The age of 50 appears to be the midpoint for muscle health in humans, with aging of these tissues accelerating from this point in life onward. It is sobering to think that the rate of decline runs, on average, around 2% loss in muscle mass per annum. A mostly inescapable aspect of getting older, declines in muscle bulk generally lead to loss of function and, with that, loss of independence.
Gueugneau et al. (2014) took a top-down proteomics approach to characterize our mortality, examining differential muscle protein expression in mature and aged post-menopausal women to clarify the changes seen anatomically.1 As muscle ages, fibers atrophy and lose the ability to regenerate. This is accompanied by motor neuron denervation and metabolic changes within the muscle tissue cells themselves. Although molecular changes in muscle aging are well studied, the scientists felt that characterizing protein expression would give greater insight into the mechanisms at work, explaining physiological alterations in addition to contributing useful biomarkers for further research.
The researchers used two approaches to prepare proteins from vastus lateralis muscle biopsies taken pre-surgery in two groups of post-menopausal women (56 years old, n = 11; 78 years old, n =13). The first preparation method examined total proteins extracted from muscle lysates, whereas the second involved low salt extraction or low ionic strength (LIS) to remove abundant myofibrillar proteins and expose less abundant proteins of interest. Following tissue lysate preparation, the researchers used two-dimensional gel electrophoresis (2DGE) with IPG (immobilized pH gradient) overlapped through three different pH ranges to separate individual proteins. Gueugneau and co-authors found that the LIS approach improved results, uncovering proteins obscured in liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of total lysate preparations.
Following gel separation, the researchers digested the individual protein spots of interest using trypsin and then examined them via LC-MS/MS utilizing a Dionex UltiMate 3000 LC system coupled to an LTQ Orbitrap Velos hybrid ion trap-Orbitrap mass spectrometer (both Thermo Scientific). They compared the resultant data against the UniProt human database to identify proteins of interest.
Overall analysis of the total lysate preparations revealed 1,919 protein spots, with 95 showing differential expression. The researchers further characterized these using MS and found differential expression of 67 individual proteins in the older women as compared to the younger group. Results from the LIS preparations showed 699 protein spots, with 86 of these differentially expressed between the two groups of women. Further analysis using LC-MS/MS identified 55 peptides, corresponding to 37 unique proteins. The research team used Western immunoblotting to confirm and quantify the changes seen in the differentially expressed proteins identified during 2DGE.
Once identified, the research team turned to protein profiling, using the Search Tool for the Retrieval of Interacting Genes (STRING) 9.0 database to establish physiological pathways altered by muscle tissue aging. Proteins identified as altered in the team’s comparison of the two groups contributed to cellular metabolism, calcium signal transduction, cytoskeletal maintenance and proteostasis pathways, among others.
In conclusion, Gueugneau et al. note that of the proteins identified in their study, 34 are novel in this field of research. They suggest that these new biomarkers represent further opportunities for investigating the chronology of muscle aging.
1. Gueugneau, M. et al. (2014) “Proteomics of muscle chronological ageing in post-menopausal women,” BioMed Central Genomics, 15 (p. 1165), doi: 10.1186/1471-2164-15-1165.
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