Comparative Proteomes of the Proliferating C2C12 Myoblasts and Fully Differentiated Myotubes Reveal the Complexity of the Skeletal Muscle Differentiation Program
2004; Elsevier BV; Volume: 3; Issue: 11 Linguagem: Inglês
10.1074/mcp.m400020-mcp200
ISSN1535-9484
AutoresNilesh S. Tannu, Vamshi K. Rao, Ritcha M. Chaudhary, Francesco Giorgianni, Abdelwahab E. Saeed, Yong Gao, Rajendra Raghow,
Tópico(s)Ubiquitin and proteasome pathways
ResumoWhen cultured in low serum-containing growth medium, the mouse C2C12 cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and phospho-proteins from the proliferating C2C12 cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS. Quantification of more than 2,000 proteins from C2C12 myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C2C12 myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phospho-proteins also occurred during conversion of C2C12 myoblasts into myotubes. We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESI-quadrupole ion trap MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate inter- and intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation. When cultured in low serum-containing growth medium, the mouse C2C12 cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and phospho-proteins from the proliferating C2C12 cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS. Quantification of more than 2,000 proteins from C2C12 myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C2C12 myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phospho-proteins also occurred during conversion of C2C12 myoblasts into myotubes. We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESI-quadrupole ion trap MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate inter- and intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation. The de novo myogenesis from mesoderm-derived committed muscle precursor cells has been studied in the embryos of mouse, chicken, frog, and zebra fish, and in a number of cell and tissue culture models of muscle differentiation. As a result of these studies, key anatomic, genetic, and molecular aspects of this multi-step process have been elucidated (1Buckingham M. Skeletal muscle formation in vertebrates..Curr. Opin. Genet. Dev. 2001; 11: 440-448Crossref PubMed Scopus (345) Google Scholar, 2Pownall M.E. Gustafsson M.K. Emerson Jr., C.P. Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos..Annu. Rev. Cell Dev. Biol. 2002; 18: 747-783Crossref PubMed Scopus (464) Google Scholar, 3Tannu, N. S., Rao, V. K., and Raghow, R.(2003) Cellular and molecular paradigms of myogenesis, inRecent Research Developments in Molecular Biology (Pandlai, S. G., ed)Vol. 1, pp.73–95, Research Signpost, Trivendrum, IndiaGoogle Scholar). The final step of myogenesis in vivo entails that the proliferating myoblasts withdraw from cell cycle, elicit a muscle-specific gene expression program and fuse to become multinucleated myotubes.The induction of muscle-specific genes during myogenic differentiation is regulated by basic helix-loop-helix (bHLH) 1The abbreviations used are: bHLH, basic helix-loop-helix; MRF, muscle-specific regulatory factor; 2D, two-dimensional; QIT, quadrupole ion trap; GM, growth medium; DMEM, Dulbecco's modified Eagle's medium; DM, differentiation medium; MHC, myosin heavy chain; RIPA, radio-immunoprecipitation assay; TBST, TBS-Tween 20; LIMK1, LIM kinase 1; PKA, protein kinase A; MKK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; HSP, heat shock protein; CRE, cAMP-responsive element; TIF, transcription intermediary factor; HAT, histone acetyl transferase; FGF, fibroblast growth factor.1The abbreviations used are: bHLH, basic helix-loop-helix; MRF, muscle-specific regulatory factor; 2D, two-dimensional; QIT, quadrupole ion trap; GM, growth medium; DMEM, Dulbecco's modified Eagle's medium; DM, differentiation medium; MHC, myosin heavy chain; RIPA, radio-immunoprecipitation assay; TBST, TBS-Tween 20; LIMK1, LIM kinase 1; PKA, protein kinase A; MKK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; HSP, heat shock protein; CRE, cAMP-responsive element; TIF, transcription intermediary factor; HAT, histone acetyl transferase; FGF, fibroblast growth factor. transcription factors such as MyoD, Myf-5, myogenin, and MRF4. The known muscle-specific regulatory factors (MRFs) exhibit distinct but somewhat overlapping spatio-temporal patterns of expression during development of the skeletal muscle. For example, Myf5 is the first of the myogenic bHLH factors to be expressed in the developing embryo followed by MyoD and myogenin. The expression of myogenin and MRF4 occurs later, and apparently the two MRFs directly control transcription of muscle-specific genes prior to the formation of multinucleated myotubes (4Charbonnier F. Gaspera B.D. Armand A.S. Van der Laarse W.J. Launay T. Becker C. Gallien C.L. Chanoine C. Two myogenin-related genes are differentially expressed in Xenopus laevis myogenesis and differ in their ability to transactivate muscle structural genes..J. Biol. Chem. 2002; 277: 1139-1147Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 5Smith 2nd, C.K. Janney M.J. Allen R.E. Temporal expression of myogenic regulatory genes during activation, proliferation, and differentiation of rat skeletal muscle satellite cells..J. Cell. Physiol. 1994; 159: 379-385Crossref PubMed Scopus (223) Google Scholar). In contrast, MyoD and Myf-5 are not only expressed in the proliferating myoblasts but may also be needed in an early step of myogenesis (i.e. muscle cell fate specification). Varying degrees of defects in muscle development are caused by loss-of-function mutations in the individual MRF genes (6Lassar A.B. Skapek S.X. Novitch B. Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal..Curr. Opin. Cell Biol. 1994; 6: 788-794Crossref PubMed Scopus (309) Google Scholar, 7Molkentin J.D. Olson E.N. Combinatorial control of muscle development by basic helix-loop-helix and MADS-box transcription factors..Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9366-9373Crossref PubMed Scopus (372) Google Scholar). Consistent with their unique roles, the combined mutations in two MRFs in mice (e.g. MyoD −/−: MRF4 −/−) result in more severe defects in muscle differentiation than those seen in either MyoD −/− or MRF4 −/− mice (2Pownall M.E. Gustafsson M.K. Emerson Jr., C.P. Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos..Annu. Rev. Cell Dev. Biol. 2002; 18: 747-783Crossref PubMed Scopus (464) Google Scholar). Additionally, MEF2 and regulators of cell cycle such as p21CIP1, p27, and p57KIP2 are also coordinately regulated during muscle differentiation (1Buckingham M. Skeletal muscle formation in vertebrates..Curr. Opin. Genet. Dev. 2001; 11: 440-448Crossref PubMed Scopus (345) Google Scholar, 2Pownall M.E. Gustafsson M.K. Emerson Jr., C.P. Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos..Annu. Rev. Cell Dev. Biol. 2002; 18: 747-783Crossref PubMed Scopus (464) Google Scholar, 3Tannu, N. S., Rao, V. K., and Raghow, R.(2003) Cellular and molecular paradigms of myogenesis, inRecent Research Developments in Molecular Biology (Pandlai, S. G., ed)Vol. 1, pp.73–95, Research Signpost, Trivendrum, IndiaGoogle Scholar).Mouse C2C12 cells have been used extensively to study the process of myogenic differentiation in culture. The application of DNA microarray technology to the differentiating C2C12 cells has led to the identification of numerous differentially expressed messenger RNAs (8Delgado I. Huang X. Jones S. Zhang L. Hatcher R. Gao B. Zhang P. Dynamic gene expression during the onset of myoblast differentiation in vitro..Genomics. 2003; 82: 109-121Crossref PubMed Scopus (64) Google Scholar, 9Shen X. Collier J.M. Hlaing M. Zhang L. Delshad E.H. Bristow J. Bernstein H.S. Genome-wide examination of myoblast cell cycle withdrawal during differentiation..Dev. Dyn. 2003; 226: 128-138Crossref PubMed Scopus (87) Google Scholar). Shen et al. (9Shen X. Collier J.M. Hlaing M. Zhang L. Delshad E.H. Bristow J. Bernstein H.S. Genome-wide examination of myoblast cell cycle withdrawal during differentiation..Dev. Dyn. 2003; 226: 128-138Crossref PubMed Scopus (87) Google Scholar) reported that the differentially expressed genes of C2C12 cells grown in low serum medium represented regulators of cell cycle (e.g. cyclin D1, p27Kip1, PP2A, and Rb), apoptosis such as DAD1, BAK, Caspase 11, and glycogen synthase kinase-3β, and muscle-specific genes (e.g. MyoD, myogenin, dystroglycan, troponin c, and creatine kinase). These authors showed that expression of Cyclin D1 was readily detected in the proliferating myoblasts while p21WAF1/Cip1 expression increased only when <40% of cells were fused into multinucleated myotubes. In a related study, assessment of global gene expression in the differentiating C2C12 cells up to the stage of myogenin induction showed that <1,500 genes, which could be classified into 12 coordinately regulated group of genes, were significantly altered (8Delgado I. Huang X. Jones S. Zhang L. Hatcher R. Gao B. Zhang P. Dynamic gene expression during the onset of myoblast differentiation in vitro..Genomics. 2003; 82: 109-121Crossref PubMed Scopus (64) Google Scholar). Similar to what was demonstrated by Shen et al. (9Shen X. Collier J.M. Hlaing M. Zhang L. Delshad E.H. Bristow J. Bernstein H.S. Genome-wide examination of myoblast cell cycle withdrawal during differentiation..Dev. Dyn. 2003; 226: 128-138Crossref PubMed Scopus (87) Google Scholar), Dalgado and colleagues found that numerous cell cycle signaling-, apoptosis-, cell architecture-, and transcriptional control-specific genes were significantly altered during early phase of myogenic differentiation (8Delgado I. Huang X. Jones S. Zhang L. Hatcher R. Gao B. Zhang P. Dynamic gene expression during the onset of myoblast differentiation in vitro..Genomics. 2003; 82: 109-121Crossref PubMed Scopus (64) Google Scholar).Although the genomics-based analyses of myogenesis in C2C12 cells have been highly instructive, we believe that the molecular mechanisms by which proliferating myoblasts leave cell cycle, initiate a program of myogenic gene expression, and become fused into multinucleated myotubes cannot be fully understood from the analysis of the transcriptome alone. This is because the signal transduction pathways mediating the phenotypic conversion of myoblasts into myotubes utilize proteins and the analysis of transcriptome informs us little about the dynamic changes in the rates of translation of various mRNAs or about proteins produced by translation of alternately spliced mRNAs. Therefore, it is desirable to complement the global gene expression analyses with studies examining the proteomes of C2C12 cells undergoing myogenesis in vitro. With a goal to compare the proteomes of the C2C12 cells undergoing differentiation, we analyzed the total cellular, membrane-, and nuclear-enriched proteins from proliferating myoblasts and fully differentiated myocytes by two-dimensional (2D)-PAGE. The differentially regulated protein spots were identified by PDQuest image analysis of the silver nitrate-stained 2D gels followed by MALDI-TOF-MS and LC-ESI-quadrupole ion trap (QIT)-MS/MS. Furthermore, because the status of phosphorylation, a key modification of proteins that regulates numerous signaling cascades, cannot be discerned from analyses of the protein abundance, we also compared the phospho-proteomes of proliferating C2C12 cells and myotubes by using the Pro-Q ® Diamond phospho-protein gel staining. We demonstrate that in addition to many well-known proteins involved with myogenesis, the expression of a number of new proteins capable of regulating inter- and intracellular signaling, cell cycle and apoptosis, cell shape, and transcription is also altered during skeletal muscle differentiation.EXPERIMENTAL MATERIALS AND METHODSCell Culture—C2C12 cells were bought from American Type Culture Collection (ATCC-CRL 1772; Bethesda, MD). Cells were cultured in growth medium (GM; Dulbecco's modified Eagle's medium [DMEM] containing 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin) in a humidified incubator at 37 °C with 5% CO2. Cells cultivated in GM were subcultured after they became 70–80% confluent, and the cell passage number was not allowed to exceed 10. To induce differentiation, nearly confluent C2C12 cells were incubated in DMEM containing 2% heat-inactivated horse serum (differentiation medium; DM) for varying lengths of time. The fraction of cells converted into myotubes was assessed by light microscopy of unstained cells or after staining with a monoclonal myosin heavy chain (MHC)-specific primary antibody. The primary antibody was diluted 1:1 with 1% BSA/PBS-Tween 20 followed by secondary reaction with a goat anti-mouse IgG conjugated with FITC. The detailed methods for staining of C2C12 cells with antibody and detection of FITC fluorescence have been outlined previously (10Mehra-Chaudhary R. Matsui H. Raghow R. Msx3 protein recruits histone deacetylase to down-regulate the Msx1 promoter..Biochem. J. 2001; 353: 13-22Crossref PubMed Scopus (0) Google Scholar).Extraction of Proteins—Cell monolayers (∼107 cells/15-cm diameter Petri dish) were washed twice with 10 ml of 0.35 m ice-cold sucrose, scraped in 4 ml of 0.35 m ice-cold sucrose, and collected by centrifugation (4,000 rpm for 5 min at 4 °C). Whole-cell proteins were extracted in radio-immunoprecipitation assay (RIPA) buffer [50 mm Tris-HCl at pH 7.5, 150 mm NaCl, 1% (w/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mm EDTA, and 100-fold diluted fresh cocktails of phosphatase inhibitor I [microcystin LR, cantharidin, and (-)-p-bromotetramisole; catalog no. p2850, Sigma-Aldrich, St. Louis, MO], phosphatase inhibitor II [sodium vanadate, sodium molybdate, sodium tartrate, and imidazole; catalog no. p5726, Sigma-Aldrich], and 40 μl of protease inhibitor mixture (catalog no. 1697498, Roche, Indianapolis, IN). Proteins were precipitated in acetone (final concentration of 80%) at −20 °C overnight, pelleted by centrifugation (14,000 rpm for 20 min at 4 °C), and pellets were air-dried. The protein pellet obtained from cells harvested from a single 15-cm diameter dish was taken up in 200 μl of rehydration buffer [7 m urea, 2 m thiourea, 4% (w/v) CHAPS. The protein solution in rehydration buffer was supplemented with immobilized pH gradient buffer (8 μl/ml), 1 μl (0.5%) of bromphenol blue, and DTT (10 mg/ml)] and kept for 1 h at room temperature. These samples were centrifuged (14,000 rpm for 20 min at 25 °C), and supernatant containing the proteins in complete solution was used for 2D-PAGE.We extracted crude membrane fraction according to the published protocol (11Simpson R.J. Connolly L.M. Eddes J.S. Pereira J.J. Moritz R.L. Reid E.G. Proteomic analysis of the human colon carcinoma cell line (LIM 1215): Development of a membrane protein database..Electrophoresis. 2000; 21: 1707-1732Crossref PubMed Scopus (212) Google Scholar). Cells were suspended in HES buffer [20 mm HEPES, 1 mm EDTA at pH 7.4, and freshly added mixture of protease and phosphatase inhibitors] and broken by freezing and thawing (−80 °C for 30 min) and 30 passes in a Dounce homogenizer (clearance of 0.1016–0.1524 mm). Unlysed cells and nuclei were removed from the cell homogenate by centrifugation (900 × g for 10 min at 4 °C). The crude plasma membranes were recovered as a pellet by centrifuging the post-nuclear supernatant at 100,000 × g for 45 min at 4 °C. Membrane proteins were extracted in RIPA buffer, precipitated by acetone as above and dissolved in re-hydration buffer (12Sanchez J.C. Hochstrasser D. Rabilloud T. In-gel sample rehydration of immobilized pH gradient..Methods Mol. Biol. 1999; 112: 221-225PubMed Google Scholar).Cells harvested from 15-cm diameter dishes as above were used to isolate nuclei according to the previously reported protocol (13Mirkovitch J. Mirault M.E. Laemmli U.K. Organization of the higher-order chromatin loop: Specific DNA attachment sites on nuclear scaffold..Cell. 1984; 39: 223-232Abstract Full Text PDF PubMed Scopus (852) Google Scholar). Cells were sequentially washed three times in 3 ml of ice-cold isolation buffer [3.75 mm Tris-Cl pH 7.4, 0.05 mm spermine, 0.125 mm spermidine, 0.5 mm EDTA, 1% thiodiglycol, 20 mm KCl] containing protease and phosphatase inhibitors, and centrifuged (900 × g for 5 min at 2 °C) after each wash. The supernatant containing the membrane and cytoplasm was removed after each spin. The crude nuclear pellets were then taken up in 3 ml of ice-cold Triton X-100 lysis buffer [isolation buffer + 0.5% Triton X-100] containing protease and phosphatase inhibitors and washed three times by stepwise resuspension and centrifugation (900 × g for 5 min at 2 °C). Finally, nuclear proteins were extracted in 500 μl of RIPA buffer, precipitated in 80% acetone at −20 °C overnight, and made soluble in 100 μl of rehydration buffer. Concentration of proteins from total cell, membrane, or nuclear extracts was determined by a modified Bradford assay kit (14Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding..Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213462) Google Scholar) as described by the manufacturer (Pierce, Rockford, IL).2D-PAGE—One hundred-microgram aliquots of whole-cell, membrane, or nuclear proteins, taken up in 360 μl of rehydration buffer, were electrofocused in Immobiline™ DryStrips (180 × 3×0.5 mm, pH 3–10/4–7 linear) with the IPGphor (Amersham Pharmacia Biotech, Piscataway, NJ) (15Bjellqvist B. Ek K. Righetti P.G. Gianazza E. Gorg A. Westermeier R. Postel W. Isoelectric focusing in immobilized pH gradients: Principle, methodology and some applications..J. Biochem. Biophys. Methods. 1982; 6: 317-339Crossref PubMed Scopus (804) Google Scholar). The IPG strip was rehydrated for 12 h and subjected to sequential IEF at 100 V for 200 V-h, at 500 V for 500 V-h, at 1,000 V for 1,000 V-h, and at 8,000 V for 80,000 V-h. The platform temperature was maintained at 20 °C, and 50-μA current was passed per strip. The IPG strips were equilibrated to reduce the disulfide bonds in a tray containing 3 ml of equilibrating solution per strip [6 m urea, 1.5 m Tris-HCl, pH 8.8, 30% (v/v) glycerol, 2% (w/v) SDS, and 2% (w/v) DTT] with gentle rocking for 10 min. The protein SH groups were blocked by rocking each strip for 10 min in 3 ml of solution containing 6 m urea, 1.5 m Tris-HCl, pH 8.8, 30% (v/v) glycerol, 2% (w/v) SDS, and 2.5% (w/v) iodoacetamide.Solubilization of proteins, equilibration of first-dimension strips, and electrophoresis in the second dimension were done in the Protean Dodeca Cell (Bio-Rad, Hercules, CA) apparatus capable of running 12 gels simultaneously. After equilibration, according to the published method (16Rabilloud T. Adessi C. Giraudel A. Lunardi J. Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients..Electrophoresis. 1997; 18: 307-316Crossref PubMed Scopus (403) Google Scholar), the IPG strips were transferred onto vertical 10% SDS-PAGE slab gels (1,800 × 1,800 × 1 mm), using 1% melted agarose as stacking gel. One microliter of the molecular mass marker (Amersham Rainbow marker RPN 800) mixed with 4 μl of running buffer was loaded on 2 mm2 filter paper, which was placed on the acidic end of the IPG strip (17Weber K. Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis..J. Biol. Chem. 1969; 244: 4406-4412Abstract Full Text PDF PubMed Google Scholar). Electrophoresis was carried out for 400 min at a steady voltage of 200 V (12Sanchez J.C. Hochstrasser D. Rabilloud T. In-gel sample rehydration of immobilized pH gradient..Methods Mol. Biol. 1999; 112: 221-225PubMed Google Scholar).Staining of 2D-PAGE and Image Analysis—Proteins separated by 2D gels were visualized after staining with Mann's modified silver staining method, which is compatible with trypsin digestion and MALDI-TOF-MS (18Merril C.R. Switzer R.C. Van Keuren M.L. Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain..Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4335-4339Crossref PubMed Scopus (353) Google Scholar, 19Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels..Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7771) Google Scholar). The staining of 2D gels for phospho-proteins was done according to the instructions provided by the manufacturer (Molecular Probes, Eugene, OR). Gels were fixed in 250 ml of 50% methanol and 10% TCA overnight. The fixed gels were then sequentially washed with 250 ml of distilled water for 15 min, incubated with 250 ml of Pro-Q ® Diamond phospho-protein stain for 3 h in the dark and destained with 20% ACN, 50 mm sodium acetate (pH 4) for 3 h. After scanning the images of the gels for phospho-proteins, the gels were silver stained for visualization of the spots for MS analyses. The stained 2D gels were scanned (Hewlett Packard 4470c scanner) and saved as TIFF files using Adobe Photoshop software. The images of the scanned 2D gels were analyzed by PDQuest (version 6.2.1) software (Bio-Rad) (20Beresini M.H. Sugarman B.J. Shepard H.M. Epstein L.B. Synergistic induction of polypeptides by tumor necrosis factor and interferon-gamma in cells sensitive or resistant to tumor necrosis factor: Assessment by computer based analysis of two-dimensional gels using the PDQUEST system..Electrophoresis. 1990; 11: 232-241Crossref PubMed Scopus (15) Google Scholar). To identify valid spots, PDQuest spot detection software was used with appropriate selection of the faintest and the smallest spots and a large representative section of the image containing spots, streaks, and background gradations to make corrections for noise filter. Absorbance of individual protein spots from the replicate gel images was combined to make "master gels" representing proteins from C2C12 cells at different stages of differentiation. The differentially regulated protein spots were identified by quantitative comparisons of master gels. The reproducibility of PDQuest-based quantification of 2D gel images was ensured by three complementary approaches. First, at least three independent sets of synchronized cultures of C2C12 cells were grown in GM or DM to extract myoblast and myotube-specific proteins. All Petri dishes were individually assessed for cell morphology, density of culture, and myotube formation (in DM) on a daily basis to select two to three uniform replicates for each condition of growth. Second, in the initial tests of reproducibility of 2D gels, protein extracts from individual replicate cultures were analyzed by 2D-PAGE. Subsequently, extracts from cells harvested from three to four Petri dishes incubated under identical conditions were mixed and subject to 2D-PAGE to "normalize" dish-to-dish variability. Finally, regardless of the source of the protein extract, either from a single dish or from multiple dishes grown under identical conditions, all samples were run on three to four replicate gels in the Protein Dodeca Cell (Bio-Rad). Unpaired Student's t test was used to determine if the averages of the myoblast or myotube-specific samples were significantly different using the Microsoft Excel®. Protein spots of interest were subject to MALDI-TOF-MS or LC-ESI-MS/MS (21Patterson S.D. Aebersold R. Mass spectrometric approaches for the identification of gel-separated proteins..Electrophoresis. 1995; 16: 1791-1814Crossref PubMed Scopus (368) Google Scholar).In-gel Trypsin Digestion—The individual protein spots from the 2D gels were excised with pipette tips, minced using a 0.5-ml pestle (Nalge Nunc, Rochester, NY), and destained in 1:1 (v/v) of 30 mm potassium ferricyanide and 100 mm sodium thiosulfate (22Gharahdaghi F. Weinberg C.R. Meagher D.A. Imai B.S. Mische S.M. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity..Electrophoresis. 1999; 20: 601-605Crossref PubMed Scopus (840) Google Scholar). Gel spots were dehydrated by sonication for 20 min in 100 μl of solution containing 50% (v/v) ACN and 100 mm ammonium bicarbonate until the gel turned opaque white. Twenty micrograms of lyophilized trypsin (883 pmol; Promega, Madison, WI) was reconstituted in 100 μl of 50 mm acetic acid to form the stock solution that was diluted [1:12 (v/v)] in 50 mm ammonium bicarbonate and incubated for 15 min at 37 °C. The gel fragments were dried by vacuum centrifugation for 30 min and incubated overnight with 50 μl of trypsin (16 ng/μl) at 37 °C.The supernatant from trypsin digest was transferred to a siliconized microcentrifuge tube. Peptides from the gel pieces were sequentially extracted three times in 50 μl of extraction buffer [60% (v/v) ACN, 5% (v/v) TFA in water]. Each extraction involved 20 min of sonication, followed by centrifugation and removal of the supernatants. The original supernatant and the supernatants from three sequential extractions were combined and dried in a vacuum centrifuge for 3–4 h. The dried peptides were dissolved in 3 μl of 12.5 mg/ml of α-cyano-4-hydroxy-cinnamic acid in 60% (v/v) ACN in water and deposited on paraffin wax-coated stainless-steel MALDI plate (23Tannu N.S. Wu J. Rao V.K. Gadgil H.S. Pabst M.J. Gerling I.C. Raghow R. Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting..Anal. Biochem. 2004; 327: 222-232Crossref PubMed Scopus (58) Google Scholar). Alternately, dried peptides were dissolved in 15 μl of 0.1% (v/v) TFA in water and taken up in siliconized microcentrifuge tube for LC-ESI-QIT-MS/MS.MALDI-TOF-MS—Mass spectrometer analyses were performed using the PerSeptive Biosystems (Framingham, MA) MALDI-TOF Voyager DE™-RP BioSpectrometry™ Workstation operated in the delayed extraction and reflector mode for positive ion detection (24Jensen O.N. Podtelejnikov A. Mann M. Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionization peptide maps..Rapid Commun. Mass Spectrom. 1996; 10: 1371-1378Crossref PubMed Scopus (279) Google Scholar). The laser wavelength and the repetition rate were 337 nm and 3Hz, respectively. The parameters set were as follows: maximum accelerating voltage, 20,000 V; grid voltage, 57%; mirror voltage ratio, 1.08; guide wire, 0.07% and the extraction delay time of 150 ns. The masses were calibrated internally with the masses of two trypsin auto-digest products: fragment 108–115 ([M+H]+ = 842.509 Da) and fragment 58–77 ([M+H]+ = 2211.104 Da). The results were analyzed with Data Explorer software (Applied Biosystems). The protein identification was carried out using the PeptIdent search engine (us.expasy.org/tools/peptident.html), and the Swiss-Prot/TrEMBL databases were used for the protein search (25Henzel W.J. Billeci T.M. Stults J.T. Wong S.C. Grimley C. Watanabe C. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases..Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5011-5015Crossref PubMed Scopus (1180) Google Scholar). The search parameters used were: pI range of ± 1.0, molecular mass range of ± 40%, mass tolerance of ± 100 ppm, one allowed missed cleavage, cysteine treated with iodoacetamide to form carbamidomethyl-cysteine and methionine as the oxidized form.LC-ESI-QIT-MS/MS—To remove the residual gel and to desalt the peptides, the mixtures were purified with ZipTipC18 micro-columns (Millipore, Bedford, MA) and eluted into 3 μl of 50% ACN in water. To the eluate, 3 μl of 0.01% hepatofluorobutyric acid was added and the sample mixture was injected onto the column of LC-nanoESI-QIT MS on the LCQDeca instrument (ThermoFinnigan, San Jose, CA). Picofrit™ columns (360-μm outer diameter, 75-μm inner diameter, 15-μm tip inner diameter) from New Objective (Woburn, MA) were used for LC. Samples were analyzed using a gradient program consisting of initial 5-min isocratic elution with 0% B, followed by linear gradient 0–70% B in 50 min, 5-min isocratic elution with 70% B and a linear gradient 70–0% B in 15 min (A =
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