Trust T. Razunguzwa

Mitochondrial dysfunction in the type 2 diabetic heart is associated with alterations in spatially distinct mitochondrial proteomes.

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.

Proteomic alterations of distinct mitochondrial subpopulations in the type 1 diabetic heart: contribution of protein import dysfunction.

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.

Reversal of mitochondrial proteomic loss in Type 1 diabetic heart with overexpression of phospholipid hydroperoxide glutathione peroxidase

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.

Nanophotonic Ion Production from Silicon Microcolumn Arrays

Nanoantennas for ions: Silicon microcolumn arrays harvest light from a laser pulse to produce ions. The system behaves like a quasi-periodic antenna array with ion yields that show profound dependence on the plane of polarization and the angle of incidence of the laser beam. Photonic ion sources promise to enable enhanced control of ion production on a micro- and nanometer scale and direct integration with miniaturized analytical devices.

Molecular Imaging of Biological Samples on Nanophotonic Laser Desorption Ionization Platforms

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 80 putatively annotated molecular species are determined with 40 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.

An electrokinetic/hydrodynamic flow microfluidic CE-ESI-MS interface utilizing a hydrodynamic flow restrictor for delivery of samples under low EOF conditions

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.

Laser desorption ionization (LDI) silicon nanopost array chips fabricated using deep UV projection lithography and deep reactive ion etching

Deep UV projection lithography (DUV-PL) and deep reactive ion etching (DRIE) processes are used to fabricate silicon nanopost surfaces for laser desorption ionization mass spectrometry (LDI-MS). Described here is a fabrication process that is amenable to mass production of silicon nanopost array (NAPA) devices optimized for laser desorption ionization mass spectrometry of small molecules less than 2 kDa, suitable for pharmaceutical and metabolomics applications. The resulting devices exhibit excellent performance for analysis and quantitation of pharmaceutical drugs over at least four orders of magnitude dynamic range, with very good limits of detection and lower limits of quantitation. For metabolite analysis, these devices also exhibit improved spectral quality over MALDI-MS which suffers from noise from the chemical matrix. With the ability to perform a one-step sample spotting, these devices become extremely useful for high throughput workflows afforded by MALDI mass spectrometry platforms.

Direct analysis of drugs in forensic applications using laser ablation electrospray ionization-tandem mass spectrometry (LAESI-MS/MS)

Laser ablation electrospray ionization tandem mass spectrometry (LAESI-MS/MS) was applied to the analysis of scheduled drugs in a variety of forensically relevant media including solutions, hair and botanic matter. LAESI-MS/MS was generally able to identify unreacted drugs directly from solutions in which common presumptive color tests had been performed. A significant correlation of 0.7 was found between the pKa of the drugs and the frequency of a positive identification in the solutions indicating that basic drugs are more favorably ionized. Basic drugs like amphetamine and methamphetamine were readily identified at 0.01 mg mL−1, well below the normal limits of detection of the color test results. For hair analysis, LAESI-MS/MS could directly identify the presence of morphine, codeine and cocaine in human hair samples at biologically relevant levels of ∼10 ng mg−1 of drug in hair. This detection was possible without any hydrolysis, extraction, derivatization, or separation of the drugs. LAESI-MS/MS could also identify the presence of tetrahydrocannabinol (THC) or cannabidiol (CBD) in cannabis leaves, in addition to mapping the spatial abundance of THC/CBD across the different leaves. The simplicity and lack of sample preparation for hair and plant analyses are noteworthy benefits, but the current detection limits are close to biologically relevant levels. These preliminary studies indicate that with some additional optimization and validation, LAESI-MS/MS could provide a direct confirmation of color spot test results at an average analysis time of 20 seconds per sample, which is considerably faster than any GC or LC run and could be a major benefit for large caseloads or backlog reduction.

A General Approach to Anionic Acid-Labile Surfactants with Tunable Properties

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.

Large-Scale Metabolite Analysis of Standards and Human Serum by Laser Desorption Ionization Mass Spectrometry from Silicon Nanopost Arrays

The unique challenges presented by metabolomics have driven the development of new mass spectrometry (MS)-based techniques for small molecule analysis. We have previously demonstrated silicon nanopost arrays (NAPA) to be an effective substrate for laser desorption ionization (LDI) of small molecules for MS. However, the utility of NAPA-LDI-MS for a wide range of metabolite classes has not been investigated. Here we apply NAPA-LDI-MS to the large-scale acquisition of high-resolution mass spectra and tandem mass spectra from a collection of metabolite standards covering a range of compound classes including amino acids, nucleotides, carbohydrates, xenobiotics, lipids, and other classes. In untargeted analysis of metabolite standard mixtures, detection was achieved for 374 compounds and useful MS/MS spectra were obtained for 287 compounds, without individual optimization of ionization or fragmentation conditions. Metabolite detection was evaluated in the context of 31 metabolic pathways, and NAPA-LDI-MS was found to provide detection for 63% of investigated pathway metabolites. Individual, targeted analysis of the 20 common amino acids provided detection of 100% of the investigated compounds, demonstrating that improved coverage is possible through optimization and targeting of individual analytes or analyte classes. In direct analysis of aqueous and organic extracts from human serum samples, spectral features were assigned to a total of 108 small metabolites and lipids. Glucose and amino acids were quantitated within their physiological concentration ranges. The broad coverage demonstrated by this large-scale screening experiment opens the door for use of NAPA-LDI-MS in numerous metabolite analysis applications.