Stephen Naylor
Medical College of Wisconsin, Pediatric Surgery, Faculty Member
Aqueous mixtures of formaldehyde and glutathione react to form a variety of cyclized adducts in addition to S-hydroxymethylglutathione. The adducts are in labile equilibrium with each other and are not readily separated. The structures of... more
Aqueous mixtures of formaldehyde and glutathione react to form a variety of cyclized adducts in addition to S-hydroxymethylglutathione. The adducts are in labile equilibrium with each other and are not readily separated. The structures of two of the other major adducts were determined by concerted application of 13C-1H two-dimensional chemical-shift correlation, fast-atom-bombardment mass spectrometry and tandem mass spectrometry to the adduct mixtures in aqueous solution.
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Pyrazoloacridine (PZA) is an experimental antitumor agent presently under investigation for treatment of solid tumors on the basis of its unique mechanism of action and selectivity for human solid tumor xenograft in mice. Using capillary... more
Pyrazoloacridine (PZA) is an experimental antitumor agent presently under investigation for treatment of solid tumors on the basis of its unique mechanism of action and selectivity for human solid tumor xenograft in mice. Using capillary electrophoresis coupled with electrospray ionization mass spectrometry, we have identified three oxidative PZA metabolites, 9-desmethyl-PZA, N-demethyl-PZA, and PZA N-oxide. The cytochrome P450 (CYP) isoforms involved in PZA metabolism were characterized by studies with CYP chemical inhibitors, correlation of marker activities for selected CYPs with formation of the metabolites using a human liver panel, and PZA metabolism by cDNA-expressed CYPs. 9-Desmethyl-PZA formation was catalyzed by CYP1A2, whereas N-demethyl-PZA formation was catalyzed by CYP3A4. PZA N-oxide formation was catalyzed by flavin monooxygenase (FMO) rather than CYP, as determined by studies with chemical inhibitors of FMO and metabolism by cDNA-expressed human flavin monooxygenase...
Research Interests: Chemistry, Catalysis, Kinetics, Capillary electrophoresis, Medicine, and 14 moreCell Division, Humans, Mice, Animals, High Pressure Liquid Chromatography, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Oxygen, Time Factors, Protein isoforms, Pyrazoles, Recombinant Proteins, Antineoplastic Agents, Minimum inhibitory concentration, and Ultraviolet Rays
ABSTRACT. Objective. To determine the chemical structure of a contaminant, X 1 , previously found in eosinophilia myalgia syndrome case-implicated 5-hydroxytryptophan (5-OHTrp), and also present in over-thecounter (OTC) commercially... more
ABSTRACT. Objective. To determine the chemical structure of a contaminant, X 1 , previously found in eosinophilia myalgia syndrome case-implicated 5-hydroxytryptophan (5-OHTrp), and also present in over-thecounter (OTC) commercially available 5-OHTrp. Methods. Case-implicated 5-OHTrp as well as 6 OTC samples were subjected to accurate mass HPLCmass spectrometry and HPLC-electrochemical detection, and reacted with reduced glutathione. Peak X 1 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, X 1 was determined to be 4,5-tryptophan-dione, a putative neurotoxin. The presence of X 1 in OTC samples varied from 0.5 to 10.3% of the amount of Trp-4,5D present in...
The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA and elsewhere in 1989 was caused by the ingestion of Showa Denko K.K. (SD) L-tryptophan (L-Trp). "Six compounds" detected in the L-Trp were reported as... more
The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA and elsewhere in 1989 was caused by the ingestion of Showa Denko K.K. (SD) L-tryptophan (L-Trp). "Six compounds" detected in the L-Trp were reported as case-associated contaminants. Recently the final and most statistically significant contaminant, "Peak AAA" was structurally characterized. The "compound" was actually shown to be two structural isomers resulting from condensation reactions of L-Trp with fatty acids derived from the bacterial cell membrane. They were identified as the indole C-2 anteiso (AAA-343) and linear (AAA-343) aliphatic chain isomers. Based on those findings, we utilized a combination of on-line HPLC-electrospray ionization mass spectrometry (LC-MS), as well as both precursor and product ion tandem mass spectrometry (MS/MS) to facilitate identification of a homologous family of condensation products related to AAA-343 and AAA-343. We structurally characteriz...
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The eosinophilia-myalgia syndrome (EMS) outbreak of 1989 that occurred in the USA and elsewhere was caused by the ingestion of L-tryptophan (L-Trp) solely manufactured by the Japanese company Showa Denko K.K. (SD). Six compounds present... more
The eosinophilia-myalgia syndrome (EMS) outbreak of 1989 that occurred in the USA and elsewhere was caused by the ingestion of L-tryptophan (L-Trp) solely manufactured by the Japanese company Showa Denko K.K. (SD). Six compounds present in the SD L-Trp were reported to be case-associated contaminants. However, "one" of these compounds, Peak AAA has remained structurally uncharacterized, despite the fact that it was described as "the only statistically significant (p=0.0014) contaminant". Here, we employ on-line microcapillary-high performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS), and tandem mass spectrometry (MS/MS) to determine that Peak AAA is in fact two structurally related isomers. Peak AAA1 and Peak AAA2 differed in LC retention times, and were determined by accurate mass-LC-MS to both have a protonated molecular ion (MH(+)) of mass 343.239 Daltons (Da), corresponding to a molecular formula of C21H30N2O2, and possessing ei...
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Ten reactive metabolites of five polycyclic aromatic hydrocarbons and styrene were investigated to determine the generality of ester adduct formation with human hemoglobin in the form of RBC and hydrolysis to the corresponding... more
Ten reactive metabolites of five polycyclic aromatic hydrocarbons and styrene were investigated to determine the generality of ester adduct formation with human hemoglobin in the form of RBC and hydrolysis to the corresponding tetrahydrotetrols or dihydrodiols. No exceptions were noted among the compounds tested, which included the anti-diol epoxides of benzo[a]pyrene (BaP), chrysene, and benz[a]anthracene; the syn-diol epoxide of BaP; a mixture of syn- and anti-diol epoxides of benzo[e]pyrene; and epoxides of styrene, benzo[e]pyrene, BaP, and cyclopenta[c,d]pyrene. A test of the propensity of the simplest benzylic epoxide, styrene oxide, to form esters that hydrolyze via a BAL1 mechanism was performed. Hydrolysis of styrene oxide-adducted hemoglobin in H2(18)O at neutral pH yielded 18O incorporation results that suggest this mechanism of hydrolysis is operant to a minor degree in styrene oxide-hemoglobin ester adducts. A method was developed for the isolation and quantification of ...
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In this work we describe the use of a modular multidimensional chromatography-tandem mass spectrometry approach for rapid identification of proteins. In particular we highlight the use of a strong cation exchange cartridge in conjunction... more
In this work we describe the use of a modular multidimensional chromatography-tandem mass spectrometry approach for rapid identification of proteins. In particular we highlight the use of a strong cation exchange cartridge in conjunction with a membrane postconcentration cartridge and nano-HPLC on-line with tandem mass spectrometry to characterize the eosinophil granule organelle proteome. Details are provided of the analytical approach we have developed and we discuss some of the advantages compared with previously reported analyses, as well as providing some specific examples of novel proteins identified.
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We are all perplexed that current medical practice often appears maladroit in curing our individual illnesses or disease. However, as is often the case, a lack of understanding, tools and technologies are the root cause of such... more
We are all perplexed that current medical practice often appears maladroit in curing our individual illnesses or disease. However, as is often the case, a lack of understanding, tools and technologies are the root cause of such situations. Human individuality is an often-quoted term but, in the context of human biology, it is poorly understood. This is compounded when there is a need to consider the variability of human populations. In the case of the former, it is possible to quantify human complexity as determined by the 35,000 genes of the human genome, the 1-10 million proteins (including antibodies) and the 2000-3000 metabolites of the human metabolome. Human variability is much more difficult to assess, since many of the variables, such as the definition of race, are not even clearly agreed on. In order to accommodate human complexity, variability and its influence on health and disease, it is necessary to undertake a systematic approach. In the past decade, the emergence of a...
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The human vitamin D receptor (VDR) and retinoid X receptor-(RXR ) modulate gene activity by forming homodimeric or heterodimeric complexes with specific DNA sequences and interaction with other elements of the transcriptional apparatus in... more
The human vitamin D receptor (VDR) and retinoid X receptor-(RXR ) modulate gene activity by forming homodimeric or heterodimeric complexes with specific DNA sequences and interaction with other elements of the transcriptional apparatus in the presence of their known ...
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Background: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that produce increased serum carbohydrate-deficient transferrin (CDT) isoforms. Methods to resolve CDT from fully glycosylated transferrin (Trf)... more
Background: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that produce increased serum carbohydrate-deficient transferrin (CDT) isoforms. Methods to resolve CDT from fully glycosylated transferrin (Trf) have been based on a neutral shift in the isoelectric focusing (IEF) pattern or on a reduction in the negative charge, allowing resolution by anion-exchange chromatography. Our purpose was to develop a method of resolution and relative quantification of Trf isoforms using online immunoaffinity liquid chromatography–mass spectrometry (LC-MS). Methods: Serum (25 μL) was diluted with 100 μL of water before application to an immunoaffinity column that sequestered Trf isoforms. Trf isoforms were eluted from the immunoaffinity column, concentrated on a C4 column, eluted from the C4 column, and introduced into the mass spectrometer. Analysis of the Trf isoforms was entirely automated and completed in <10 min per sample. Results: The LC-MS method demonstrat...
Research Interests: Mass Spectrometry, Sample Preparation, Clinical Chemistry, Electrospray, Medical Biotechnology, and 14 moreClinical, Transferrin, Affinity chromatography, Carbohydrate-deficient transferrin, Clinical Sciences, Qualitative Analysis, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Liquid Chromatography, Very high throughput, Sialic Acid, Autosomal Recessive, Immunoaffinity Chromatography, Mass Spectrometer, and Medical biochemistry and metabolomics
The current paradigm of modern healthcare is a reactive response to patient symptoms, subsequent diagnosis and corresponding treatment of the specific disease(s). This approach is predicated on methodologies first espoused by the Cnidean... more
The current paradigm of modern healthcare is a reactive response to patient symptoms, subsequent diagnosis and corresponding treatment of the specific disease(s). This approach is predicated on methodologies first espoused by the Cnidean School of Medicine approximately 2500years ago. More recently escalating healthcare costs and relatively poor disease treatment outcomes have fermented a rethink in how we carry out medical practices. This has led to the emergence of "P-Medicine" in the form of Personalized and Precision Medicine. The terms are used interchangeably, but in fact there are significant differences in the way they are implemented. The former relies on an "N-of-1" model whereas the latter uses a "1-in-N" model. Personalized Medicine is still in a fledgling and evolutionary phase and there has been much debate over its current status and future prospects. A confounding factor has been the sudden development of Precision Medicine, which has cu...
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Research Interests: Mass Spectrometry, Humans, Animals, Peptides, Time Resolved, and 12 moreHigh Performance Liquid Chromatography, Amyloid Beta, High Pressure Liquid Chromatography, Liquid Chromatography, Swine, Guinea Pig, Sensitivity and Specificity, Brain Chemistry, Quantitative Method, Enzyme Linked Immunosorbent Assay, Biochemistry and cell biology, and Phosphate Buffer Saline
... 25 F. Van Gastel, NJ Taylor and AJ Carly, Inorg. Chem., 28 (1989) 384. 26 D. Braga, K. Henrick, BFG Johnson, J. Lewis, M. McPartlin, WJH Nelson and MD Vargas, J. Chem. Soc., Dalton Trans., (1986) 975. 27 K. Wade, Adv. lnorg. Chem. ...
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Research Interests: Mass Spectrometry, Cancer, In Vitro, Animals, Male, and 14 moreAffinity chromatography, High Pressure Liquid Chromatography, In Vivo, Isolation, Rats, Rat, Molecular Conformation, Metabolic pathway, Amino Acid Sequence, Retention Time, Carcinogen, Hemoglobins, HPLC Chromatography, and In Vitro Techniques
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Page 1. SHORT CO ~ ~ UNICATION Residually Coupled Attached Proton Test in the 13C NMR Assignment of Natural Products Phillip Crews,* Steve Naylor, Barbara L. Myers, James Loo and Lawrence V. Manes Thimann Laboratories ...
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Research Interests: Mass Spectrometry, Cancer, In Vitro, Animals, Male, and 14 moreAffinity chromatography, High Pressure Liquid Chromatography, In Vivo, Isolation, Rats, Rat, Molecular Conformation, Metabolic pathway, Amino Acid Sequence, Retention Time, Carcinogen, Hemoglobins, HPLC Chromatography, and In Vitro Techniques
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Research Interests: Calcium, NMR Spectroscopy, Magnetic Resonance Spectroscopy, Manganese, Signal Transduction, and 15 moreProtein Kinases, Magnesium, Copper, Phosphorylation, Bacillus subtilis, Biometals, Metal ion, Protein Conformation, Sporulation, Amino Acid Sequence, Protein Binding, Biochemistry and cell biology, Molecular Sequence Data, Reference standards, and In Vitro Techniques
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Research Interests: Biochemistry, Mass Spectrometry, Magnetic Resonance Spectroscopy, DNA, Humans, and 14 moreCircular Dichroism, C2H2 zinc fingers, Zinc, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Molecular cloning, Analytical Ultracentrifugation, Protein Conformation, Amino Acid Sequence, Base Sequence, DNA binding proteins, Biochemistry and cell biology, Molecular Sequence Data, binding sites, and Medical biochemistry and metabolomics
Human hemoglobin was alkylated with (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and then treated with aqueous (+/-)-3-amino-1,2-propanediol to convert alkylated carboxyl side chains to... more
Human hemoglobin was alkylated with (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and then treated with aqueous (+/-)-3-amino-1,2-propanediol to convert alkylated carboxyl side chains to N-(2,3-dihydroxypropyl) amides. Tryptic peptides produced from the modified protein were subjected to affinity chromatography on phenylboronic acid. The bound fraction was further purified by HPLC on C-4 reverse-phase medium to yield one modified peptide, which was identified as the Thr(41)-Lys(56) peptide of the alpha chain by amino acid analysis, Edman sequencing analysis, and FAB-MS. Limited direct evidence from this study and further indirect evidence from previous work identify Asp(47) alpha as the amino acid reacting with BPDE. The only other likely sites would be the C-terminal carboxyl groups of either the alpha or beta chain. Possible reasons for the site selectivity of the alkylation of human hemoglobin by BPDE are discussed.
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... Following the dive to single cells, Kevin Chen (BD Biosciences, CA, USA) brought us back to the macro scale with a presentation detailing ... Matrix-assisted laser desorp-tion/ionization mass spectrometry (MALDI-MS) has revolutionized... more
... Following the dive to single cells, Kevin Chen (BD Biosciences, CA, USA) brought us back to the macro scale with a presentation detailing ... Matrix-assisted laser desorp-tion/ionization mass spectrometry (MALDI-MS) has revolutionized our ability to analyze peptides and proteins ...
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(40) Shiemke, AK; Loehr, T. M.; Sanders-Loehr, J. J . Am. Chem. Soc. (41) Adman, E.; Watenpaugh, KD; Jensen, LH Proc. Natl. Acad. Sci. USA 1975, 72, 4854. (42) Tsukihara, T.; Fukuyama, K.; Nakamura, M.; Katsube, Y.; Tanaka, N.; Kakudo,... more
(40) Shiemke, AK; Loehr, T. M.; Sanders-Loehr, J. J . Am. Chem. Soc. (41) Adman, E.; Watenpaugh, KD; Jensen, LH Proc. Natl. Acad. Sci. USA 1975, 72, 4854. (42) Tsukihara, T.; Fukuyama, K.; Nakamura, M.; Katsube, Y.; Tanaka, N.; Kakudo, M.; Wada, K.; Hase, T.; ...
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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... more
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.