S1 and S2 are soluble fraction replicates; S3 and S4 are total heart lysate, S5 and S6 are contractile fractions.

O-GlcNAc enrichment Hearts were fractionated into soluble and insoluble heart lysates using the modified InSequence protocol ​[13]​. Alternatively, total heart lysates were made in 9M urea. Heart proteins (200g) were desalted and concentrated by Methanol Chloroform precipitation as previously described ​[24]​. Samples were reduced with 50 mM dithiothreitol in 20 mM ammonium bicarbonate (1 h, 60°C) and alkylated with 100 mM iodoacetamide (15 min, room temperature, dark). Proteins were methanol chloroform precipitated and subjected to proteolysis with LysC (Promega, Madison WI) at a 1:30 enzyme to protein ratio. Peptides were purified away from contaminants and salts using a C18 Sep-Pak (Waters; Milford MA; Cat # 186004618), eluted in 40% acetonitrile with 0.1% TFA, and lyophilized for 3 days. O-GlcNAc enrichment was achieved using the PTM Scan O-GlcNAc antibodies (Cell Signaling Technologies, Cat/ #95220) following the manufacturer’s instructions. Briefly, peptides were resuspended in 1x IAP Buffer containing control O-GlcNAc modified peptides (Cell Signaling Technologies, Cat. #34200). The peptide solution was clarified at 5000xg (10min, 4oC) and applied to the O-GlcNAc enrichment resin and incubated (2h, 4oC). Beads were subsequently washed in 1x IAP buffer (x2) and MilliQ water (x3) before being eluted in 0.15% TFA (3x 10 min). Peptides were concentrated by lyophilization. Mass-spectrometry Analysis was performed using an Orbitrap Exploris 480 mass spectrometer interfaced with a Neo Vanquish nanoflow liquid chromatography system (Thermo Scientific). Peptides were loaded on a trap column (Reprosil C18AQ, 5mm) using solvent A (0.1% v/v formic acid) with a flow rate 4 mL/min. Peptides were separated on a house packed nanoLC column (ESI Source Solutions packed with 2.4mm Reprosil C18AQ, 100A, 150mm x 75mm) using a 90 min gradient at a flow rate of 300 nL/min. The spray voltage was set to 2.2 kV while ion transfer tube temperature was set to 250℃. The mass spectrometer was operated in data-dependent acquisition mode. A survey full scan MS (m/z 350–1550) was acquired in the Orbitrap analyzer with resolution 120,000 at m/z 200. The AGC target for MS1 was set as 2 x 105 with ion injection time as 50 ms. The most intense ions with charge state ≥ 2 were isolated in 3 sec cycle and fragmented using higher energy collisional dissociation (HCD) fragmentation with 32% normalized collision energy (NCE) and detected at a mass resolution of 30,000 at 200 m/z. The AGC target for MS/MS was set as 5 x 104 and ion filling time set 100 ms dynamic exclusion was set for 30 s with a ±7 ppm mass window. .Raw files are publically accessible on Figshare. Data analysis To obtain protein identification and peptide modification sites, all spectra were searched against the UniProt Mus musculus database version UP589. The following search parameters were used: 2 missed cleavages; carboxyamidomethylation of cysteines (fixed); oxidation of methionine (variable); deamidation of NQ (variable); HexNAc (203.08; variable); and Phospho ST (variable). Mass tolerances of ± 5 ppm for precursors and a +/- 10ppm for fragment ions was used. The data were filtered with a 1% false discovery rate as calculated using the Target Decoy PSM Validator node. Spectra containing HexNAc oxonium ions were manually validated as previously reported ​[25]​. .MSF files are publically available on Figshare.