PRIDE Assigned Tags:Biological Dataset
Dataset Belongs to:Skeletal Muscle Proteome (B/D-HPP)
Proteomics of Human Skeletal Muscle
The skeletal muscle proteomics study of vastus lateralis muscle biopsies which were collected from participants from the Genetic and Epigenetic Study of Aging and Laboratory Testing (GESTALT). The participants were free of major diseases, except for controlled hypertension or a history of cancer that had been clinically silent for at least 10 years, were not chronically medicated (except one on antihypertensive medication), had no physical or cognitive impairments, had a BMI less than 30 kg/m2, and were not professional athletes.
Sample Processing Protocol
Muscle samples were snap frozen in liquid nitrogen immediately after collection and stored at 80 °C. On average, 8 mg of muscle tissue was pulverized in liquid nitrogen and lysed (8 M urea, 2 M thiourea, 4% CHAPS, 1% Triton X-100, 50 mM Tris (Sigma), pH 8.5) and sonicated. Protein concentration was determined using a 2-D Quant Kit (GE Healthcare Life Sciences). Detergents and lipids were removed using a methanol/chloroform extraction protocol (Bligh and Dyer 1959). Proteins were resuspended in urea buffer (8 M urea, 2 M thiourea, 150 mM NaCl (Sigma)), reduced (50 mM DTT), and alkylated (100 mM iodoacetamide), then diluted 12 times with 50 mM ammonium bicarbonate, and digested (18 hours at 36 °C using a trypsin/LysC mixture (Promega, 1:50 (w/w))). Peptides were desalted, speed vacuum dried, and stored at -80 °C. Tandem Mass Tag (TMT) labeling was performed according to the manufacturer’s instructions (TMT6plex, Thermo Fisher, Cat# 90066). Donor IDs were blinded and randomized to prevent TMT bias. Each TMT set included one donor from each of the 5 age groups and one reference sample. Each sample was spiked with 200 fM of bacterial beta-galactosidase digest (SCIEX) prior to TMT labeling. Labeled peptides were combined and fractionated.
Data Processing Protocol
Raw data from each sample fraction was converted to mascot generic format (.mgf) using MSConvert (ProteoWizard 3.0.6002), producing a list of MS ions with retention times and MS/MS spectra. This list of ions was searched with Mascot 2.4.1 and X!Tandem CYCLONE (2010.12.01.1) using SwissProt Human sequences from the Uniprot database (Version Year 2015, 20,200 sequences, appended with 115 contaminants). The search engine uses each protein sequence from the database to produce every possible peptide from it according to the following search parameters: TMT6plex lysine and n-terminus as fixed modifications and variable modifications of carbamidomethyl cysteine, deamidation of asparagine and glutamate, and carbamylation of lysine, the N-terminus, and oxidized methionine. These latter were frequently set as variable modifications because they are often generated by sample preparation. A peptide mass tolerance of 20 ppm and 0.08 Da were allowed, according to the known mass accuracy of the instrument, for precursor and fragment ions, as well as two missed cleavages. The TMT channels’ isotopic purity was corrected according to the TMT Kit instructions.
Ubaida-Mohien C, Gonzalez-Freire M, Lyashkov A, Moaddel R, Chia CW, Simonsick EM, Sen R, Ferrucci L. Physical Activity Associated Proteomics of Skeletal Muscle: Being Physically Active in Daily Life May Protect Skeletal Muscle From Aging. Front Physiol. 2019 10:312 PubMed: 30971946