RESUMO
Analysis and quantification of analytes in biological systems is a critical component of metabolomic investigations of cell function. The most widely used methods employ chromatographic separation followed by mass spectrometric analysis, which requires significant time for sample preparation and sequential chromatography. We introduce a novel high-throughput, separation-free methodology based on MALDI mass spectrometry that allows for the parallel analysis of targeted metabolomes. Proof-of-concept is demonstrated by analysis of prostaglandins and glyceryl prostaglandins. Derivatization to incorporate a charged moiety into ketone-containing prostaglandins dramatically increases the signal-to-noise ratio relative to underivatized samples. This resulted in an increased dynamic range (15-2000 fmol on plate) and improved linearity (r(2) = 0.99). The method was adapted for high-throughput screening methods for enzymology and drug discovery. Application to cellular metabolomics was also demonstrated.
Assuntos
Lipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Ensaios de Triagem em Larga Escala , Cetonas/química , Metaboloma , Camundongos , Prostaglandinas/análiseRESUMO
Mass spectrometry-based tissue profiling and imaging are technologies that allow identification and visualization of protein signals directly on thin sections cut from fresh frozen tissue specimens. These technologies were utilized to evaluate protein expression profiles in the normal mouse prostate during development (1-5 weeks of age), at sexual maturation (6 weeks of age), and in adult prostate (at 10, 15, or 40 weeks of age). The evolution of protein expression during normal prostate development and maturation were subsequently compared with 15-week prostate tumors derived from genetically engineered mice carrying the Large T antigen gene under regulation of the prostate-specific probasin promoter (LPB-Tag mouse model for prostate cancer). This approach identified proteins differentially expressed at specific time points during prostate development. Furthermore expression of some of these proteins, for example probasin and spermine-binding protein, were associated with prostate maturation, and prostate tumor formation resulted in their loss of expression. Cyclophilin A, a protein found in other cancers, was differentially alpha-acetylated on the N terminus, and both isoforms appeared during normal prostate and prostate tumor development. Imaging mass spectrometry localized the protein signals to specific prostatic lobes or regions. Thus, tissue profiling and imaging can be utilized to analyze the ontogeny of protein expression during prostate morphogenesis and tumorigenesis and identify proteins that could potentially serve as biomarkers for prostate cancer.
Assuntos
Próstata/crescimento & desenvolvimento , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Animais , Western Blotting , Ciclofilina A/química , Ciclofilina A/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Dados de Sequência Molecular , Próstata/citologia , Próstata/patologia , Neoplasias da Próstata/químicaRESUMO
We report the quantitative, label-free analysis of protein-protein interactions in free solution within picoliter volumes using backscatter interferometry (BSI). Changes in the refractive index are measured for solutions introduced on a PDMS microchip allowing determination of forward and reverse rate constants for two-mode binding. Time-dependent BSI traces are directly fit using a global analysis approach to characterize the interaction of the small heat-shock protein alpha-Crystallin with two substrates: destabilized mutants of T4 lysozyme and the in vivo target betaB1-Crystallin. The results recapitulate the selectivity of alphaB-Crystallin differentially binding T4L mutants according to their free energies of unfolding. Furthermore, we demonstrate that an alphaA-Crystallin mutant linked to hereditary cataract has activated binding to betaB1-Crystallin. Binding isotherms obtained from steady-state values of the BSI signal yielded meaningful dissociation constants and establishes BSI as a novel tool for the rapid identification of molecular partners using exceedingly small sample quantities under physiological conditions. This work demonstrates that BSI can be extended to screen libraries of disease-related mutants to quantify changes in affinity and/or kinetics of binding.
Assuntos
Interferometria/métodos , Chaperonas Moleculares/química , Coloração e Rotulagem/métodos , alfa-Cristalinas/química , Cristalinas/química , Interações Medicamentosas , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Mitochondrial disease is a family of genetic disorders characterized by defects in the generation and regulation of energy. Epilepsy is a common symptom of mitochondrial disease, and in the vast majority of cases, refractory to commonly used antiepileptic drugs. Ferroptosis is a recently-described form of iron- and lipid-dependent regulated cell death associated with glutathione depletion and production of lipid peroxides by lipoxygenase enzymes. Activation of the ferroptosis pathway has been implicated in a growing number of disorders, including epilepsy. Given that ferroptosis is regulated by balancing the activities of glutathione peroxidase-4 (GPX4) and 15-lipoxygenase (15-LO), targeting these enzymes may provide a rational therapeutic strategy to modulate seizure. The clinical-stage therapeutic vatiquinone (EPI-743, α-tocotrienol quinone) was reported to reduce seizure frequency and associated morbidity in children with the mitochondrial disorder pontocerebellar hypoplasia type 6. We sought to elucidate the molecular mechanism of EPI-743 and explore the potential of targeting 15-LO to treat additional mitochondrial disease-associated epilepsies. METHODS: Primary fibroblasts and B-lymphocytes derived from patients with mitochondrial disease-associated epilepsy were cultured under standardized conditions. Ferroptosis was induced by treatment with the irreversible GPX4 inhibitor RSL3 or a combination of pharmacological glutathione depletion and excess iron. EPI-743 was co-administered and endpoints, including cell viability and 15-LO-dependent lipid oxidation, were measured. RESULTS: EPI-743 potently prevented ferroptosis in patient cells representing five distinct pediatric disease syndromes with associated epilepsy. Cytoprotection was preceded by a dose-dependent decrease in general lipid oxidation and the specific 15-LO product 15-hydroxyeicosatetraenoic acid (15-HETE). CONCLUSIONS: These findings support the continued clinical evaluation of EPI-743 as a therapeutic agent for PCH6 and other mitochondrial diseases with associated epilepsy.
Assuntos
Carbolinas/farmacologia , Epilepsia/tratamento farmacológico , Ferroptose/efeitos dos fármacos , Doenças Mitocondriais/tratamento farmacológico , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , Ubiquinona/análogos & derivados , Araquidonato 15-Lipoxigenase/metabolismo , Linhagem Celular , Epilepsia/metabolismo , Epilepsia/patologia , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Ubiquinona/farmacologiaRESUMO
Ferroptosis is a form of programmed cell death associated with inflammation, neurodegeneration, and ischemia. Vitamin E (alpha-tocopherol) has been reported to prevent ferroptosis, but the mechanism by which this occurs is controversial. To elucidate the biochemical mechanism of vitamin E activity, we systematically investigated the effects of its major vitamers and metabolites on lipid oxidation and ferroptosis in a striatal cell model. We found that a specific endogenous metabolite of vitamin E, alpha-tocopherol hydroquinone, was a dramatically more potent inhibitor of ferroptosis than its parent compound, and inhibits 15-lipoxygenase via reduction of the enzyme's non-heme iron from its active Fe3+ state to an inactive Fe2+ state. Furthermore, a non-metabolizable isosteric analog of vitamin E which retains antioxidant activity neither inhibited 15-lipoxygenase nor prevented ferroptosis. These results call into question the prevailing model that vitamin E acts predominantly as a non-specific lipophilic antioxidant. We propose that, similar to the other lipophilic vitamins A, D and K, vitamin E is instead a pro-vitamin, with its quinone/hydroquinone metabolites responsible for its anti-ferroptotic cytoprotective activity.
Assuntos
Apoptose/efeitos dos fármacos , Araquidonato 15-Lipoxigenase/metabolismo , Ferro/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Vitaminas/farmacologia , alfa-Tocoferol/análogos & derivados , Animais , Linhagem Celular , Citoproteção/efeitos dos fármacos , Camundongos , alfa-Tocoferol/farmacologiaRESUMO
Imaging mass spectrometry is becoming a key technology for the investigation of the molecular content of biological tissue sections in direct correlation with the underlying histology. Much of our work has been done with fresh-frozen tissue sections that has undergone minimal protein degradation between the time a tissue biopsy is sampled and the time it is snap-frozen so that no preserving or fixing agents need to be added to the frozen biopsy. However, in many sampling environments, immediate flash freezing may not be possible and so we have explored the use of ethanol-preserved, paraffin-embedded tissue specimens for proteomic analyses. Solvent-only preserved tissue specimens provide long-term preservation at room temperature, generation of high quality histological sections and little if any chemical alteration of the proteins. Using mouse organs, several key steps involved in the tissue dehydration process have been investigated to assess the potential of such preserved specimens for profiling and imaging mass spectrometry investigations.
Assuntos
Proteoma/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Dessecação , Eletroforese , Etanol , Fixadores , Formaldeído , Rim/citologia , Rim/metabolismo , Fígado/citologia , Fígado/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Camundongos , Inclusão em Parafina , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Preservação de TecidoRESUMO
Free-solution, label-free molecular interactions were investigated with back-scattering interferometry in a simple optical train composed of a helium-neon laser, a microfluidic channel, and a position sensor. Molecular binding interactions between proteins, ions and protein, and small molecules and protein, were determined with high dynamic range dissociation constants (Kd spanning six decades) and unmatched sensitivity (picomolar Kd's and detection limits of 10,000s of molecules). With this technique, equilibrium dissociation constants were quantified for protein A and immunoglobulin G, interleukin-2 with its monoclonal antibody, and calmodulin with calcium ion Ca2+, a small molecule inhibitor, the protein calcineurin, and the M13 peptide. The high sensitivity of back-scattering interferometry and small volumes of microfluidics allowed the entire calmodulin assay to be performed with 200 picomoles of solute.