Separation and identification of rat skeletal muscle proteins using two-dimensional gel electrophoresis and mass spectrometry.

Skeletal muscle plays a major role in whole body protein metabolism, and changes in the rates of synthesis and degradation of proteins are likely to lead to characteristic changes in the amounts of different proteins in muscle under various physiological and pathological conditions. This paper demonstrates the feasibility of a proteomic approach to analyzing the protein composition of skeletal muscle. We report here the initial establishment of two-dimensional gel electrophoresis (2-D PAGE) reference maps for mixed skeletal muscle taken from the abdominal wall of a normal adult rat. We used immobilized pH gradients of 3-10 (non-linear) and 4-7 (linear), and matrix assisted laser desorption/ionization--time of flight mass spectrometry for protein identification by peptide mass fingerprinting. More than 600 protein spots were detected on each gel, of which 100 were excised and characterized. In-gel digestion followed by peptide mass fingerprinting enabled tentative identification of 74 of these, which included a wide range of myofibrillary and sarcoplasmic proteins. This database should provide the nucleus of a valuable resource for investigation of the biochemical basis of skeletal muscle pathologies in general and such specific disorders as alcoholic myopathy and injury.

A reference map of human lung MRC-5 fibroblast proteins using immobilized pH gradient-isoelectric focusing-based two-dimensional electrophoresis.

We report the first protein map of human adult lung MRC-5 fibroblasts using isoelectric focusing-immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis. MRC-5 is an immortalised cell line used in a wide range of investigations. The two-dimensional gel pattern of proteins generated from any given cell system provides a fingerprint that is unique to those cells. Therefore, the establishment of a protein map for a particular cell system provides a useful reference tool as a "master map" for subsequent studies using those cells. In this map a total of 98 protein spots were identified by comparative searches of the nucleotide and protein database using peptide masses obtained by matrix-assisted laser desorption/ionization time of flight following trypsin digestion. To increase the utility of the reference map, cells were cultured in both Dulbecco's modified Eagle medium (DMEM), the standard medium, and Roswell Park Memorial Institute (RPMI)-1640. Two-dimensional gel protein patterns of MRC-5 cultures were shown to be largely unaffected by the use of RPMI compared to DMEM, respectively. In combination with the reference map, the standardised protocol described provides a tool for comparative studies involving MRC-5 cells in which nonspecific variation is minimized.

Zooming-in on the proteome: very narrow-range immobilised pH gradients reveal more protein species and isoforms.

Two-dimensional gel electrophoresis (2-DE) enables separation of complex mixtures of proteins on a single polyacrylamide gel according to isoelectric point, molecular weight, solubility, and relative abundance. For this reason, 2-DE together with mass spectrometry (MS) has become a key technology in proteome analysis. The introduction of immobilised pH gradients (IPGs) for isoelectric focusing of proteins affords improved reproducibility and permits full-scale proteome analyses to be undertaken. Whilst broad-range IPGs are useful for investigating simple proteomes (e.g. Mycoplasma genitalium) it is becoming clear that additional resolving power is needed for separating the more complex proteomes of eukaryotic organisms. The use of narrow-range and very narrow-range IPGs provides the means with which to dissect a complex proteome. We have compared very narrow-range IPGs (3.5-4.5L, 4-5L, 4.5-5.5L, 5-6L, and 5.5-6.7L) with broad- (3-10NL) and narrow-range IPGs (4-7L and 6-9L) for the visualisation of the human heart proteome. The superior ability of very narrow-range IPGs to separate different protein species and isoforms, compared with 3-10NL and 4-7L 2-D gels is demonstrated. The results are supported by MS identifications which further show that reduction of the number of comigrating protein species results in less ambiguous and more reliable database search results.

Identification and mapping of human saphenous vein medial smooth muscle proteins by two-dimensional polyacrylamide gel electrophoresis.

Changing smooth muscle phenotype and abnormal cell proliferation are important features of vascular pathology, including the failure of saphenous vein bypass grafts. We have characterised and mapped protein expression in human saphenous vein medial smooth muscle, using two-dimensional (2-D) polyacrylamide gel electrophoresis. The 2-D system comprised a nonlinear immobilised pH 3-10 gradient in the first dimension (separating proteins with isoelectric point values between pH 3-10), and 12%T total gel concentration sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension (separating proteins in the range 14,000-200,000 Daltons). Using a combination of peptide mass fingerprinting by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and partial amino acid sequencing by nanospray tandem mass spectrometry, a subset of 149 protein spots was analysed, with 129 protein spots being identified and mapped. The data presented here are an important addition to the limited knowledge of venous medial smooth muscle protein expression in vivo. Our protein map will facilitate the identification of proteins differentially expressed in human saphenous vein bypass grafts. In turn, this may lead to the elucidation of molecular events involved in saphenous vein bypass graft failure. The map should also provide a basis for comparative studies of protein expression in vascular smooth muscle of varying origins.