RESUMO
RATIONALE: Using a proteomic-based approach we have investigated possible altered expression of a range of cerebral spinal fluid (CSF) proteins following exposure to the neurotoxicant carbonyl sulfide (COS). CSF is ideal for the investigation of markers of brain injury or disease since it is secreted from several central nervous system structures and changes in the CSF composition may reflect brain insult and many pathological processes. METHODS: Animals were placed in exposure chambers and were exposed to 0 ppm or 500 ppm COS for 1, 2 or 3 days, 6 h per day. After the last inhalation exposure, 50-70 µL CSF sample was obtained by lumbar puncture. CSF samples were analyzed by electrospray ionization mass spectrometry (ESI-MS) on either a Premier quadrupole time-of-flight (QTOF) or an Agilent 6340 ion trap and by matrix-assisted laser desorption/ionization (MALDI)-MS on a 4800 MALDI-TOF/TOF analyzer. RESULTS: The dynamic range of abundance of the identified proteins spanned over more than three orders of magnitude. The four most abundant proteins identified (albumin, cystatin C, serotransferrin, transthyretin) are major proteins that are present in both CSF and blood at high levels but the fifth most abundant protein identified (prostaglandin H2D isomerase) is the second most abundant protein in human CSF and is secreted and synthesized in the rat central nervous system. No significant differences were observed between COS-treated CSF samples and the control CSF samples because of blood contamination. CONCLUSIONS: Quantitative MS protein analyses of rat CSF is limited by the low sample volumes that can practicably be obtained from rats and the low protein concentrations in rat CSF. Results of this work suggest a clear need for CSF collection that would minimize blood contamination. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.
Assuntos
Síndromes Neurotóxicas/líquido cefalorraquidiano , Síndromes Neurotóxicas/etiologia , Proteoma/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Óxidos de Enxofre/toxicidade , Animais , Proteínas do Líquido Cefalorraquidiano/análise , Exposição por Inalação , Masculino , Análise de Componente Principal , Proteoma/química , Proteômica , RatosRESUMO
BACKGROUND: Recent immunological data demonstrated that dendritic cells preferentially recognize advanced glycation end product (AGE)-modified proteins, upregulate expression of the receptor for AGE (RAGE), and consequently bias the immune response toward allergy. METHODS: Peanut extract was characterized by mass spectrometry (MS) to elucidate the specific residues and specific AGE modifications found in raw and roasted peanuts and on rAra h 1 that was artificially glycated by incubation with glucose or xylose. The binding of the RAGE-V1C1 domain to peanut allergens was assessed by PAGE and Western analysis with anti-Ara h 1, 2, and 3 antibodies. IgE binding to rAra h 1 was also assessed using the same methods. RESULTS: AGE modifications were found on Ara h 1 and Ara h 3 in both raw and roasted peanut extract. No AGE modifications were found on Ara h 2. Mass spectrometry and Western blot analysis demonstrated that RAGE binds selectively to Ara h 1 and Ara h 3 derived from peanut extract, whereas the analysis failed to demonstrate Ara h 2 binding to RAGE. rAra h 1 with no AGE modifications did not bind RAGE; however, after AGE modification with xylose, rAra h 1 bound to RAGE. CONCLUSIONS: AGE modifications to Ara h 1 and Ara h 3 can be found in both raw and roasted peanuts. Receptor for AGE was demonstrated to selectively interact with AGE-modified rAra h 1. If sensitization to peanut allergens occurs in dendritic cells via RAGE interactions, these cells are likely interacting with modified Ara h 1 and Ara h 3, but not Ara h 2.
Assuntos
Alérgenos/química , Arachis/química , Produtos Finais de Glicação Avançada/metabolismo , Reação de Maillard , Alérgenos/imunologia , Sequência de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Antígenos de Plantas/metabolismo , Arachis/imunologia , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/imunologia , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Glicosilação , Humanos , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Proteínas de Membrana , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica , Espectrometria de Massas em TandemRESUMO
Liver tumors from a previous National Toxicology Program study were examined using global gene expression and mutation analysis to define the mechanisms of carcinogenesis in mice exposed to oxazepam. Five hepatocellular adenomas and 5 hepatocellular carcinomas from male B6C3F1 mice exposed to 5000 ppm oxazepam and 6 histologically normal liver samples from control animals were examined. One of the major findings in the study was upregulation of the Wnt/ß-catenin signaling pathway. Genes that activate ß-catenin, such as Sox4, were upregulated, whereas genes that inhibit Wnt signaling, such as APC and Crebbp, were downregulated. In addition, liver tumors from oxazepam-exposed mice displayed ß-catenin mutations and increased protein expression of glutamine synthetase, a downstream target in the Wnt signaling pathway. Another important finding in this study was the altered expression of oxidative stress-related genes, specifically increased expression of cytochrome p450 genes, including Cyp1a2 and Cyp2b10, and decreased expression of genes that protect against oxidative stress, such as Sod2 and Cat. Increased oxidative stress was confirmed by measuring isoprostane expression using mass spectrometry. Furthermore, global gene expression identified altered expression of genes that are associated with epigenetic mechanisms of cancer. There was decreased expression of genes that are hypermethylated in human liver cancer, including tumor suppressors APC and Pten. Oxazepam-induced tumors also exhibited decreased expression of genes involved in DNA methylation (Crebbp, Dnmt3b) and histone modification (Sirt1). These data suggest that formation of hepatocellular adenomas and carcinomas in oxazepam-exposed mice involves alteration of the Wnt signaling pathway, oxidative stress, and potential epigenetic alterations.
Assuntos
Carcinógenos/toxicidade , Epigênese Genética/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/induzido quimicamente , Oxazepam/toxicidade , Animais , Feminino , Genoma , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Mutação , Estresse Oxidativo , Reação em Cadeia da Polimerase/métodos , Análise Serial de Proteínas , Reprodutibilidade dos Testes , Transdução de Sinais , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
BACKGROUND: Dietary (n-6)-polyunsaturated fatty acids influence cancer development, but the mechanisms have not been well characterised in gastric carcinoma. METHODS: We used two in vivo models to investigate the effects of these common dietary components on tumour metastasis. In a model of experimental metastasis, immunocompromised mice were fed diets containing linoleic acid (LA) at 2% (LLA), 8% (HLA) or 12% (VHLA) by weight and inoculated intraperitoneally (i.p.) with human gastric carcinoma cells (OCUM-2MD3). To model spontaneous metastasis, OCUM-2MD3 tumours were grafted onto the stomach walls of mice fed with the different diets. In in vitro assays, we investigated invasion and ERK phosphorylation of OCUM-2MD3 cells in the presence or absence of LA. Finally, we tested whether a cyclooxygenase (COX) inhibitor, indomethacin, could block peritoneal metastasis in vivo. RESULTS: Both the HLA and VHLA groups showed increased incidence of tumour nodules (LA: 53%; HLA: 89%; VHLA: 100%; P<0.03); the VHLA group also displayed increased numbers of tumour nodules and higher total volume relative to LLA group in experimental metastasis model. Both liver invasion (78%) and metastasis to the peritoneal cavity (67%) were more frequent in VHLA group compared with the LLA group (22% and 11%, respectively; P<0.03) in spontaneous metastasis model. We also found that the invasive ability of these cells is greatly enhanced when exposed to LA in vitro. Linoleic acid also increased invasion of other scirrhous gastric carcinoma cells, OCUM-12, NUGC3 and MKN-45. Linoleic acid effect on OCUM-2MD3 cells seems to be dependent on phosphorylation of ERK. The data suggest that invasion and phosphorylation of ERK were dependent on COX. Indomethacin decreased the number of tumours and total tumour volume in both LLA and VHLA groups. Finally, COX-1, which is known to be an important enzyme in the generation of bioactive metabolites from dietary fatty acids, appears to be responsible for the increased metastatic behaviour of OCUM-2MD3 cells in the mouse model. CONCLUSION: Dietary LA stimulates invasion and peritoneal metastasis of gastric carcinoma cells through COX-catalysed metabolism and activation of ERK, steps that compose pathway potentially amenable to therapeutic intervention.
Assuntos
Carcinoma/patologia , Movimento Celular/efeitos dos fármacos , Gorduras Insaturadas na Dieta/farmacologia , Ácido Linoleico/efeitos adversos , Ácido Linoleico/farmacologia , Neoplasias Gástricas/patologia , Animais , Gorduras Insaturadas na Dieta/efeitos adversos , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Previous studies have shown that short-wavelength blue visible light induces retinal injury and may be a risk factor for age related macular degeneration. A2E is a blue light absorbing retinal chromophore that accumulates with age. Our previous in vitro studies have determined that, although A2E itself has a low phototoxic efficiency, the oxidation products of A2E that are formed in the presence of visible light can contribute to observed retinal pigment epithelial photodamage. The purpose of this study was to investigate the effects of blue light on retinal phototoxicity and its relationship to A2E, oxidized A2E and its isomers. Sprague-Dawley albino rats were dark adapted for 24 h. Control rats remained in the dark while experimental rats were exposed to blue light (λ = 450 nm, 3.1 mW cm(-2)) for 6 h. Isolated retinas were homogenized in Folch extraction mixture and then in chloroform. The dried extracts were reconstituted and divided for determination of organic soluble compound. Esters of fatty acids were determined with GC-MS, A2E and other chromophores using HPLC, and A2E oxidation products with LC-MS. Exposure of rat eyes to blue light did not significantly change the fatty acid composition of the retina. The A2E concentration (normalized to fatty acid content) in blue light exposed animals was found to be lower than the A2E concentration in control rats. The concentrations of all-trans-retinal-ethanolamine adduct and iso-A2E a precursor and an isomer of A2E respectively, were also lower after blue-light exposure than in the retinas of rats housed in the dark. On the other hand, the amount of oxidized forms of A2E was higher in the animals exposed to blue light. We conclude that in the rat eye, blue-light exposure promotes oxidation of A2E and iso-A2E to the products that are toxic to retinal tissue. Although high concentrations of A2E may be cytotoxic to the retina, the phototoxicity associated with blue light damage to the retina is in part a result of the formation of toxic A2E oxides. This effect may partially explain the association between blue light induced retinal injury and macular degeneration.
Assuntos
Modelos Animais de Doenças , Luz/efeitos adversos , Degeneração Macular/etiologia , Compostos de Piridínio/efeitos adversos , Retinoides/efeitos adversos , Animais , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Degeneração Macular/patologia , Masculino , Oxirredução , Compostos de Piridínio/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/patologia , Retina/efeitos da radiação , Retinoides/metabolismoRESUMO
RXR is a nuclear receptor that plays a central role in cell signaling by pairing with a host of other receptors. Previously, 9-cis-retinoic acid (9cRA) was defined as a potent RXR activator. Here we describe a unique RXR effector identified from organic extracts of bovine serum by following RXR-dependent transcriptional activity. Structural analyses of material in active fractions pointed to the saturated diterpenoid phytanic acid, which induced RXR-dependent transcription at concentrations between 4 and 64 microM. Although 200 times more potent than phytanic acid, 9cRA was undetectable in equivalent amounts of extract and cannot be present at a concentration that could account for the activity. Phytanic acid, another phytol metabolite, was synthesized and stimulated RXR with a potency and efficacy similar to phytanic acid. These metabolites specifically displaced [3H]-9cRA from RXR with Ki values of 4 microM, indicating that their transcriptional effects are mediated by direct receptor interactions. Phytol metabolites are compelling candidates for physiological effectors, because their RXR binding affinities and activation potencies match their micromolar circulating concentrations. Given their exclusive dietary origin, these chlorophyll metabolites may represent essential nutrients that coordinate cellular metabolism through RXR-dependent signaling pathways.
Assuntos
Fitol/metabolismo , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células CHO , Bovinos , Cricetinae , Gorduras na Dieta/metabolismo , Ácidos Graxos Essenciais/isolamento & purificação , Ácidos Graxos Essenciais/metabolismo , Técnicas In Vitro , Ligantes , Ácido Fitânico/análogos & derivados , Ácido Fitânico/isolamento & purificação , Ácido Fitânico/metabolismo , Ácido Fitânico/farmacologia , Receptores do Ácido Retinoico/efeitos dos fármacos , Doença de Refsum/metabolismo , Receptores X de Retinoides , Transdução de Sinais , Fatores de Transcrição/efeitos dos fármacos , Tretinoína/sangue , Tretinoína/metabolismoRESUMO
Plasma and urinary levels of malondialdehyde-like products (MDA) and isoprostanes were identified as markers of in vivo lipid peroxidation in an animal model of CCl4 poisoning. We sought to determine the extent to which the formation of these oxidation products is influenced by inhibition of the cyclooxygenase enzymes which catalytically generate proinflammatory lipid peroxidation products known as prostaglandins and thromboxane. In the present studies, after induction of oxidant stress in rats with CCl4, lipid peroxidation products measured in plasma and urine demonstrate that isoprostanes and MDA can be partially inhibited by cyclooxygenase inhibitors, albeit to different extents. The lowering of isoprostane and MDA formation, however, may not to due primarily to the diminution of catalytic generation of isoprostanes or MDA by the cyclooxygenases but, rather, may be the result of the suppression of nonenzymatic lipid peroxidation. This is suggested since 8,12-iso-iPF2alpha-VI is also reduced by indomethacin, yet, unlike other isoprostanes and MDA, it is not generated catalytically by the cyclooxygenase. Thus, although the two cyclooxygenase inhibitors we tested have statistically significant effects on the measurements of both isoprostanes and MDA in this study, the results provide evidence that these lipid-degradation products primarily constitute markers of oxidative stress.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Biomarcadores/metabolismo , Intoxicação por Tetracloreto de Carbono/tratamento farmacológico , Tetracloreto de Carbono/toxicidade , Indometacina/farmacologia , Metabolismo dos Lipídeos , Ácido Meclofenâmico/farmacologia , Estresse Oxidativo , Animais , Cromatografia Líquida de Alta Pressão , Radicais Livres , Cromatografia Gasosa-Espectrometria de Massas , Imunoensaio , Indometacina/metabolismo , Inflamação , Peroxidação de Lipídeos , Espectrometria de Massas , Oxigênio/metabolismo , Prostaglandinas/metabolismo , Isoformas de Proteínas , Ratos , Ratos Endogâmicos F344 , Tromboxano A2/metabolismo , Fatores de TempoRESUMO
Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.
Assuntos
Intoxicação por Tetracloreto de Carbono/metabolismo , Tetracloreto de Carbono/toxicidade , DNA/metabolismo , Desoxiguanosina/análogos & derivados , Metabolismo dos Lipídeos , Estresse Oxidativo , 8-Hidroxi-2'-Desoxiguanosina , Animais , Ensaio Cometa , Dano ao DNA , Desoxiguanosina/farmacologia , Radicais Livres , Cromatografia Gasosa-Espectrometria de Massas , Peróxido de Hidrogênio/metabolismo , Imunoensaio , Immunoblotting , Fígado/metabolismo , Masculino , Malondialdeído/farmacologia , Metionina/metabolismo , Oxigênio/metabolismo , Ratos , Ratos Endogâmicos F344 , Espectrofotometria , Substâncias Reativas com Ácido Tiobarbitúrico , Fatores de Tempo , Tirosina/química , Tirosina/metabolismoRESUMO
BACKGROUND: Although alcohol intake has been positively associated with breast cancer risk in epidemiologic studies, the mechanisms mediating this association are speculative. OBJECTIVE: The Postmenopausal Women's Alcohol Study was designed to explore the effects of moderate alcohol consumption on potential risk factors for breast cancer. In the present analysis, we evaluated the relationship of alcohol consumption with antioxidant nutrients and a biomarker of oxidative stress. DESIGN: Participants (n=53) consumed a controlled diet plus each of three treatments (15 or 30 g alcohol/day or a no-alcohol placebo beverage), during three 8-week periods in random order. We measured the antioxidants, vitamin E (alpha (alpha)- and gamma (gamma)-tocopherols), selenium, and vitamin C in fasting blood samples which were collected at the end of diet periods, treated and frozen for assay at the end of the study. We also measured 15-F(2t)-IsoP isoprostane, produced by lipid peroxidation, which serves as an indicator of oxidative stress and may serve as a biomarker for conditions favorable to carcinogenesis. RESULTS: After adjusting for BMI (all models) and total serum cholesterol (tocopherol and isoprostane models) we observed a significant 4.6% decrease (P=0.02) in alpha-tocopherol and a marginally significant 4.9% increase (P=0.07) in isoprostane levels when women consumed 30 g alcohol/day (P=0.06 and 0.05 for overall effect of alcohol on alpha-tocopherol and isoprostanes, respectively). The other antioxidants were not significantly modified by the alcohol treatment. CONCLUSIONS: These results suggest that moderate alcohol consumption increases some biomarkers of oxidative stress in postmenopausal women.
Assuntos
Consumo de Bebidas Alcoólicas , Antioxidantes/metabolismo , Neoplasias da Mama/epidemiologia , Etanol/administração & dosagem , Isoprostanos/sangue , Estresse Oxidativo/efeitos dos fármacos , Pós-Menopausa/sangue , Consumo de Bebidas Alcoólicas/efeitos adversos , Ácido Ascórbico/sangue , Biomarcadores/sangue , Estudos Cross-Over , Feminino , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Pessoa de Meia-Idade , Estresse Oxidativo/fisiologia , Fatores de Risco , Selênio/sangue , Vitamina E/sangueRESUMO
We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.
Assuntos
Bombesina/imunologia , Mapeamento de Epitopos/métodos , Peptídeos/imunologia , Sequência de Aminoácidos , Aminopeptidases/metabolismo , Anticorpos Monoclonais , Complexo Antígeno-Anticorpo/metabolismo , Quimotripsina/metabolismo , Epitopos/imunologia , Peptídeo Liberador de Gastrina , Lasers , Espectrometria de Massas , Dados de Sequência Molecular , Termolisina/metabolismo , Tripsina/metabolismoRESUMO
A combination of epitope excision, epitope extraction, and differential chemical modification followed by mass spectrometric peptide mapping was used for the characterization of a discontinuous epitope that is recognized by the mouse anti-HIV-p24 monoclonal antibody 5E2.A3. In epitope excision, the protein is first bound to an immobilized antibody and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the antibody. After epitope excision of the p24-5E2.A3 complex with endoproteinase Lys-C, a large fragment remained affinity bound corresponding to amino acids 1-158 of HIV-p24 (fragment 1-158). Further digestion, however, resulted in loss of affinity. Moreover, no affinity-bound fragments were observed after an epitope extraction experiment. These data from the epitope excision and extraction experiments suggest that the epitope is discontinuous. For the further characterization of the epitope, amino groups in the epitope-containing fragment were acetylated in both the affinity bound and free states followed by mass spectrometric analysis. Two successive acetylation reactions were performed: (1) the first used a low molar excess of acetic anhydride, and (2) the second, after separation from the antibody, a high molar excess of its hexadeuteroderivative. This isotopic labeling procedure, in combination with high resolution mass spectrometry, allowed the precise determination of relative reactivities of amino groups. In this study, no differences were observed in the ranking of the relative reactivities of five lysine residues. However, the N-terminal amino group was found to be part of the discontinuous epitope.
Assuntos
Epitopos , Proteína do Núcleo p24 do HIV/química , Fragmentos de Peptídeos/química , Acetilação , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Complexo Antígeno-Anticorpo/química , Cromatografia Líquida , Proteína do Núcleo p24 do HIV/imunologia , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mapeamento de Peptídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The aim of the work is to identify and characterize the hemoglobins found in B6C3F1 mice using mass spectrometry. The primary structures are compared to those reported for BALB/c mice. Individual hemoglobin chains were isolated by reverse-phase high performance liquid chromatography (RP-HPLC). The molecular masses of the globins were determined using electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The purified globin chains were enzymatically cleaved and the resulting peptides were separated by RP-HPLC. The chains were identified by N-terminal sequencing and mass spectrometry (MALDI). Selected peptides were analysed by Edman degradation. ESI analysis indicates that B6C3F1 mice have two alpha-globin chains (alpha-1 and alpha-2) and at least three beta-globin chains, beta-1, beta-2 and beta-3. This is one additional alpha- and one additional beta-globin chain than reported in the literature for BALB/c mice. Mass and sequence analysis of enzymatically generated peptides showed variations in the amino acid sequence in the alpha-1, alpha-2, beta-2 and beta-3 chains compared to the BALB/c mouse hemoglobins (alpha, beta (minor) and beta (major)). The study showed that mass spectrometry in combination with traditional protein chemistry is able to identify and locate minor protein sequence variations.
Assuntos
Hemoglobinas/análise , Hemoglobinas/genética , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Globinas/genética , Globinas/isolamento & purificação , Hemoglobinas/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mapeamento de Peptídeos , Análise de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Our laboratory recently described a new human cytochrome P450 arachidonic acid epoxygenase (CYP2J2) and the corresponding rat homolog (CYP2J3). Immunoblotting studies using a polyclonal antibody raised against recombinant human CYP2J2 confirmed CYP2J protein expression in human and rat pancreatic tissues. Immunohistochemical staining of formalin-fixed paraffin-embedded rat and human pancreas using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection revealed that CYP2J2 protein expression was highly localized to cells in the islets of Langerhans, with minimal staining in pancreatic exocrine cells. Colocalization studies using antibodies to the glucagon, insulin, somatostatin, and pancreatic polypeptide as markers for alpha-, beta-, delta-, and PP cells, respectively, showed that CYP2J protein expression was abundantly present in all four cell types, but was highest in the glucagon-producing alpha-cells. Direct evidence for the epoxidation of arachidonic acid by pancreatic cytochrome P450 was provided by documenting, for the first time, the presence of epoxyeicosatrienoic acids in vivo in human and rat pancreas by gas chromatography/mass spectrometry. Importantly, the levels of immunoreactive CYP2J2 in different human pancreatic tissues were highly correlated with endogenous epoxyeicosatrienoic acid concentrations. We conclude that human and rat pancreas contain an arachidonic acid epoxygenase belonging to the CYP2J subfamily that is highly localized to islet cells. These data together with previous work showing effects of epoxyeicosatrienoic acids in stimulating insulin and glucagon secretion from isolated rat pancreatic islets support the hypothesis that epoxygenase products may be involved in stimulus-secretion coupling in the pancreas.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Ilhotas Pancreáticas/enzimologia , Oxigenases/metabolismo , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Animais , Citocromo P-450 CYP2J2 , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Immunoblotting , Imuno-Histoquímica , Pâncreas/metabolismo , Ratos , Distribuição TecidualRESUMO
Cytosolic sulfotransferases sulfate steroids such as estrogens and hydroxysteroids. The enzymes, including human estrogen sulfotransferase (hEST) and hydroxysteroid sulfotransferase (hHST), are generally homodimers in solution with mouse estrogen sulfotransferase (mEST) being one of few exceptions. To identify the amino acid residues responsible for the dimerization, eight residues on the surface of hEST were mutated to their counterparts in mEST and mutated hESTs were then analyzed by gel filtration chromatography. A single mutation of Val(269) to Glu was sufficient to convert hEST to a monomer and the corresponding mutation of Val(260) also altered hHST to a monomer. The hHST crystal structure revealed a short stretch of peptide with the side-chains from two hHST monomers forming a hydrophobic zipper-like structure enforced by ion pairs at both ends. This peptide consisted of 10 residues near the C-terminus that, including the critical Val residue, is conserved as KXXXTVXXXE in nearly all cytosolic sulfotransferases. When mEST underwent the double mutations Pro269Thr/Glu270Val dimerization resulted. Thus, the KXXXTVXXXE sequence appears to be the common protein-protein interaction motif that mediates the homo- as well as heterodimerization of cytosolic sulfotransferases.
Assuntos
Citosol/enzimologia , Dimerização , Sulfotransferases/química , Sulfotransferases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Reagentes de Ligações Cruzadas/metabolismo , Cristalografia por Raios X , Análise Mutacional de DNA , Eletroforese em Gel de Poliacrilamida , Glutamina/química , Glutationa/metabolismo , Humanos , Espectrometria de Massas , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Sefarose/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Valina/químicaRESUMO
The 4-POBN[α-(4-pyridyl-l-oxide)-N-tert-butyl-nitrone] radical adducts of ethyl and pentyl radicals were determined by a combination of high performance liquid chromatography (HPLC) combined with electron paramagnetic resonance (EPR) with HPLC-electrospray (ESI)-mass spectrometry and HPLC-thermospray (TSP)-MS. The identifIcation of the peak corresponding to the spin-trapped radical was done by performing HPLC-EPR under the same chromatographic conditions as the HPLC-MS. The radical adducts could be determined by both techniques, even though for ESI only 12 µL/min of the total 1 mL/min HPLC flow rate could be directed into the ion source.
RESUMO
Collisional activation (CA) of large ions at kiloelectronvolt energies is accompanied by unexpectedly large losses of translational energy, which vary with the nature of the collision gas. Previous investigations have concentrated upon subsequent fragmentations occurring within a time window covering a few fis immediately following collision, using massanalyzed ion kinetic energy spectrometry. In the present work, survivor ions were selected for specified values of translational energy loss, and their internal energy contents assessed via their subsequent unimolecular fragmentation reactions within a later time window. Beam collimation was also applied when circumstances permitted to impose angular selection, thus minimizing cross talk between effects of collisional scattering and energy dispersion. It was shown that internal excitation of the reactant ion can account for only a small fraction of the observed loss of translational energy. The recoil energy of the target is thus the principal sink for the translational energy loss, since the latter was always chosen to be less than the lowest excitation energy of the target. This conclusion is shown to be consistent with theoretical models of the CA process. The practical implications of these conclusions for CA of large ions at kiloelectronvolt energies are discussed.
RESUMO
A variety of backbone-modified peptides were desorbed by fast atom bombardment and collisionally activated. These peptide modifications involve the replacement of a normal [CONH] peptide linkage with such groups as thiomethylene ether (CH2S), thioamide (CSNH), methyleneamine (CH2NH), and thiomethylene sulfoxide (CH2SO) moieties. Modified linear peptides decompose to give fragmentations characteristic of the modifications as well as typical peptide bond fragments. The presence of a replacement group in cyclic peptides can induce new fragmentations. The presence of other functional groups, such as an exocyclic N-terminal residue, however, can dominate the observed fragmentations. Upon collisional activation, unmodified linear peptides fragment to give N-terminal ions as the most abundant daughter ions. In comparison, ψ[CH2NH] and ψ[CH2S ] modified linear peptides decompose to give prominent C-terminal sequence ions. The ψ[CH2SO] modified linear peptides, however, fragment into both N- and C-terminal ions of high relative abundance. Depending on the modification, daughter ions or internal fragment ions are observed that are characteristic of the amide bond replacement. Useful structural information can therefore be obtained.
RESUMO
Nanoscale capillary liquid chromatography (nCLC) and capillary zone electrophoresis (CZE) have been combined with quadrupole mass spectrometry via an electrospray ionization (ESI) interface. These methodologies have been applied to the separation and determination of a variety of sulfonamides. CZE/ESI/MS is the more rapid and sensitive technique, but nCLC/ESI/MS shows promise for the analysis of dilute samples. Ultimately, the two techniques provide complementary methods of analysis. The detection limits of these techniques in the full-scan mode are in the low picomole range. Dissociation of the sulfonamides can be induced by increasing the skimmer voltage. This provides a limited means of discriminating between compounds of identical molecular weight but, more important, provides fragments that could be used to confirm the presence of analyte within a sample.
RESUMO
Consecutive enzymatic reactions of analytes which are affinity bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ionization mass spectrometry have been used for detecting phosphorylation sites. The usefulness of this method was demonstrated by analyzing two commercially available phosphoproteins, beta-casein and alpha-casein, as well as one phosphopeptide from a kinase reaction mixture. Agarose loaded with either Fe3+ or Ga3+ was used to isolate phosphopeptides from the protein digest. Results from using either metal ion were complementary. Less overall suppression effect was achieved when Ga3+-loaded agarose was used to isolate phosphopeptides. The selectivity for monophosphorylated peptides, however, was better with Fe3+-loaded agarose. This technique is easy to use and has the ability to analyze extremely complicated phosphopeptide mixtures. Moreover, it eliminates the need for prior high-performance liquid chromatography separation or radiolabeling, thus greatly simplifying the sample preparation.