Label-free quantitative protein profiling of vastus lateralis muscle during human aging

Abstract

Sarcopenia corresponds to the loss of muscle mass occurring during aging, and is associated with a loss of muscle functionality. Proteomic links the muscle functional changes with protein expression pattern. To better understand the mechanisms involved in muscle aging, we performed a proteomic analysis of Vastus lateralis muscle in mature and older women. For this, a shotgun proteomic method was applied to identify soluble proteins in muscle, using a combination of high performance liquid chromatography and mass spectrometry. A label-free protein profiling was then conducted to quantify proteins and compare profiles from mature and older women. This analysis showed that 35 of the 366 identified proteins were linked to aging in muscle. Most of the proteins were under-represented in older compared with mature women. We built a functional interaction network linking the proteins differentially expressed between mature and older women. The results revealed that the main differences between mature and older women were defined by proteins involved in energy metabolism and proteins from the myofilament and cytoskeleton. This is the first time that label-free quantitative proteomics has been applied to study of aging mechanisms in human skeletal muscle. This approach highlights new elements for elucidating the alterations observed during aging and may lead to novel sarcopenia biomarkers.

Fig. 1.

Identified proteins from LIS fraction of vastus lateralis muscle of mature and old women (Supplemental Table 1) analyzed with the PANTHER bioinformatics tool (http://www.pantherdb.org) using the Gene Ontology categories “Molecular function,” “Biological Process,” and “Cellular Component.” The molecular functions “Catalytic activity” and “Binding” are detailed. For each category, the number of identified proteins and their percentage of the total number of proteins in the pie chart are indicated for each Gene Ontology term.

Fig. 2.

Quantitative analysis with LCProgenesis software. A, Total ion chromatograms of LIS protein fraction from mature (pink) and old (green) women. B, Detected feature of carbonic anhydrase 3, according to the liquid chromatography retention time and the m/z. C, Alignment grid according to the liquid chromatography retention time and the m/z of all the detected peptides. D, Representation of the abundance of one peptide from carbonic anhydrase 3 protein within mature (pink) and old (blue) women representative samples.

Fig. 3.

Score plot and loadings of principal component analysis from quantified proteins in LIS fraction of vastus lateralis from mature and old women. Discriminations are on principal component 1, and revealed the protein expressions related to muscle aging. The pink line delineates the mature women (pink circles) and the blue line delineates the old women (blue circles). The differentially expressed proteins are marked by abbreviations: ACADM, medium-chain specific acyl-CoA dehydrogenase, mitochondrial; ACTB, actin, cytoplasmic 1; AHSG, alpha-2-HS glycoprotein; ANKRD2, ankyrin repeat domain-containing protein 2; ANXA2, annexin 2; APEH, acylamino-acid-releasing enzyme; ATP5A1, ATP synthase subunit alpha, mitochondrial; ATP5B, ATP synthase subunit beta, mitochondrial; BLVRB, flavin reductase (NADPH); C4A, complement C4-A; CA3, carbonic anhydrase 3; CFL1, cofilin-1; CRAT, carnitine O-acetyltransferase; EEF2, elongation factor 2; FABP4, fatty acid-binding protein, adipocyte; FGA, fibrinogen alpha chain; FH, fumarate hydratase, mitochondrial; FLNC, filamin-C; GOT2, aspartate aminotransferase, mitochondrial; HBG1, hemoglobin subunit gamma-1; HSPA4, heat shock 70 kDa protein 4; HV304, Ig heavy chain V-III region TEI; KV402, Ig kappa chain V-IV region Len; LDHB, l-lactate dehydrogenase B chain; MYH1, myosin-1; MYL1, myosin light chain 1/3, skeletal muscle isoform; OAS2, 2′-5′-oligoadenylate synthase 2; PFKL, 6-phosphofructokinase, liver type; PRKAR2A, cAMP-dependent protein kinase type II-alpha regulatory subunit; RPS27A, ubiquitin-40S ribosomal protein 27A; SLC25A4, ADP/ATP translocase 1; TAGLN, transgelin; TALDO1, transaldolase; TTN, titin; YWHAE, 14–3-3 protein epsilon.

Fig. 4.

Functional interactions network linking the significantly differentially expressed proteins between mature and old women (String database) (20). K-mean classification revealed three substructures mainly represented by contractile and energy metabolism proteins. The proteins involved in the network are marked by abbreviations: ACADM, medium-chain specific acyl-CoA dehydrogenase, mitochondrial; ACTB, actin, cytoplasmic 1; AHSG, alpha-2-HS glycoprotein; ANKRD2, ankyrin repeat domain-containing protein 2; ANXA2, annexin 2; ATP5A1, ATP synthase subunit alpha, mitochondrial; ATP5B, ATP synthase subunit beta, mitochondrial; CFL1, cofilin-1; CRAT, carnitine O-acetyltransferase; EEF2, elongation factor 2; FABP4, fatty acid-binding protein, adipocyte; FH, fumarate hydratase, mitochondrial; GOT2, aspartate aminotransferase, mitochondrial; HSPA4, heat shock 70 kDa protein 4; LDHB, l-lactate dehydrogenase B chain; MYH1, myosin-1; MYL1, myosin light chain 1/3, skeletal muscle isoform; PFKL, 6-phosphofructokinase, liver type; PRKAR2A, cAMP-dependent protein kinase type II-alpha regulatory subunit; SLC25A4, ADP/ATP translocase 1; TAGLN, transgelin; TALDO1, transaldolase; TTN, titin.

Fig. 5.

Representative Western blot of aspartate aminotransferase (GOT2), medium-chain specific acyl-CoA dehydrogenase (ACADM), alpha-2-HS glycoprotein (AHSG), ATP synthase subunit alpha (ATP5A1), carbonic anhydrase 3 (CA3), and transgelin (TAGLN), between mature (pink) and old (blue) women. Results are indicated as mean ± standard deviation. Significance: *: p value < 0.05 and ***: p value < 0.001.