PXD064177-1

PXD064177 is an original dataset announced via ProteomeXchange.

Title	Longevity of cardiac and skeletal muscle proteins in MYH7 transgenic mice
Description	Mice with transgenic expression of MYH7 with a myc-tag at the C-terminus generated in the Leinwand lab were analyzed. The transgene is expressed under control of a skeletal muscle specific MCK promoter, as previously described in: https://pmc.ncbi.nlm.nih.gov/articles/PMC11060730/ 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 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.
HostingRepository	jPOST
AnnounceDate	2026-04-26
AnnouncementXML	Submission_2026-04-26_12:10:49.925.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Edward Lau
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide; alpha-amino acetylated residue
Instrument	Q Exactive HF

Revision	Datetime	Status	ChangeLog Entry
0	2025-05-21 19:30:40	ID requested
⏵ 1	2026-04-26 12:10:50	announced

Gugel J, Currie J, Alamillo L, Flint J, Kim KY, Debliqui M, Ellisman MH, Lam MPY, Lau E, Arrojo E Drigo R, Leinwand L, Longevity of cardiac and skeletal muscle proteins is dependent on tissue and subcellular compartmentation patterns. Cell Rep, 45(1):116768(2026) [pubmed]

submitter keyword: mouse, transgenic, heart, EDL, turnover, deuterium, heavy water, Riana, mass spectrometry

Edward Lau
lab head
Edward Lau
contact affiliation	University of Colorado
dataset submitter

jPOST dataset URI

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