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

<strong>O-GlcNAc enrichment</strong> 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 5000<em>xg</em> (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. <br> <strong>Mass-spectrometry</strong> 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℃. <br> 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. <br> <strong>Data analysis</strong> 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.