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Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

Deshmukh AS, Murgia M, Nagaraj N, Treebak JT, Cox J, Mann M - Mol. Cell Proteomics (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany;

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Functional differences between C2C12 myotubes and adult skeletal muscle.A, Histogram of total proteins identified in skeletal muscle and C2C12 myotubes (gray) and proteins that are significantly changing between the skeletal muscle and C2C12 myotubes (blue). B, Hierarchical clustering of significantly changing proteins. Cluster 1 (3,308 proteins) represents significantly down-regulated and Cluster 2 (1,002 proteins) significantly up-regulated proteins in skeletal muscle. Annotation categories below the respective clusters are representative examples from significantly enriched categories.
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Figure 3: Functional differences between C2C12 myotubes and adult skeletal muscle.A, Histogram of total proteins identified in skeletal muscle and C2C12 myotubes (gray) and proteins that are significantly changing between the skeletal muscle and C2C12 myotubes (blue). B, Hierarchical clustering of significantly changing proteins. Cluster 1 (3,308 proteins) represents significantly down-regulated and Cluster 2 (1,002 proteins) significantly up-regulated proteins in skeletal muscle. Annotation categories below the respective clusters are representative examples from significantly enriched categories.

Mentions: To gain insights into biological differences between the cell line and tissue systems, we analyzed the label-free quantification values of close to 10,000 proteins. Examination of the complete data set by two sample t test extracted 4,310 proteins (44% of total identified) that are significantly different between skeletal muscle and C2C12 myotubes (FDR 0.05, supplemental Table S4). As depicted in Fig. 3A, the majority of them were of high abundance, presumably at least in part because it is more difficult to reach statistical significance for the low expressed proteins. The statistical test will tend to more easily identify statistically significant differences in proteins abundant in one or the other systems. Low abundance proteins can be expressed differently without passing the significance threshold of our test. Note also that we do not claim that proteins that we do not detect are not present, even when we use “match between runs.” Fig. 3B shows the heat map of significantly different proteins, with Cluster 1 representing 3,308 proteins, which were significantly down-regulated and Cluster 2 representing 1,002 proteins, which were significantly up-regulated in skeletal muscle (Fig. 3C). Fischer's exact test for the enrichment of Gene Ontology (GO) protein annotations in the set of significantly changing proteins returned many significantly changing protein categories (FDR 0.05; Fig. 3C). As expected, glycolysis, tricarboxylic acid (TCA) cycle, mitochondrion, contractile proteins, respiratory chain, and calcium signaling were significantly enriched in skeletal muscle (Cluster 2). In fact, members of GOCC category “mitochondrial part” were the most significantly up-regulated proteins in skeletal muscle (p < 10−72) and collectively this category was expressed about twofold higher in muscle. Conversely, cell lines lacked the transverse tubular system (GOCC category “t-tubules”; p < 10−6). Tables of all significantly enriched protein annotations for Cluster 1 and Cluster 2 are presented in supplemental Table S5.


Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

Deshmukh AS, Murgia M, Nagaraj N, Treebak JT, Cox J, Mann M - Mol. Cell Proteomics (2015)

Functional differences between C2C12 myotubes and adult skeletal muscle.A, Histogram of total proteins identified in skeletal muscle and C2C12 myotubes (gray) and proteins that are significantly changing between the skeletal muscle and C2C12 myotubes (blue). B, Hierarchical clustering of significantly changing proteins. Cluster 1 (3,308 proteins) represents significantly down-regulated and Cluster 2 (1,002 proteins) significantly up-regulated proteins in skeletal muscle. Annotation categories below the respective clusters are representative examples from significantly enriched categories.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4390264&req=5

Figure 3: Functional differences between C2C12 myotubes and adult skeletal muscle.A, Histogram of total proteins identified in skeletal muscle and C2C12 myotubes (gray) and proteins that are significantly changing between the skeletal muscle and C2C12 myotubes (blue). B, Hierarchical clustering of significantly changing proteins. Cluster 1 (3,308 proteins) represents significantly down-regulated and Cluster 2 (1,002 proteins) significantly up-regulated proteins in skeletal muscle. Annotation categories below the respective clusters are representative examples from significantly enriched categories.
Mentions: To gain insights into biological differences between the cell line and tissue systems, we analyzed the label-free quantification values of close to 10,000 proteins. Examination of the complete data set by two sample t test extracted 4,310 proteins (44% of total identified) that are significantly different between skeletal muscle and C2C12 myotubes (FDR 0.05, supplemental Table S4). As depicted in Fig. 3A, the majority of them were of high abundance, presumably at least in part because it is more difficult to reach statistical significance for the low expressed proteins. The statistical test will tend to more easily identify statistically significant differences in proteins abundant in one or the other systems. Low abundance proteins can be expressed differently without passing the significance threshold of our test. Note also that we do not claim that proteins that we do not detect are not present, even when we use “match between runs.” Fig. 3B shows the heat map of significantly different proteins, with Cluster 1 representing 3,308 proteins, which were significantly down-regulated and Cluster 2 representing 1,002 proteins, which were significantly up-regulated in skeletal muscle (Fig. 3C). Fischer's exact test for the enrichment of Gene Ontology (GO) protein annotations in the set of significantly changing proteins returned many significantly changing protein categories (FDR 0.05; Fig. 3C). As expected, glycolysis, tricarboxylic acid (TCA) cycle, mitochondrion, contractile proteins, respiratory chain, and calcium signaling were significantly enriched in skeletal muscle (Cluster 2). In fact, members of GOCC category “mitochondrial part” were the most significantly up-regulated proteins in skeletal muscle (p < 10−72) and collectively this category was expressed about twofold higher in muscle. Conversely, cell lines lacked the transverse tubular system (GOCC category “t-tubules”; p < 10−6). Tables of all significantly enriched protein annotations for Cluster 1 and Cluster 2 are presented in supplemental Table S5.

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.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany;

Show MeSH