Abstract
Myogenesis is a well-characterized program of cellular differentiation that is exquisitely sensitive to the extracellular milieu. Systematic characterization of the myogenic secretome (i.e. the ensemble of secreted proteins) is, therefore, warranted for the identification of novel secretome components that regulate both the pluripotency of these progenitor mesenchymal cells, and also their commitment and passage through the differentiation program. Previously, we have successfully identified 26 secreted proteins in the mouse skeletal muscle cell line C2C12 (1). In an effort to attain a more comprehensive picture of the regulation of myogenesis by its extracellular milieu, quantitative profiling employing stable isotope labeling by amino acids in cell culture was implemented in conjunction with two parallel high throughput online reverse phase liquid chromatography-tandem mass spectrometry systems. In summary, 34 secreted proteins were quantified, 30 of which were shown to be differentially expressed during muscle development. Intriguingly, our analysis has revealed several novel up- and down-regulated secretome components that may have critical biological relevance for both the maintenance of pluripotency and the passage of cells through the differentiation program. In particular, the altered regulation of secretome components, including follistatin-like protein-1, osteoglycin, spondin-2, and cytokine-induced apoptosis inhibitor-1, along with constitutively expressed factors, such as fibulin-2, illustrate dynamic changes in the secretome that take place when differentiation to a specific lineage occurs.
Highlights
34 secreted proteins were quantified, 30 of which were shown to be differentially expressed during muscle development
Myogenesis has proven to be a paradigm for cellular differentiation that has led to many discoveries concerning lineage commitment and the molecular control of tissue-specific gene activation
Differential protein expression analyses using SILAC in tandem with high-throughput online RPLC-MS/MS were implemented in an effort to identify and quantify secreted proteins
Summary
For determining the thresholds for differential expression, cell lysates from MBs cultured in light- and heavy-labeled GM for 192 h (see Step 1 of Fig. 1) were prepared in two identical sets. Mass spectra were analyzed with Analyst QS 1.1 (Applied Biosystems/MDS SCIEX) and submitted to ProteinPilot 2.0.1 [96] for analysis with the following search parameters: sample type, SILAC (Lysϩ6); cysteine alkylation, iodoacetamide; digestion, trypsin; instrument, QSTAR electrospray ionization; species, Mus musculus; quantitate, checked; ID focus, biological modifications; database, NCBInr; search effort, thorough ID; detected protein threshold [unused ProtScore (Conf)], Ͼ1.3 (95%). 20 g of CM proteins derived from light-labeled MBs or heavy-labeled MTs cultured in serum-free DM for 24 h and 120 h, respectively, were resolved by 1D-SDS PAGE, followed by an overnight electrophoretic transfer to Immobilon-P membranes (Millipore) at 20 V and a 1-h transfer at 50 V. The cells were developed with AEC Chromogen kit as the chromogenic substrate (Sigma). 0 nM OGN and 8 nM bone morphogenetic protein-2 (BMP-2) was used as positive control for MyHCand ALP-positive cells, respectively; the former were stained brown, whereas the latter purple
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