Protein O-GlcNAcylation and hexokinase mitochondrial dissociation drive heart failure with preserved ejection fraction_Proteomics data
Description
For proteomics with pulled down biotinylated samples, MS-grade trypsin (Promega, Madison WI) was added each sample at an enzyme-to-substrate ratio of 1:50 for overnight digestion at 37 ⁰C. Digestion was halted with the addition of 10% formic acid to a final concentration of 0.5%. The peptides were de-salted with C18 spin columns and completely dried prior to running on the mass-spectrometer. LC-MS analysis was performed on Vanquish Neo UHPLC (Thermo Fisher, VN-S10-A-01) coupled to Orbitrap Exploris 240 mass spectrometer (Thermo Fisher, BRE725535). Analytical separation was conducted using a UHPLC C18 column (Ion Opticks, AUR3-15075C18-CSI). For each run, 1ug equivalent of sample was injected. The flow rate was set at 0.2 µl/min. Elution of peptides from analytical separation column was performed using a 120 min gradient between buffer A (0.1% Formic acid and 99.9% Optima LC/MS grade water) and buffer B (80% Acetonitrile, 19.9% Optima LC/MS grade water, and 0.1% formic acid): 0 % B at the beginning, 8 % B at 1 min, 28 % B at 86 min, 50 % B at 106 min, 100 % B from 107 to 110 min, 0 % B from 111 to 120 min. Electrospray ionization was performed using a Nanospray Flex Ion Source (Thermo Fisher, ES071) and positive static spray voltage was set at 2200 V. For full Scan range was set to 350-1600 m/z with RF lens: 60%, Orbitrap resolution: 120,000, Normalized AGC Target (%): 300, Maximum injection time (ms): 25, microscans: 1, and Intensity threshold: 5.0e3. Data-dependent acquisition (DDA) by TopN was performed through fragmentation isolated precursor ions with charges between +2 to +5. The following parameters were used for DDA: Dynamic exclusion mode: exclusion duration (s) = 30, mass tolerance: 5 ppm (low) and 5 ppm (high). Data dependent mode: cycle time (s): 2, ddMS2 parameters isolation window (m/z) = 1.5, Normalized collisional energy = 30%, Orbitrap resolution 15,000, scan range mode: define first mass, first mass (m/z) 200, Normalized AGC target (%): 100, Maximum injection time (ms) = 50. Proteins were identified from the MS raw files using the Mascot search engine (Matrix Science, London, UK. version 2.5.1). MS/MS spectra were searched against the SwissProt Human database. All searches included oxidized Methionine, deamidated asparagine and aspartic Acid, and acetylated N-term as variable modifications. Two missed tryptic cleavages were allowed. 1% false discovery rate cutoff was applied at the peptide level. Only proteins with a minimum of two peptides above the cutoff were considered for further study. Identified peptides/protein were visualized by Scaffold software (version 5.0, Proteome Software Inc., Portland, OR)."