The S-glutathionylation and phosphorylation proteomes of mouse hearts were analyzed using shotgun proteomics methods in order to assess the effects of aging and the ability of mitochondrion-targeted drug SS-31 to reverse age-related changes. There was a nearly universal increase in S-gluthationylation of cysteine residues on proteins taken from Old (24 months old at the start of the study) mouse hearts compared to Young (5-6 months old). This shift in proteome oxidation state was almost completely reversed by 8-weeks of SS-31 treatment. Many of the significant changes that occurred were in mitochondrial pathways. Changes in phosphorylation did not show a clear pattern, as there was a mix of enhancement and suppression of phosphorylation with age among different proteins. SS-31 showed some effect at restoring the phosphorylation state of proteins that had increased phosphorylation with age but it had no effect on those that had decreased phosphorylation with age. A subset of phosphorylation sites was also analyzed by parallel reaction monitoring, which revealed that Myot S231 had a change in the proportion of phosphorylated and unphosphorylated proteins and that cMyBP-C S307 had a proportional increase in phosphorylated and unphosphorylated protein with age, but that SS-31 treatment did not affect these changes.

Sample Lysis and Digestion (performed by Jeremy Whitson) Powdered heart tissue was solubilized in 50 mM triethylammonium bicarbonate (TEAB) pH 7.55 buffer with 5% SDS, 2 mM MgCl2, and HALT phosphatase and protease inhibitors (Thermo Fisher Scientific). 800 ng enolase was added to each 50 µg sample for normalization. Lysates were bound to S-Trap mini columns, washed with TEAB-buffered methanol and a 1:1 mix of chloroform and methanol, digested with trypsin, and eluted following the manufacturer’s protocol (Profiti, Farmingdale, NY). Eluents were dried using a CentriVap Concentrator (LABCONCO) and reconstituted in 0.1% formic acid. Liquid Chromatography and Mass Spectrometry One ug of each sample with 50 femtomole of heavy labeled Peptide Retention Time Calibrant (PRTC) mixture (Thermo, cat # 88321) was loaded onto a 30 cm fused silica picofrit (New Objective) 75 μm column and 4 cm 150 μm fused silica Kasil1 (PQ Corporation) frit trap loaded with 3 μm Reprosil-Pur C18 (Dr. Maisch) reverse-phase resin analyzed with a Thermo Easy-nLC 1200. The PRTC mixture is used to assess system suitability (QC) before and during analysis. Buffer A was 0.1% formic acid in water and buffer B was 0.1% formic acid in 80% acetonitrile. The 40-minute system suitability gradient consisted of a 0 to 16% B in 5 minutes, 16 to 35% B in 20 minutes, 35 to 75% B in 1 minute, 75 to 100% B in 5 minutes, followed by a wash of 9 minutes and a 30-minute column equilibration. The 110-minute sample LC gradient consists of a 2 to 7% for 1 minutes, 7 to 14% B in 35 minutes, 14 to 40% B in 55 minutes, 40 to 60% B in 5 minutes, 60 to 98% B in 5 minutes, followed by a 9-minute wash and a 30-minute column equilibration. Peptides were eluted from the column with a 50°C heated source (CorSolutions) and electrosprayed into a Thermo Orbitrap Fusion Lumos Mass Spectrometer with the application of a distal 3 kV spray voltage. For the sample digest, a full-scan mass spectrum at 60,000 resolution with a mass range of 400 to 2000 m/z, AGC target of 4e5, 50 ms maximum injection time was followed by 81 unscheduled PRM scans at 15,000 resolution with a mass range of 150 to 2000 m/z, AGC target of 5e5, 22 ms maximum injection time and 27% NCE. Application of the mass spectrometer and LC solvent gradients are controlled by the ThermoFisher XCalibur (version 3.3.2782.34) data system. PRM Data Analysis Thermo RAW files were converted to mzML format using Proteowizard (version 3.0.19113) and imported into a Skyline (daily version 20.1.9.234) document configured with inclusion peptides (Appendix B). A DDA with MS1 filtering search was performed using MSAmanda (with built-in Percolator) in Skyline filtering for PRM peptides with a default cut-off of 0.95. Parameters used included a fixed carbamidomethyl modification of 57 Da on Cysteine, up to 3 variable phosphorylation modifications of 80 Da on Serine and Threonine, MS1 settings of precursor charge state of 2 and a mass accuracy of 10 ppm with centroided peaks. MS2 settings were fully tryptic allowing for 2 missed cleavages, fragment b- and y-ions, retention time filtering of scans within 5 minutes of MS2 IDs at a tolerance of 10 ppm using the Uniprot mouse canonical FASTA. MSAmanda mzid output files were imported with default cut-off of 0.95 to build a PRM spectral library. Data was then normalized to TIC and mean normalized areas of peptides were exported from Skyline.

Hearts from young, old and SS-31 treated mice were used in this experiment. Mouse ID and digestion batch were provided as metadata but no information on the mouse category was provided. The samples analyzed included 48 experimental samples and 2 universal mouse references. Samples were randomized for mass spectrometry.