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ABSTRACT: Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were "skinned," and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56alpha. Immunoblot analysis of myocyte fractions confirmed that beta-adrenergic stimulation by isoproterenol decreased the B56alpha content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56alpha from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following beta-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.
Molecular & Cellular Proteomics 03/2010; 9(3):497-509. · 7.40 Impact Factor
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Yuqin Wang,
Kyle M Sousa,
Karl Bodin,
Spyridon Theofilopoulos,
Paola Sacchetti, Martin Hornshaw,
Gary Woffendin,
Kersti Karu,
Jan Sjövall,
Ernest Arenas,
William J Griffiths
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ABSTRACT: In this study two regions of embryonic (E11) mouse central nervous system (CNS) have been profiled for their unesterified sterol content. Using high-performance liquid chromatography (HPLC)-mass spectrometry (MS) and tandem mass spectrometry (MS(n)) low levels of oxysterols (estimated 2-165 ng g(-1) wet weight) were identified in cortex (Ctx) and spinal cord (Sc). The identified oxysterols include 7 alpha-, 7 beta-, 22R-, 24S-, 25- and 27-hydroxycholesterol; 24,25- and 24,27-dihydroxycholesterol; and 24S,25-epoxycholesterol. Of these, 24S-hydroxycholesterol is biosynthesised exclusively in brain. In comparison to adult mouse where the 24S-hydroxycholesterol level is about 40 microg g(-1) in brain the level of 24S-hydroxycholesterol reported here (estimated 26 ng g(-1) in Ctx and 13 ng g(-1) in Sc) is extremely low. Interestingly, the level of 24S,25-epoxycholesterol in both CNS regions (estimated 165 ng g(-1) in Ctx and 91 ng g(-1) in Sc) is somewhat higher than the levels of the hydroxycholesterols. This oxysterol is formed in parallel to cholesterol via a shunt of the mevalonate pathway and its comparatively high abundance may be a reflection of a high rate of cholesterol synthesis at this stage of development. Levels of cholesterol (estimated 1.25 mg g(-1) in Ctx and 1.15 mg g(-1) in Sc) and its precursors were determined by gas chromatography-mass spectrometry (GC-MS). In both CNS regions cholesterol levels were found to be lower than those reported in the adult, but in relation to cholesterol the levels of cholesterol precursors were higher than found in adult indicating a high rate of cholesterol synthesis. In summary, our data provide evidence for the presence of endogenous oxysterols in two brain regions of the developing CNS. Moreover, while most of the enzymes involved in hydroxysterol synthesis are minimally active at E11, our results suggest that the mevalonate pathway is significantly active, opening up the possibility for a function of 24S,25-epoxycholesterol during brain development.
Molecular BioSystems 06/2009; 5(5):529-41. · 3.53 Impact Factor
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ABSTRACT: While the proteome defines the expressed gene products, the metabolome results from reactions controlled by such gene products. Plasma represents an accessible "window" to the metabolome both in regard of availability and content. The wide range of the plasma metabolome, in terms of molecular diversity and abundance, makes its comprehensive analysis challenging. Here we demonstrate an analytical method designed to target one region of the metabolome, that is, oxysterols. Since the discovery of their biological activity as ligands to nuclear receptors there has been a reawakening of interest in oxysterols and their analysis. In addition, the oxysterols, 24S- and 27-hydroxycholesterol, are currently under investigation as potential biomarkers associated with neurodegenerative disorders such as Alzheimer's disease and multiple sclerosis; widespread analysis of these lipids in clinical studies will require the development of robust, sensitive and rapid analytical techniques. In this communication we present results of an investigation of the oxysterols content of human plasma using a newly developed high-performance liquid chromatography-mass spectrometry (HPLC-MS) method incorporating charge-tagging and high-resolution MS. The method has allowed the identification in plasma of monohydroxylated cholesterol molecules, 7alpha-, 24S-, and 27-hydroxycholesterol; the cholestenetriol 7alpha,27-dihydroxycholesterol; and 3beta-hydroxycholest-5-en-27-oic acid and its metabolite 3beta,7alpha-dihydroxycholest-5-en-27-oic acid. The methodology described is also applicable for the analysis of other sterols in plasma, that is, cholesterol, 7-dehydrocholesterol, and desmosterol, as well as cholesterol 5,6- seco-sterols and steroid hormones. Although involving derivatization, sample preparation is straightforward and chromatographic analysis rapid (17 min), while the MS method offers high sensitivity (ng/mL of sterol in plasma, or pg on-column) and specificity. The methodology is suitable for targeted metabolomic analysis of sterols, oxysterols, and steroid hormones opening a "window" to view this region of the metabolome.
Journal of Proteome Research 08/2008; 7(8):3602-12. · 5.11 Impact Factor
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ABSTRACT: Smith-Lemli-Opitz syndrome (SLOS), a severe disorder of cholesterol synthesis, is classically diagnosed prenatally by GC-MS analysis of sterols in amniotic fluid. Considering the current trend toward tandem mass spectrometry (MS/MS) methodologies, we developed prototype LC-MS/MS methods for accurate diagnosis of the disorder.
3beta-Hydroxysterols in amniotic fluid are oxidized with cholesterol oxidase to their corresponding 3-ketones, which are then derivatized with Girard P (GP) hydrazine in a "one-pot" reaction. The resulting GP-hydrazones give an improved response in electrospray (ES)-MS/MS owing to the presence of a charged quaternary nitrogen and are analyzed by reversed-phase LC-ES-MS/MS. Both capillary and conventional LC-MS/MS formats are suitable, and the method is also applicable to paper-absorbed blood spots.
In a double-blind analysis of 18 amniotic fluid samples comprising 6 SLOS and 12 controls, the ratio of 7 + 8-dehydrocholesterol (7 + 8-DHC) to cholesterol was <0.02 [range 0.00-0.02, mean (SD) 0.01 (0.007)] in all control samples (intraassay variation 5.91%) and >0.20 [0.20-1.13, 0.79 (0.35)] in SLOS (intraassay variation 4.56%), corresponding to a difference in ratios between the 2 groups of at least a factor of 10. The limit of quantification was equivalent to that of 2 nL amniotic fluid injected on-column.
We describe a proof-of-concept for the prenatal diagnosis of SLOS. Further developments will be necessary to automate sample handling and reduce chromatographic time for the methodology to be used in pre- and postnatal diagnosis.
Clinical Chemistry 06/2008; 54(8):1317-24. · 7.91 Impact Factor
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ABSTRACT: In humans, the brain accounts for about 20% of the body's free cholesterol, most of which is synthesized de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolized to 24S-hydroxycholesterol, principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover; however, the route by which the remainder is turned over has yet to be elucidated. Here, we describe a novel liquid chromatography (LC) multi-stage fragmentation mass spectrometry (MS(n)) methodology for the identification, with high sensitivity (low pg), of cholesterol metabolites in rat brain. The methodology includes derivatization to enhance ionization, exact mass analysis at high resolution to identify potential metabolites, and LC-MS(n) (n=3) to allow their characterization. 24S-hydroxycholesterol was confirmed as a major oxysterol in rat brain, and other oxysterols identified for the first time in brain included 24,25-, 24,27-, 25,27-, 6,24,- 7alpha,25-, and 7alpha,27-dihydroxycholesterols. In addition, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterized in rat brain.
The Journal of Lipid Research 05/2007; 48(4):976-87. · 5.56 Impact Factor
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ABSTRACT: Neutral steroids have traditionally been analyzed by gas chromatography/mass spectrometry (GC/MS) after necessary derivatization reactions. However, GC/MS is unsuitable for the analysis of many conjugated steroids and those with unsuspected functional groups. Here we describe an alternative analytical method specifically designed for the analysis of oxosteroids and those with a 3beta-hydroxy-delta5 or 5alpha-hydrogen-3beta-hydroxy structure. Steroids were derivatized with Girard P (GP) hydrazine to give GP hydrazones, which are charged species and readily analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The resulting [M]+ ions were then subjected to high-energy collision-induced dissociation on a tandem time-of-flight instrument. The product ion spectra give structurally informative fragment ion patterns. The sensitivity of the analytical method is such that steroid structures can be determined from low-picogram (low-femtomole) amounts of sample. The utility of the method has been demonstrated by the analysis of oxysterols extracted from rat brain.
Analytical Chemistry 02/2006; 78(1):164-73. · 5.86 Impact Factor
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ABSTRACT: To reveal the peptide contents of the visually nonidentifiable neurons from a neuronal circuit of interest, we combined retrograde labeling of neurons with mass spectrometric single cell analysis. We used the neuronal circuit involved in the copulation behavior of a freshwater snail, Lymnaea stagnalis, as a model. Central neurons that control this behavior are known to send their axons to the penis nerve and innervate the penis complex. By retrograde filling from the penis nerve with nickel-lysine, these neurons were selectively labeled darkish blue. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometric analyses of single stained neurons in the parietal ganglion from different animals reveal consistently the presence of several molecular ion species in the range of 800-1200 Da. From a single neuron, six molecular ion species were further characterized with MALDI time-of-flight/time-of-flight mass spectrometry, which demonstrates that the peptides are derived from a previously reported -FLRFamide precursor.
Analytical Chemistry 08/2003; 75(13):2996-3000. · 5.86 Impact Factor
