Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.
Bottom Line: Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins.Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue.This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.
Affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany;Show MeSH
Mentions: In previous attempts at very deep proteome coverage of cell lines, we have employed protein level fractionation as well as several proteolytic enzymes (15). However, recent experience in our laboratory suggested that single enzyme digestion and fractionation only at the peptide level, when coupled to advanced MS instrumentation, would be sufficient to achieve great depth of protein identification (31). We therefore developed a streamlined workflow, consisting of cell line or tissue homogenization, filter-aided sample preparation (FASP) (23), followed by peptide separation into twelve fractions according to their isoelectric point as described (24). Peptides were measured by a linear quadrupole-Orbitrap mass analyzer, which achieves sub or low parts per million mass accuracy for peptide ions and their fragments (25). This streamlined method required only 1 day of measurement time for a single analysis each of the proteomes and computational analysis followed by automated computational analysis in the MaxQuant environment (26) (Fig. 1A–1C).
Affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany;