Label-free Quantitative Protein Profiling of vastus lateralis Muscle During Human Aging

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.A gradual degenerative loss of skeletal muscle mass and function is one of the most consistent hallmarks of normal aging. When it reaches defined thresholds, this condition is referred to as sarcopenia (1, 2), and can be associated with disability, poor quality of life, frailty, and increased mortality (3). Aging impacts the morphology, function and biochemical properties of skeletal muscle, but the mechanisms leading to the changes in muscle tissue remain unclear.Proteomics links the muscle functional changes with the protein expression pattern. Several proteomic approaches have already been used to study sarcopenia. Protein profiling of whole tissue homogenates has been performed using two-dimensional gel electrophoresis (2DGE)¹ and mass spectrometry to identify the proteins differentially expressed during aging in rat (4–6) and human muscle (7, 8). Other studies have focused on specific fractions such as mitochondrial proteins (9), phosphoproteins (10), glycoproteins (11), basic proteins (12), or calpain interacting proteins (13). The few proteomic studies available on human skeletal muscle are mostly based on the 2DGE approach, which implies focusing on a specific pH range (7, 8). Despite its power of high-resolution, 2DGE presents a limited dynamic range and scarcely resolves low abundance regulatory proteins, hydrophobic proteins, and proteins with extreme pI and/or M_r (14).To circumvent these limitations, we propose in the present study to apply a label-free protein profiling based on a shotgun proteomics approach. This technique permits to identify proteins in a complex mixture after trypsin hydrolysis, using a combination of high performance liquid chromatography and mass spectrometry. In a shotgun analysis previously performed on whole muscle extracts, the major isoforms of myosin heavy-chain comprise ∼42% of the total spectra (15). Because these major isoforms may hamper identification of other proteins, we decided to precipitate myofibrils at low ionic strength (16, 17) and to focus on the soluble fraction. In this paper, we present the analytical steps of label-free quantitation, which resulted in the identification and quantitation of 255 muscle proteins common to all ten individuals. The comparison of protein profiling between mature and older women highlighted 35 differentially expressed proteins during aging, 25 proteins that have not previously been related to muscle aging. The functional interactions network linking these proteins showed that the two main biological processes were represented by proteins involved in energy metabolism and contractile proteins.