JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST003787/files/20230909_JC_Boulder_ATR_time20.rawhttps://storage.jpostdb.org/JPST003787/files/20230906_JC_Boulder_EDL_time3.rawhttps://storage.jpostdb.org/JPST003787/files/20230909_JC_Boulder_ATR_time0.rawhttps://storage.jpostdb.org/JPST003787/files/20230909_JC_Boulder_ATR_time3.rawhttps://storage.jpostdb.org/JPST003787/files/20230909_JC_Boulder_ATR_time1.rawhttps://storage.jpostdb.org/JPST003787/files/20230906_JC_Boulder_EDL_time10.rawhttps://storage.jpostdb.org/JPST003787/files/20230906_JC_Boulder_EDL_time1.rawhttps://storage.jpostdb.org/JPST003787/files/20230909_JC_Boulder_ATR_time25.rawhttps://storage.jpostdb.org/JPST003787/files/20230909_JC_Boulder_ATR_time8.rawhttps://storage.jpostdb.org/JPST003787/files/20230906_JC_Boulder_EDL_time6.rawhttps://storage.jpostdb.org/JPST003787/files/20230906_JC_Boulder_EDL_time4.rawhttps://storage.jpostdb.org/JPST003787/files/20230909_JC_Boulder_ATR_time10.rawhttps://storage.jpostdb.org/JPST003787/files/20230906_JC_Boulder_EDL_time2.rawhttps://storage.jpostdb.org/JPST003787/files/20230909_JC_Boulder_ATR_time6.rawhttps://storage.jpostdb.org/JPST003787/files/20230906_JC_Boulder_EDL_time8.rawhttps://storage.jpostdb.org/JPST003787/files/20230909_JC_Boulder_ATR_time15.rawhttps://storage.jpostdb.org/JPST003787/files/20230909_JC_Boulder_ATR_time2.rawhttps://storage.jpostdb.org/JPST003787/files/20230909_JC_Boulder_ATR_time4.rawhttps://storage.jpostdb.org/JPST003787/files/20230909_JC_Boulder_ATR_time30.rawhttps://storage.jpostdb.org/JPST003787/files/20230906_JC_Boulder_EDL_time0.rawprimaryOK200ProteomicsEdward LauMus Musculus (mouse)https://repository.jpostdb.org/entry/JPST003787University of ColoradojPOSTMyocytes are exceptionally long-lived cells that must maintain proteome integrity over decades while adjusting for changes in functional output and metabolic demand. We used in vivo stable isotope labeling combined with mass spectrometry proteomics and correlated multi-isotope imaging mass spectrometry to quantify and visualize protein turnover across cardiac, fast-twitch, and slow-twitch skeletal muscles, creating a resource of hundreds of individual protein turnover rates from each tissue. We found that cardiac muscle has the highest rate of protein turnover, followed by slow-twitch skeletal muscle and then fast-twitch skeletal muscle, and that these different rates of protein turnover are driven by different levels of muscle use, rather than myosin isoform composition. We also identified protein age heterogeneity at the myofiber and sarcomere levels. These findings uncover fundamental principles of muscle protein maintenance and have broad implications for understanding cellular aging, muscle disease, and the design of therapeutic strategies targeting muscle protein turnover.Longevity of cardiac and skeletal muscle proteins is dependent on tissue and subcellular compartmentation patterns.Gugel Jack J, Currie Jordan J, Alamillo Lorena L, Flint Jason J, Kim Keun-Young KY, Debliqui Marc M, Ellisman Mark H MH, Lam Maggie P Y MPY, Lau Edward E, Arrojo E Drigo Rafael R, Leinwand Leslie LfalseLongevity of cardiac and skeletal muscle proteins Mice were injected with two 500μL injections of 0.15M NaCl dissolved in 99.9% D2O, separated by a four-hour interval. Then, the mice were given ad libitum access to 8% D2O drinking water for the remainder of the experiment. Mice were euthanized and dissected on days 0, 1, 2, 3, 4, 6, 8, 10, 15, 20, 25, and 30 after the start of labelling. Left ventricle, left atria, soleus, and extensor digitorum longus were dissected, flash frozen in liquid nitrogen, and stored at -80°C. Minced tissues were resuspended in 500 μL or 1 mL (volume dependent on wet mass) RIPA buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitor (Thermo Scientific). Tissues were homogenized in prefilled tubes containing 2.8 mm ceramic beads using an Omni Bead Ruptor for 20 seconds at speed 5. Protein from the homogenized lysate was extracted with sonication in a Biorupter (Diagenode) with settings 10× 30 sec on 30 sec off at 4°C. Insoluble debris was removed from all samples by centrifugation at 14,000 × g, 5 minutes. Protein concentration of all samples was measured with Rapid Gold BCA. The samples were digested in randomized batches using a modified version of the filter-aided sample preparation approach. Pierce Protein Concentrators PES, 10K MWCO (Thermo Scientific) were prewashed with 100 mM ammonium bicarbonate (Ambic). 50 ug extracted protein per sample in 250 uL 8M urea was loaded onto each filter. The samples were washed with 8 M urea to denature proteins and remove SDS followed by two washes of 300 uL 100 mM ambic. The samples were reduced and alkylated with final concentrations 5 mM dithiothreitol (DTT) and 18 mM iodoacetamide (IAA) for 30 minutes at 37°C in the dark. DTT and IAA were removed with centrifugation and the samples were washed 3x with 100 mM ambic. Samples were digested atop the filters overnight at 37°C with mass spectrometry grade trypsin (Promega) at a ratio of 1:50 enzyme:protein. The following day samples were cleaned with Pierce C18 spin columns (Thermo Scientific) according to the manufacturer’s protocol. Eluted peptides were dried with speed-vac and resuspended in 0.1% formic acid. Peptides were analyzed on a Thermo Q-Exactive HF quadrupole-Orbitrap high-resolution mass spectrometer, connected to a Thermo nanoflow Easy-nLC 1200 UPLC with the Thermo EasySpray electrospray ionization source. The peptides were separated with an Easy-Spray C18 column 75 μm × 15 cm, 2 μm particle size (Thermo ES-904) with a 90 minute gradient from 0 to 100% pH 2 solvent B (0.1% formic acid in 80% v/v LC-MS grade acetonitrile). Mass spectra were acquired in data-dependent acquisition (DDA) mode with the following settings: MS1 resolution: 60,000; max IT: 20 ms; AGC target: 3e6; scan range: 200 to 1650 m/z, profile; MS2 resolution: 60,000; AGC target: 2e5; max IT: 110 ms; top 15; isolation width: 2.0 m/z; NCE: 25, 27; dynamic exclusion: 30 s. Sun Apr 26 00:00:00 BST 2026PXD0635791009041477761