by Francesco Maria Del Re '21
I conducted a bottom-up large-scale shot-gun proteomic study comparing age and diet. I used 20 Sprague Dawley rat soleus samples fed different diets and divided into four different groups (n=5 for each group): young controls (6 mo); aged controls (22 mo); and young and aged rats fed an ω-3 supplemented diet. The proteomic analysis extracted solei, followed by the splitting of each extract into a pellet (contractile cellular components) and supernatant (soluble proteins) fractions so as to increase the dynamic range of the protein identifications. The protein concentration of each of the 40 aliquots (supernatant and pellets) was estimated by the Lowry protein procedure (Lowry et al., 1951). Then, the proteins were reduced with dithiothreitol, irreversibly alkylated with iodoacetamide, and finally digested with trypsin. The resulting peptide mixture was quantified in order to label peptides from each sample with tandem mass tags (TMT). Ultimately, the differentially expressed proteins among the groups were identified using a nano liquid chromatography-coupled tandem Q Exactive HF-X Orbitrap Mass Spectrometer and Protein Discoverer 2.4 software.
Presentation deriving from research with Stylianos P. Scordilis, professor of biological sciences.