Mass Spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays. These measurements are typically performed by timeconsuming LC-MS to eliminate buffer salts that interfere with electrospray ionization MS. However, matrix-assisted laser desorption ionization, time-of-flight MS (MALDI-TOF MS) offers a label-free and direct readout of substrate and product, a fast sampling rate, and is tolerant of many buffer salts, reagents, and compounds that are typically found in enzyme reaction mixtures. In this report, a demonstration of how MALDI-TOF MS can be used to directly measure ratios of substrates and products to produce IC 50 curves for rapid enzyme assays and compound screening is provided. Typical reproducibility parameters were Ͻ7% RSD-a value comparable to ESI MS quantitative assays and well within the acceptable limits for screening assays. The speed of the MALDI readout is currently about 10 s per sample, thus allowing for over 7500 samples/day. From a simplicity standpoint, the enzymatic reaction mixtures are prepared by liquid handling robots, the reactions are stopped by addition of a 10 times volume of acidic matrix solution, and the samples are simultaneously transferred to MALDI target plate for analysis. Importantly, the ratios of substrate to product are of sufficient reproducibility to eliminate the need for internal standards and, thus, minimize the cost and increasing the speed of assay development. (J Am Soc Mass Spectrom 2006, 17, 815-822)
Several GTP-binding protein (G-protein)-coupled receptors that signal through Galphas (GTP-binding protein alpha stimulatory) and the cyclic adenosine monophosphate (cAMP) pathway increase skeletal muscle mass. In order to further evaluate the role of the cAMP pathway in the regulation of skeletal muscle mass, we utilized inhibitors of phosphodiesterase 4 (PDE 4), the major cAMP-modifying PDE found in skeletal muscle, to modulate skeletal muscle cAMP levels. We found that PDE 4 inhibitors reduced the loss of muscle mass and force resulting from denervation and casting in rats and mice. These studies indicate that PDE 4 inhibitors may have a role in the treatment of skeletal muscle-wasting diseases.
Background: Duchenne muscular dystrophy results from mutation of the dystrophin gene, causing skeletal and cardiac muscle loss of function. The mdx mouse model of Duchenne muscular dystrophy is widely utilized to evaluate the potential of therapeutic regimens to modulate the loss of skeletal muscle function associated with dystrophin mutation. Importantly, progressive loss of diaphragm function is the most consistent striated muscle effect observed in the mdx mouse model, which is the same as in patients suffering from Duchenne muscular dystrophy.
Skeletal muscle atrophy is a process in which protein degradation exceeds protein synthesis, resulting in a decrease of the muscle's physiological cross-sectional area and mass, and is often a serious consequence of numerous health problems. We used the isotope-coded affinity tag (ICAT) labelling approach and MS-MS to protein profile cytosolic subcellular fractions from mouse tibialis anterior skeletal muscle undergoing 0, 4, 8, or 16 days of immobilisation-induced atrophy. For the validation of peptide and protein identifications statistical algorithms were applied to the sequence database search results in order to obtain consistent sensitivity/error rates for protein and peptide identifications at each immobilisation time point. In this study, we identified and quantified a large number of mouse skeletal muscle proteins. At a protein probability (P) of P> or = 0.9 (corresponding to a false positive error rate of less than 1%) 807 proteins were identified (231, 226, 217 for 4, 8, 16 days of immobilisation and 133 for the control sample, respectively), from which 51 displayed altered protein abundance with atrophy. Due to randomness of data acquisition, a full time course could be generated only for 62 proteins, most of which displayed unchanged protein abundance. In spite of this, useful information about dataset characteristics and underlying biological processes could be obtained through gene over-representation analysis. 20 gene categories-mainly but not exclusively encoded by the subset of overlapping proteins--were consistently found to be significantly (p < 0.05) over-represented in all 4 sub-datasets.
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