RESUMEN
Mass spectrometry imaging (MSI) is a comprehensive tool for the analysis of a wide range of biomolecules. The mainstream method for molecular MSI is matrix-assisted laser desorption ionization, however, the presence of a matrix results in spectral interferences and the suppression of some analyte ions. Herein we demonstrate a new matrix-free MSI technique using nanophotonic ionization based on laser desorption ionization (LDI) from a highly uniform silicon nanopost array (NAPA). In mouse brain and kidney tissue sections, the distributions of over 80â putatively annotated molecular species are determined with 40â µm spatial resolution. Furthermore, NAPA-LDI-MS is used to selectively analyze metabolites and lipids from sparsely distributed algal cells and the lamellipodia of human hepatocytes. Our results open the door for matrix-free MSI of tissue sections and small cell populations by nanophotonic ionization.
Asunto(s)
Rayos Láser , Imagen Molecular , Fotones , Animales , Ratones , Microscopía Electrónica de Rastreo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Mitochondrial dysfunction is a contributor to diabetic cardiomyopathy. Previously, we observed proteomic decrements within the inner mitochondrial membrane (IMM) and matrix of diabetic cardiac interfibrillar mitochondria (IFM) correlating with dysfunctional mitochondrial protein import. The goal of this study was to determine whether overexpression of mitochondria phospholipid hydroperoxide glutathione peroxidase 4 (mPHGPx), an antioxidant enzyme capable of scavenging membrane-associated lipid peroxides in the IMM, could reverse proteomic alterations, dysfunctional protein import, and ultimately, mitochondrial dysfunction associated with the diabetic heart. MPHGPx transgenic mice and controls were made diabetic by multiple low-dose streptozotocin injections and examined after 5 wk of hyperglycemia. Five weeks after hyperglycemia onset, in vivo analysis of cardiac contractile function revealed decreased ejection fraction and fractional shortening in diabetic hearts that was reversed with mPHGPx overexpression. MPHGPx overexpression increased electron transport chain function while attenuating hydrogen peroxide production and lipid peroxidation in diabetic mPHGPx IFM. MPHGPx overexpression lessened proteomic loss observed in diabetic IFM. Posttranslational modifications, including oxidations and deamidations, were attenuated in diabetic IFM with mPHGPx overexpression. Mitochondrial protein import dysfunction in diabetic IFM was reversed with mPHGPx overexpression correlating with protein import constituent preservation. Ingenuity Pathway Analyses indicated that oxidative phosphorylation, tricarboxylic acid cycle, and fatty acid oxidation processes most influenced in diabetic IFM were preserved by mPHGPx overexpression. Specific mitochondrial networks preserved included complex I and II, mitochondrial ultrastructure, and mitochondrial protein import. These results indicate that mPHGPx overexpression can preserve the mitochondrial proteome and provide cardioprotective benefits to the diabetic heart.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Cardiomiopatías Diabéticas/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Glutatión Peroxidasa/metabolismo , Mitocondrias Cardíacas/metabolismo , Animales , Transporte Biológico , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 1/complicaciones , Cardiomiopatías Diabéticas/complicaciones , Femenino , Glutatión Peroxidasa/genética , Peroxidación de Lípido , Masculino , Espectrometría de Masas , Ratones , Ratones Transgénicos , Mitocondrias Cardíacas/enzimología , Mitocondrias Cardíacas/genética , Estrés Oxidativo , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Proteómica , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Finding insights into how viruses hijack metabolic processes and biomarkers for viral diseases often require hypotheses about target compounds and/or labelling techniques. Here we present a method based on laser ablation electrospray ionization mass spectrometry to rapidly identify potential protein and metabolite biomarkers of oncovirus infection in B lymphocytes.
Asunto(s)
Linfocitos B/metabolismo , Herpesvirus Humano 8 , Fosfatidilcolinas/metabolismo , Infecciones por Retroviridae/metabolismo , Timosina/metabolismo , Biomarcadores/metabolismo , Línea Celular , Humanos , Rayos Láser , Infecciones por Retroviridae/diagnóstico , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Diabetic cardiomyopathy is associated with increased risk of heart failure in type 1 diabetic patients. Mitochondrial dysfunction is suggested as an underlying contributor to diabetic cardiomyopathy. Cardiac mitochondria are characterized by subcellular spatial locale, including mitochondria located beneath the sarcolemma, subsarcolemmal mitochondria (SSM), and mitochondria situated between the myofibrils, interfibrillar mitochondria (IFM). The goal of this study was to determine whether type 1 diabetic insult in the heart influences proteomic make-up of spatially distinct mitochondrial subpopulations and to evaluate the role of nuclear encoded mitochondrial protein import. Utilizing multiple proteomic approaches (iTRAQ and two-dimensional-differential in-gel electrophoresis), IFM proteomic make-up was impacted by type 1 diabetes mellitus to a greater extent than SSM, as evidenced by decreased abundance of fatty acid oxidation and electron transport chain proteins. Mitochondrial phosphate carrier and adenine nucleotide translocator, as well as inner membrane translocases, were decreased in the diabetic IFM (P < 0.05 for both). Mitofilin, a protein involved in cristae morphology, was diminished in the diabetic IFM (P < 0.05). Posttranslational modifications, including oxidations and deamidations, were most prevalent in the diabetic IFM. Mitochondrial heat shock protein 70 (mtHsp70) was significantly decreased in diabetic IFM (P < 0.05). Mitochondrial protein import was decreased in the diabetic IFM with no change in the diabetic SSM (P < 0.05). Taken together, these results indicate that mitochondrial proteomic alterations in the type 1 diabetic heart are more pronounced in the IFM. Further, proteomic alterations are associated with nuclear encoded mitochondrial protein import dysfunction and loss of an essential mitochondrial protein import constituent, mtHsp70, implicating this process in the pathogenesis of the diabetic heart.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Cardiomiopatías Diabéticas/metabolismo , Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Transporte de Proteínas/fisiología , Proteoma/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Acetil-CoA C-Aciltransferasa/metabolismo , Animales , Glucemia/metabolismo , Isomerasas de Doble Vínculo Carbono-Carbono/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Ciclo del Ácido Cítrico/fisiología , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/fisiopatología , Cardiomiopatías Diabéticas/fisiopatología , Regulación hacia Abajo/fisiología , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Enoil-CoA Hidratasa/metabolismo , Expresión Génica/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Corazón/fisiopatología , Insulina/sangre , Masculino , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Ratones Endogámicos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas Musculares/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteoma/genética , Proteómica , Racemasas y Epimerasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
A general approach to the synthesis of a new series of unique sulfate anionic acid-labile surfactants (AALS) was developed. In this approach, the ketal was derived from methyl pyruvate, and the sulfate motif was introduced via sulfitylation of the alcohol, oxidation, and finally conversion of the sulfate diester to the desired sodium salt. The physicochemical properties in aqueous solution of this novel series of surfactants, such as CMCs, solubility, acid lability, and stability were studied.
Asunto(s)
Ácidos Sulfúricos/química , Tensoactivos/síntesis química , Aniones/química , Estructura Molecular , Estereoisomerismo , Tensoactivos/químicaRESUMEN
Cardiac complications and heart failure are the leading cause of death in type 2 diabetic patients. Mitochondrial dysfunction is central in the pathogenesis of the type 2 diabetic heart. However, it is unclear whether this dysfunction is specific for a particular subcellular region. The purpose of this study was to determine whether mitochondrial dysfunction in the type 2 diabetic heart is specific to a spatially distinct subset of mitochondria. We investigated mitochondrial morphology, function, and proteomic composition of subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) in 18-wk-old db/db mice. Oxidative damage was assessed in subpopulations through the measurement of lipid peroxidation byproducts and nitrotyrosine residues. Proteomic profiles and posttranslational modifications were assessed in mitochondrial subpopulations using iTRAQ and multi-dimensional protein identification technologies, respectively. SSM from db/db hearts had altered morphology, including a decrease in size and internal complexity, whereas db/db IFM were increased in internal complexity. Db/db SSM displayed decreased state 3 respiration rates, electron transport chain activities, ATP synthase activities, and mitochondrial membrane potential and increased oxidative damage, with no change in IFM. Proteomic assessment revealed a greater impact on db/db SSM compared with db/db IFM. Inner mitochondrial membrane proteins, including electron transport chain, ATP synthesis, and mitochondrial protein import machinery, were predominantly decreased. We provide evidence that mitochondrial dysfunction in the type 2 diabetic heart is associated with a specific subcellular locale. Furthermore, mitochondrial morphological and functional indexes are impacted differently during type 2 diabetic insult and may result from the modulation of spatially distinct mitochondrial proteomes.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/metabolismo , Proteoma , Animales , Antioxidantes/metabolismo , Respiración de la Célula , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Transporte de Electrón , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Canales Iónicos/metabolismo , Peroxidación de Lípido , Masculino , Potencial de la Membrana Mitocondrial , Ratones , Mitocondrias Cardíacas/patología , Translocasas Mitocondriales de ADP y ATP/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Tamaño Mitocondrial , Estrés Oxidativo , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Tirosina/análogos & derivados , Tirosina/metabolismo , Proteína Desacopladora 3RESUMEN
Thin poly(ethylene glycol) silane (PEG-silane) coatings formed from N-(triethoxysilyl propyl)-O-poly(ethylene oxide) urethane with different chain lengths of poly(ethylene glycol) (MW 750 and 4000-5000) are used to modify glass microfluidic channels and fused-silica capillaries for electrophoretic separations of proteins. These coatings combine three important properties, which make them favorable for proteomic analyses including reduction of protein adsorption, compatibility with mass spectrometry due to their stability, and the ability to control the magnitude of electroosmotic flow (EOF). The coatings have been successfully used in microfluidic chips and fused-silica capillaries for separation of protein sample mixtures under low EOF conditions. The long-chain and mixed PEG-silane coatings suppress electroosmotic flow by more than 90%, whereas the short-chain PEG silane suppresses EOF by 65-75% at pH values of 3-9. The long-chain and mixed PEG-silane coatings are suitable for low EOF applications or for cases where negative effects of EOF are to be minimized. Efficient separations of unlabeled basic proteins at low pH and FITC-labeled proteins at high pH were achieved, as well as excellent stability for at least 200 electrophoretic runs. Additionally, these covalent coatings produce no detectable background ions in ESI-MS, making them compatible with on-line mass spectrometry.
Asunto(s)
Electroforesis Capilar/métodos , Vidrio , Polietilenglicoles/química , Proteínas/aislamiento & purificación , Silanos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Sensibilidad y EspecificidadRESUMEN
A hydrodynamic flow restrictor (HDR) that is used to combine electrokinetic and hydrodynamic flow streams has been fabricated in a microfluidic channel by laser micromachining. Combining electrokinetic and hydrodynamic flow streams is challenging in microfluidic devices, because the hydrodynamic flow often overpowers the electrokinetic flow, making it more difficult to use low electroosmotic flow in the electrokinetic portion of the system. The HDR has been incorporated into a capillary electrophoresis-mass spectrometry interface that provides continuous introduction of a make-up solution and negates the hydrodynamic backpressure in the capillary electrophoresis channel to the extent that low EOF can be utilized. Moreover, the hydrodynamic backpressure is sufficiently minimized to allow coatings that minimize EOF to be used in the electrokinetically driven channel. Such coatings are of great importance for the analysis of proteins and other biomolecules that adsorb to charged surfaces.
Asunto(s)
Electroforesis Capilar/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
A fritless electroosmotic pump with reduced pH dependence has been fabricated on a glass microchip and its performance characterized. The chip design consists of two 500-microm channels, one packed with anion exchange beads and the other packed with cation exchange beads, which produce convergent electroosmotic flow streams. The electroosmotically pumped solution flows away from the intersection of the two pumping channels through a field-free channel. This simple design allows for the production of a fritless electroosmotic pump and easy replacement of the ion exchange beads whose charged surfaces generate the flow. The pump was found to produce volumetric flow rates of up to 2 microL/min for an applied voltage of 3 kV at a pH of 6.8. Moreover, the electroosmotic pump can generate high flow rates over an extended pH range of at least 2-12, a significant advantage over previously fabricated electroosmotic pumps, which typically have a more limited range in which they can achieve high flow rates.