Functional lipidomics: the roles of specialized lipids and lipid-protein interactions in modulating neuronal function

Lipids fulfill multiple specialized roles in neuronal function. In brain, the conduction of electrical impulses, synaptic function, and complex signaling pathways depend on the temporally and spatially coordinated interactions of specialized lipids (e.g., arachidonic acid and plasmalogens), proteins (e.g., ion channels, phospholipases and cyclooxygenases) and integrative lipid-protein interactions. Recent technical advances in mass spectrometry have allowed unparalled insight into the roles of lipids in neuronal function. Through shotgun lipidomics and multidimensional mass spectrometry, in conjunction with the identification of new classes of phospholipases (e.g., calcium dependent and calcium independent intracellular phospholipases), new roles for lipids in cerebral function have been accrued. This review summarizes the advances in our understanding of the types of lipids and phospholipases in the brain and the role of functional lipidomics in increasing our chemical understanding of complex neuronal processes.     

Proteomic profiling of human placenta-derived mesenchymal stem cells upon transforming LIM mineralization protein-1 stimulation

Human placenta-derived mesenchymal stem cells (hPDMSCs) can differentiate into different types of cells and thus have tremendous potential for cell therapy and tissue engineering. LIM mineralization protein-1 (LMP-1) plays an important role in osteoblast differentiation, maturation and bone formation. To determine a global effect of LMP-1 on hPDMSCs, we designed a study using a proteomic approach combined with adenovirus-mediated gene transfer of LMP-1 to identify LMP-1-induced changes in hPDMSCs on proteome level. We have generated proteome maps of undifferentiated hPDMSCs and LMP-1 induced hPDMSCs. Two dimensional gel electrophoresis revealed 22 spots with at least 2.0-fold changes in expression and 15 differently expressed proteins were successfully identified by MALDI-TOF-MS. The proteins regulated by LMP-1 included cytoskeletal proteins, cadmium-binding proteins, and metabolic proteins, etc. The expression of some identified proteins was confirmed by further Western blot analyses. Our results will play an important role in better elucidating the underlying molecular mechanism in LMP-1 included hPDMSCs differentiation into osteoblasts.     

Dramatic accumulation of triglycerides and precipitation of cardiac hemodynamic dysfunction during brief caloric restriction in transgenic myocardium expressing human calcium-independent phospholipase A2gamma

Previously, we identified calcium-independent phospholipase A2gamma (iPLA2gamma) with multiple translation initiation sites and dual mitochondrial and peroxisomal localization motifs. To determine the role of iPLA2gamma in integrating lipid and energy metabolism, we generated transgenic mice containing the alpha-myosin heavy chain promoter (alphaMHC) placed proximally to the human iPLA2gamma coding sequence that resulted in cardiac myocyte-restricted expression of iPLA2gamma (TGiPLA2gamma). TGiPLA2gamma mice possessed multiple phenotypes including: 1) a dramatic approximately 35% reduction in myocardial phospholipid mass in both the fed and mildly fasted states; 2) a marked accumulation of triglycerides during brief caloric restriction that represented 50% of total myocardial lipid mass; and 3) acute fasting-induced hemodynamic dysfunction. Biochemical characterization of the TGiPLA2gamma protein expressed in cardiac myocytes demonstrated over 25 distinct isoforms by two-dimensional SDS-PAGE Western analysis. Immunohistochemistry identified iPLA2gamma in the peroxisomal and mitochondrial compartments in both wild type and transgenic myocardium. Electron microscopy revealed the presence of loosely packed and disorganized mitochondrial cristae in TGiPLA2gamma mice that were accompanied by defects in mitochondrial function. Moreover, markedly elevated levels of 1-hydroxyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-hydroxyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine were prominent in the TGiPLA2gamma myocardium identifying the production of signaling metabolites by this enzyme in vivo. Collectively, these results identified the participation of iPLA2gamma in the remarkable lipid plasticity of myocardium, its role in generating signaling metabolites, and its prominent effects in modulating energy storage and utilization in myocardium in different metabolic contexts.     

Mitochondrial single nucleotide polymorphism genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using cleavable biotinylated dideoxynucleotides

Characterization of mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) and mutations is crucial for disease diagnosis, which requires accurate and sensitive detection methods and quantification due to mitochondrial heteroplasmy. We report here the characterization of mutations for myoclonic epilepsy with ragged red fibers syndrome using chemically cleavable biotinylated dideoxynucleotides and a mass spectrometry (MS)-based solid phase capture (SPC) single base extension (SBE) assay. The method effectively eliminates unextended primers and primer dimers, and the presence of cleavable linkers between the base and biotin allows efficient desalting and release of the DNA products from solid phase for MS analysis. This approach is capable of high multiplexing, and the use of different length linkers for each of the purines and each of the pyrimidines permits better discrimination of the four bases by MS. Both homoplasmic and heteroplasmic genotypes were accurately determined on different mtDNA samples. The specificity of the method for mtDNA detection was validated by using mitochondrial DNA-negative cells. The sensitivity of the approach permitted detection of less than 5% mtDNA heteroplasmy levels. This indicates that the SPC-SBE approach based on chemically cleavable biotinylated dideoxynucleotides and MS enables rapid, accurate, and sensitive genotyping of mtDNA and has broad applications for genetic analysis.     

Shotgun lipidomics of cardiolipin molecular species in lipid extracts of biological samples

Cardiolipin is a prominent component of the mitochondrial inner membranes contributing to the regulation of multiple discrete mitochondrial functions. Here, we extend shotgun lipidomics to identify and quantitate cardiolipin molecular species directly from lipid extracts of biological samples. Three shotgun lipidomics approaches for analyses of cardiolipin molecular species were developed using either a continuous ion-transmission instrument (i.e., triple-quadrupole type) with either low or high mass resolution settings or a high mass resolution hybrid pulsed instrument [i.e., quadrupole time-of-flight (QqTOF) type]. Three chemical principles were used for the development of these approaches. These include the marked enrichment of linoleate in cardiolipin to maximize the signal-to-noise ratio, the specific neutral loss of ketenes from doubly charged cardiolipin molecular ions to yield doubly charged triacyl monolysocardiolipins, and the doubly charged character of two phosphates in each cardiolipin molecular species. Through these techniques, we identified and quantified the specific molecular species profiles of cardiolipin directly from lipid extracts of mouse heart, liver, and skeletal muscle. The accuracy ( approximately 5%) and the low end of the linear dynamic range (10 fmol/microl) for quantitation make these approaches useful for studying alterations in cardiolipin metabolism in multiple disease states using either type of mass spectrometer.     

Spin-on end-functional diblock copolymers for quantitative DNA immobilization

We demonstrate a simple means to covalently bond DNA to both hard (i.e., glass and silicon wafers) and soft (i.e., polymeric) substrates that provides quantitative and precise control of the DNA areal density. The approach is based on spin coating an alkyne-end-functional diblock copolymer, alpha-alkyne-omega-Br-poly( tBA- b-MMA), that self-assembles on both types of substrates as an ordered monolayer and thereby directs alkyne groups to the surface. Azido-functionalized DNA is covalently linked to the alkyne functionalized substrates by means of a "click" reaction between azide and alkyne groups. The density of immobilized DNA can be quantitatively controlled by varying the parameters used for spin-coating the copolymer film, that is, solution concentration and rotational speed, or by varying the copolymer molecular weight. We find the yield of the DNA coupling reaction to be dependent on the nature of the polymer underlying the reactive alkyne functional groups, being higher for more hydrophilic polymers.     

Effects of mechanical vibration on cell morphology,proliferation, apoptosis,and cytokine expression/secretion in osteocyte‐like MLO‐Y4 cells exposed to high glucose

Diabetic patients exhibit significant bone deterioration. Our recent findings demonstrate that mechanical vibration is capable of resisting diabetic bone loss, whereas the relevant mechanism remains unclear. We herein examined the effects of mechanical vibration on the activities and functions of osteocytes (the most abundant and well‐recognized mechanosensitive cells in the bone) exposed to high glucose (HG). The osteocytic MLO‐Y4 cells were incubated with 50 mM HG for 24 h, and then stimulated with 1 h/day mechanical vibration (0.5 g, 45 Hz) for 3 days. We found that mechanical vibration significantly increased the proliferation and viability of MLO‐Y4 cells under the HG environment via the MTT, BrdU, and Cell Viability Analyzer assays. The apoptosis detection showed that HG‐induced apoptosis in MLO‐Y4 cells was inhibited by mechanical vibration. Moreover, increased cellular area, microfilament density, and anisotropy in HG‐incubated MLO‐Y4 cells were observed after mechanical vibration via the F‐actin fluorescence staining. The real‐time polymerase chain reaction and western blotting results demonstrated that mechanical vibration significantly upregulated the gene and protein expression of Wnt3a, β‐catenin, and osteoprotegerin (OPG) and decreased the sclerostin, DKK1, and receptor activator for nuclear factor‐κB ligand (RANKL) expression in osteocytes exposed to HG. The enzyme‐linked immunosorbent assay assays showed that mechanical vibration promoted the secretion of prostaglandin E₂ and OPG, and inhibited the secretion of tumor necrosis factor‐α and RANKL in the supernatant of HG‐treated MLO‐Y4 cells. Together, this study demonstrates that mechanical vibration improves osteocytic architecture and viability, and regulates cytokine expression and secretion in the HG environment, and implies the potential great contribution of the modulation of osteocytic activities in resisting diabetic osteopenia/osteoporosis by mechanical vibration.     

Shotgun lipidomics identifies cardiolipin depletion in diabetic myocardium linking altered substrate utilization with mitochondrial dysfunction

Diabetic cardiomyopathy is characterized by excessive utilization of fatty acid substrate, diminished glucose transport, and mitochondrial dysfunction. However, the chemical mechanisms linking altered substrate utilization to mitochondrial dysfunction are unknown. Herein, we use shotgun lipidomics and multidimensional mass spectrometry to identify dramatic decreases in the critical mitochondrial inner membrane lipid, cardiolipin, in diabetic murine myocardium (from 7.2 +/- 0.3 nmol/mg of protein in control hearts to 3.1 +/- 0.1 nmol/mg of protein in diabetic myocardium; p < 0.001, n = 7). Moreover, the direct metabolic precursor of cardiolipin, phosphatidylglycerol, was also substantially depleted (2.5 +/- 0.2 nmol/mg of protein in control hearts vs 1.3 +/- 0.1 nmol/mg of protein in diabetic myocardium; p < 0.001, n = 7). Similarly, glycerol 3-phosphate, necessary for the penultimate step in phosphatidylglycerol production, decreased by 58% in diabetic myocardium (from 4.9 +/- 0.9 to 2.2 +/- 0.3 nmol/mg of protein; n = 4). Since Barth's syndrome (a disorder of cardiolipin metabolism) induces mitochondrial dysfunction and cardiomyopathy, and since decreases in cardiolipin content precipitate mitochondrial dysfunction, these results provide a unifying hypothesis linking altered substrate utilization and metabolic flux in diabetic myocardium with altered lipid metabolism, cardiolipin depletion, mitochondrial dysfunction, and resultant hemodynamic compromise.     

Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry

We report here the design, synthesis and evaluation of a novel photocleavable (PC) biotinylated nucleotide analog, dUTP-PC-Biotin, for DNA polymerase extension reaction to isolate DNA products for mass spectrometry (MS) analysis. This nucleotide analog has a biotin moiety attached to the 5-position of 2′-deoxyribouridine 5′-triphosphate via a photocleavable 2-nitrobenzyl linker. We have demonstrated that dUTP-PC-Biotin can be faithfully incorporated by the DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA polymerase extension reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. Therefore, the DNA extension fragments generated by using the dUTP-PC-Biotin can be efficiently isolated by a streptavidin-coated surface and recovered by near-UV light irradiation at room temperature in mild condition for further analysis without using any chemicals or heat. Single and multiple primer extension reactions were performed using the dUTP-PC-Biotin to generate DNA products for MALDI-TOF MS analysis. Such nucleotide analogs that carry a biotin and a photocleavable linker will allow the isolation and purification of DNA products under mild conditions for MS-based genetic analysis by DNA sequencing or multiplex single nucleotide polymorphism (SNP) detection. Furthermore, these nucleotide analogs should also be useful in isolating DNA–protein complexes under non-denaturing conditions.     

DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) has been explored widely for DNA sequencing. The major requirement for this method is that the DNA sequencing fragments must be free from alkaline and alkaline earth salts as well as other contaminants for accurately measuring the masses of the DNA fragments. We report here the development of a novel MS DNA sequencing method that generates Sanger-sequencing fragments in one tube using biotinylated dideoxynucleotides. The DNA sequencing fragments that carry a biotin at the 3′-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Only correctly terminated biotinylated DNA fragments are subsequently released and loaded onto a mass spectrometer to obtain accurate DNA sequencing data. Compared with gel electrophoresis-based sequencing systems, MS produces a very high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and completely eliminates the compressions associated with gel electrophoresis. The high resolution of MS allows accurate mutation and heterozygote detection. This optimized solid-phase DNA-sequencing chemistry plus future improvements in detector sensitivity for large DNA fragments in MS instrumentation will further improve MS for DNA sequencing.