues of patients treated with cefazolin prior to surgery. To do that, we took advantage of the fact that heterocyclic thiols can undergo S-methylation catalyzed by the genetically polymorphic drug-metabolizing enzyme thiopurine S-methyltransferase (TPMT). Initially, we tested recombinant human TPMT as a "reagent" to S-methylate MTD. MTD was a substrate for TPMT-catalyzed S-methylation, with an apparent K(m) value of 63 micro M. Recombinant TPMT, with [(14)C-methyl]S-adenosyl-L-methionine as a cosubstrate, was then used to radioactively label a methyl acceptor substrate present in liver and kidney cytosol preparations from patients who had been treated preoperatively with cefazolin. Pooled renal cytosol from 10 of those patients was used to purify and isolate the methylated product by reverse-phase high-performance liquid chromatography. That methylated compound coeluted with S-methyl MTD. When the methylated product was subjected to tandem mass spectrometry, it was identified as S-methyl MTD. Therefore, MTD is present in the tissues of patients treated with cefazolin. These observations also raise the possibility that the TPMT genetic polymorphism may represent a risk factor for cefazolin-induced hypoprothrombinemia since subjects who genetically lack TPMT would be unable to catalyze this MTD biotransformation pathway.

Animals↗
PubMed2002

Activation of the antitumor agent aminoflavone (NSC 686288) is mediated by induction of tumor cell cytochrome P450 1A1/1A2.

The present studies were performed to elucidate the mechanism of cytotoxicity of the aminoflavone analog (5-amino-2,3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one (AF; NSC 686288), a novel flavone with potent in vitro and in vivo antiproliferative activity against a number of human tumor cell lines and with a unique pattern of antiproliferative activity in the National Cancer Institute tumor cell line screen. AF was extensively metabolized by cytochrome P450 (P450) 1A1 and 1A2 to several metabolites, one of which was identified by mass spectrometry as a potentially reactive hydroxylamine. Radiolabeled AF was converted by rat and human microsomes, by recombinant CYP1A1 and CYP1A2, and by sensitive human tumor cell lines to species that covalently bound macromolecules. Treatment of sensitive human MCF7 cells with AF resulted in increased CYP1A1 mRNA and CYP1A1/1A2 protein followed by covalent binding of an AF metabolite to DNA, phosphorylation and stabilization of p53, and increased expression of the p53 transcriptional target p21. Covalent binding of the AF metabolite was increased by pretreatment with the CYP1A inducer 3-methylcholanthrene and decreased by coincubation with the CYP1A inhibitor alpha-naphthoflavone. In contrast, induction of CYP1A1 and covalent binding of the AF metabolite did not occur in AF-resistant M14-MEL cells. These observations suggest that AF is uniquely able to induce its own metabolic activation via CYP1A1/1A2 in duction to cytotoxic DNA-damaging species directly in tumor cells. AF, and possibly other agents, may offer a treatment strategy for tumors responsive to CYP1A1/1A2 induction, such as breast, ovarian, and renal cancers.

Animals↗