Improved determination of protein turnover rate with heavy water labeling by mass isotopomer ratio selection.

Unlabelled: The synthesis and degradation rates of proteins form an essential component of gene expression control. Heavy water labeling has been used in conjunction with mass spectrometry to measure protein turnover rates, but the optimal analytical approaches to derive turnover rates from the isotopomer patterns of deuterium labeled peptides continue to be a subject of research. Here we describe a method, which comprises a reverse lookup of numerically approximated peptide isotope envelopes, coupled to the selection of optimal isotopomer pairs based on peptide sequence, to calculate the molar fraction of new peptide synthesis in heavy water labeling mass spectrometry experiments.

A Ratiometric Catalog of Protein Isoform Shifts in the Cardiac Fetal Gene Program.

The fetal genetic program orchestrates cardiac development and the re-expression of fetal genes is thought to underlie cardiac disease and adaptation. Here, a proteomics ratio test using mass spectrometry is applied to find protein isoforms with statistically significant usage differences in the fetal vs. postnatal mouse heart.

Tissue Usage Preference and Intrinsically Disordered Region Remodeling of Alternative Splicing Derived Proteoforms in the Heart.

A computational analysis of mass spectrometry data was performed to uncover alternative splicing derived protein variants across chambers of the human heart. Evidence for 216 non-canonical isoforms was apparent in the atrium and the ventricle, including 52 isoforms not documented on SwissProt and recovered using an RNA sequencing derived database. Among non-canonical isoforms, 29 show signs of regulation based on statistically significant preferences in tissue usage, including a ventricular enriched protein isoform of tensin-1 (TNS1) and an atrium-enriched PDZ and LIM Domain 3 (PDLIM3) isoform 2 (PDLIM3-2/ALP-H).

Senescence in human AC16 cardiac cells is associated with thymidine kinase induction and histone loss.

AC16 cells are a transformed human cardiac cell line commonly used to study cardiomyocyte biology. We show that reduced proliferation and senescence markers can be robustly induced in AC16 cells cultured in low serum condition and treated with (i) low-dose doxorubicin, (ii) UV 254 nm, or (iii) H O exposure for up to 48 hours. Increased p21 (CDKN1A) and H2A.