In vitro analysis of metabolites from the untreated tissue of Torpedo californica electric organ by mid-infrared laser ablation electrospray ionization mass spectrometry (original) (raw)
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Skeletal Muscle, 2011
Background: During development, the branchial mesoderm of Torpedo californica transdifferentiates into an electric organ capable of generating high voltage discharges to stun fish. The organ contains a high density of cholinergic synapses and has served as a biochemical model for the membrane specialization of myofibers, the neuromuscular junction (NMJ). We studied the genome and proteome of the electric organ to gain insight into its composition, to determine if there is concordance with skeletal muscle and the NMJ, and to identify novel synaptic proteins. Results: Of 435 proteins identified, 300 mapped to Torpedo cDNA sequences with ≥2 peptides. We identified 14 uncharacterized proteins in the electric organ that are known to play a role in acetylcholine receptor clustering or signal transduction. In addition, two human open reading frames, C1orf123 and C6orf130, showed high sequence similarity to electric organ proteins. Our profile lists several proteins that are highly expressed in skeletal muscle or are muscle specific. Synaptic proteins such as acetylcholinesterase, acetylcholine receptor subunits, and rapsyn were present in the electric organ proteome but absent in the skeletal muscle proteome. Conclusions: Our integrated genomic and proteomic analysis supports research describing a muscle-like profile of the organ. We show that it is a repository of NMJ proteins but we present limitations on its use as a comprehensive model of the NMJ. Finally, we identified several proteins that may become candidates for signaling proteins not previously characterized as components of the NMJ.
PROTEOMICS, 2002
Proteomic analysis of secreted muscle components: Search for factors involved in neuromuscular synapse formation Denervated but not innervated skeletal muscles secrete polypeptides that are involved in neuromuscular synapse formation. With the aim of identifying such components, metabolically labeled polypeptides in extracts from denervated and innervated muscles were submitted to two-dimensional gel electrophoresis, and the abundance of individual molecular species was compared. Consistent differences between the proteomic maps from the two sources of muscles were seen. Likewise, proteomic maps of polypeptides from organ culture media conditioned by chronically denervated muscles and by control muscles revealed consistent differences, but the abundance of material within individual spots from conditioned media was not sufficient for analysis by mass spectrometry. Since it was not possible to match the patterns from muscle extracts and from conditioned media, it has been established that extract of Sol8 muscle cells was a satisfactory source of material for analysis. From 1200 spots identified on the proteomic map from Sol8 cells by image analysis, some 140 have been defined by mass spectrometric analysis. In order to identify the components that are shared by secreted molecules from denervated muscles and Sol8 cells, a mixture of extracts from the two sources was co-electrophoresed and a shared proteomic pattern was established by visualization of metabolically labeled spots from the conditioned medium and of silver stained spots from the Sol8 cells. More than 100 spots sharing x/y coordinate localization could be seen on the pattern. Of these, fourteen were among those identified by mass spectrometry. It is concluded that co-electrophoresis of radioactively labeled polypeptides from conditioned media with extracts from Sol8 cells can be used to mark in the proteome of Sol8 cells those polypeptides that are secreted at low abundance by adult muscles. Their higher abundance in Sol8 cells opens the possibility for further scrutiny of spots by mass spectrometry or by microsequencing.
Journal of The American Society for Mass Spectrometry, 2015
Skeletal muscles are composed of heterogeneous muscle fibers that have different physiological, morphological, biochemical, and histological characteristics. In this work, skeletal muscles extensor digitorum longus, soleus, and whole gastrocnemius were analyzed by matrix-assisted laser desorption/ionization mass spectrometry to characterize small molecule metabolites of oxidative and glycolytic muscle fiber types as well as to visualize biomarker localization. Multivariate data analysis such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed to extract significant features. Different metabolic fingerprints were observed from oxidative and glycolytic fibers. Higher abundances of biomolecules such as antioxidant anserine as well as acylcarnitines were observed in the glycolytic fibers, whereas taurine and some nucleotides were found to be localized in the oxidative fibers.
Bioscience Reports, 2014
While the long-term physiological adaptation of the neuromuscular system to changed functional demands is usually reflected by unilateral skeletal muscle transitions, the progressive degeneration of distinct motor neuron populations is often associated with more complex changes in the abundance and/or isoform expression pattern of contractile proteins and metabolic enzymes. In order to evaluate these intricate effects of primary motor neuronopathy on the skeletal muscle proteome, label-free MS was employed to study global alterations in the WR (wobbler) mouse model of progressive neurodegeneration. In motor neuron disease, fibre-type specification and the metabolic weighting of bioenergetic pathways appear to be strongly influenced by both a differing degree of a subtype-specific vulnerability of neuromuscular synapses and compensatory mechanisms of fibre-type shifting. Proteomic profiling confirmed this pathobiochemical complexity of disease-induced changes and showed distinct alterations in 72 protein species, including a variety of fibre-type-specific isoforms of contractile proteins, metabolic enzymes, metabolite transporters and ion-regulatory proteins, as well as changes in molecular chaperones and various structural proteins. Increases in slow myosin light chains and the troponin complex and a decrease in fast MBP (myosin-binding protein) probably reflect the initial preferential loss of the fast type of neuromuscular synapses in motor neuron disease. 2014) Intricate effects of primary motor neuronopathy on contractile proteins and metabolic muscle enzymes as revealed by label-free mass spectrometry. Biosci. Rep. 34(4), art:e00119.
PROTEOMICS - Clinical Applications, 2014
Recent years have provided clear evidence for the skeletal muscle as an endocrine organ. Muscle contraction during physical activity has emerged as an important activator of the release of the proteins and peptides called "myokines." Diverse proteomic profiling approaches were applied to rodent and human skeletal muscle cells to characterize the complete secretome, to study the regulation of the secretome during cell differentiation or the release of myokines upon contractile activity of myotubes. Several of the exercise-regulated factors have the potency to mediate an interorgan crosstalk. The paracrine function of the secreted peptides and proteins to regulate muscle regeneration, tissue remodeling, and trainability can have direct effects on whole-body glucose disposal and oxygen consumption. The overall composition and dynamic of the myokinome are still incompletely characterized. Recent advantages in metabolomics and lipidomics will add metabolites and lipids with autocrine, paracrine, or endocrine function to the contraction-induced secretome of the skeletal muscle. The identification of these metabolites will lead to a more comprehensive view described by a new myo(metabo)kinome consisting of peptides, proteins, and metabolites.
2009
A method that enables metabolomic profiling of single cells and subcellular structures is described using capillary electrophoresis coupled to electrospray ionization time-of-flight mass spectrometry. A nebulizer-free coaxial sheath-flow interface completes the circuit and provides a stable electrospray, yielding a signal with a relative standard deviation of under 5% for the total ion electropherogram. Detection limits are in the low nanomolar range (i.e., < 50 nM (< 300 amol)) for a number of cell-to-cell signaling molecules, including acetylcholine (ACh), histamine, dopamine, and serotonin. The instrument also yields high efficiency separations, e.g., ~600,000 for eluting ACh bands. The utility of this setup for single cell metabolomic profiling is demonstrated with identified neurons from Aplysia californica-the R2 neuron and metacerebral cell (MCC). Single cell electropherograms are reproducible, with a large number of metabolites detected; more than 100 compounds yield signals of over 10 4 counts from the injection of only 0.1% of the total content from a single MCC. Expected neurotransmitters are detected within the cells (ACh in R2 and serotonin in MCC), as are compounds that have molecular masses consistent with all of the naturally-occurring amino acids (except cysteine). Tandem MS using a quadrupole time-of-flight tandem mass spectrometer distinguishes ACh from isobaric compounds in the R2 neuron and demonstrates the ability of this method to characterize and identify metabolites present within single cells.
The Journal of Cell Biology, 1984
The synaptic portion of a muscle fiber's basal lamina sheath has molecules tightly bound to it that cause aggregation of acetylcholine receptors (AChRs) on regenerating myofibers . Since basal lamina and other extracellular matrix constituents are insoluble in isotonic saline and detergent solutions, insoluble detergent-extracted fractions of tissues receiving cholinergic input may provide an enriched source of the AChR-aggregating molecules for detailed characterization . Here we demonstrate that such an insoluble fraction from Torpedo electric organ, a tissue with a high concentration of cholinergic synapses, causes AChRs on cultured chick muscle cells to aggregate . We have partially characterized the insoluble fraction, examined the response of muscle cells to it, and devised ways of extracting the active components with a view toward purifying them and learning whether they are similar to those in the basal lamina at the neuromuscular junction.
Analysis of lipids in nerve tissue by MALDI tandem mass spectrometric imaging
2009
of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ANALYSIS OF LIPIDS IN NERVE TISSUE BY MALDI TANDEM MASS SPECTROMETRIC IMAGING By Rachelle Renee Landgraf May 2009 Chair: Richard A. Yost Major: Chemistry Direct analysis of tissue from both the central and peripheral nervous systems of control rats and rats administered the potential neurotoxin dichloroacetate (DCA) was investigated using an intermediate-pressure matrix-assisted laser desorption/ionization (IP-MALDI) source coupled to a linear ion trap (LIT) mass spectrometer. The MALDI matrix, 2,5-dihydroxybenzoic acid, was applied to the tissue using a novel automated inkjet printer system. The MS/MS capabilities of the LIT allowed identification of lipids desorbed directly from tissue. In some cases, a marked decrease is observed in the intensity of lipid ions in spinal cord and sciatic nerve from rats exposed to DCA. The ...