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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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Cumulative and ranked protein abundance.A, Cumulative protein mass from highest to lowest abundant proteins. B, Ranked protein abundances from highest to the lowest. C, Screenshot of the MaxQB database. Query protein for glucose transporter slc2a1 (I) returns the search results (II). Selection of protein slc2a1 displays the details on this protein (III). By clicking the expression tab in II, protein expression plots in skeletal muscle (Expt_1, 2, 3) and C2C12 myotubes (Expt_4, 5, 6) are displayed (IV).
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Figure 2: Cumulative and ranked protein abundance.A, Cumulative protein mass from highest to lowest abundant proteins. B, Ranked protein abundances from highest to the lowest. C, Screenshot of the MaxQB database. Query protein for glucose transporter slc2a1 (I) returns the search results (II). Selection of protein slc2a1 displays the details on this protein (III). By clicking the expression tab in II, protein expression plots in skeletal muscle (Expt_1, 2, 3) and C2C12 myotubes (Expt_4, 5, 6) are displayed (IV).

Mentions: Using summed MS-signals (“abundances”) of the peptides identifying each protein in the MS measurements, we estimated their contribution to the proteome. For protein isoforms, the shared peptides were counted as contributing to each of them. To obtain a global view of the dynamic range of the muscle proteome, the C2C12 myotube proteome and their differences, we plotted the cumulative contribution of each protein to total protein mass (Fig. 2A) as well as the individual, ranked abundance (Fig. 2A, 2B; supplemental Table S3). Combining the abundances of all identified heavy and light myosin isoforms results in 18% of total muscle mass, positioning myosin as the highest abundant protein in skeletal muscle. The giant protein titin (390 kDa) alone accounts for another 16% of total protein mass. The 10 most abundant proteins according to MS measurement were titin, myosin-4, nebulin, calcium ATPase 1, actin α, creatine kinase, α-actinine-3, glycogen phosphorylase, and myosin-1 and together they make up 50% of total protein mass in skeletal muscle. Actin and myosin are the key members of the contractile units (sarcomere) in muscle. The estimated ratio between actin/myosin abundances was ∼1:7. The summed abundance of proteins that are assigned to the contractile machinery by Gene Ontology Cellular Compartment (GOCC) annotation was 53.6% of total protein mass for skeletal muscle. The ranked distribution of all individual proteins revealed a much steeper decline of abundances for the tissue compared with the cell line (Fig. 2B). In skeletal muscle, the lower half of the proteome accounts for a negligible fraction of its total mass (<0.1%). Our data also allows us to determine the amino acid make up of mouse muscle proteome as the weighted contribution from all identified protein sequences. For instance, branched chain amino acids, which are important in protein synthesis, constitute 20.5% of all amino acids weighted by protein abundance, which is similar to values in other mouse tissues (supplemental Fig. S3).


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)

Cumulative and ranked protein abundance.A, Cumulative protein mass from highest to lowest abundant proteins. B, Ranked protein abundances from highest to the lowest. C, Screenshot of the MaxQB database. Query protein for glucose transporter slc2a1 (I) returns the search results (II). Selection of protein slc2a1 displays the details on this protein (III). By clicking the expression tab in II, protein expression plots in skeletal muscle (Expt_1, 2, 3) and C2C12 myotubes (Expt_4, 5, 6) are displayed (IV).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4390264&req=5

Figure 2: Cumulative and ranked protein abundance.A, Cumulative protein mass from highest to lowest abundant proteins. B, Ranked protein abundances from highest to the lowest. C, Screenshot of the MaxQB database. Query protein for glucose transporter slc2a1 (I) returns the search results (II). Selection of protein slc2a1 displays the details on this protein (III). By clicking the expression tab in II, protein expression plots in skeletal muscle (Expt_1, 2, 3) and C2C12 myotubes (Expt_4, 5, 6) are displayed (IV).
Mentions: Using summed MS-signals (“abundances”) of the peptides identifying each protein in the MS measurements, we estimated their contribution to the proteome. For protein isoforms, the shared peptides were counted as contributing to each of them. To obtain a global view of the dynamic range of the muscle proteome, the C2C12 myotube proteome and their differences, we plotted the cumulative contribution of each protein to total protein mass (Fig. 2A) as well as the individual, ranked abundance (Fig. 2A, 2B; supplemental Table S3). Combining the abundances of all identified heavy and light myosin isoforms results in 18% of total muscle mass, positioning myosin as the highest abundant protein in skeletal muscle. The giant protein titin (390 kDa) alone accounts for another 16% of total protein mass. The 10 most abundant proteins according to MS measurement were titin, myosin-4, nebulin, calcium ATPase 1, actin α, creatine kinase, α-actinine-3, glycogen phosphorylase, and myosin-1 and together they make up 50% of total protein mass in skeletal muscle. Actin and myosin are the key members of the contractile units (sarcomere) in muscle. The estimated ratio between actin/myosin abundances was ∼1:7. The summed abundance of proteins that are assigned to the contractile machinery by Gene Ontology Cellular Compartment (GOCC) annotation was 53.6% of total protein mass for skeletal muscle. The ranked distribution of all individual proteins revealed a much steeper decline of abundances for the tissue compared with the cell line (Fig. 2B). In skeletal muscle, the lower half of the proteome accounts for a negligible fraction of its total mass (<0.1%). Our data also allows us to determine the amino acid make up of mouse muscle proteome as the weighted contribution from all identified protein sequences. For instance, branched chain amino acids, which are important in protein synthesis, constitute 20.5% of all amino acids weighted by protein abundance, which is similar to values in other mouse tissues (supplemental Fig. S3).

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