Fetal heart and postnatal hearts (n=5 each) were acquired commercially at E17.5 and P1 from Zyagen (San Diego, CA), weighed, and homogenized for proteomics profiling. Tissue homogenization was performed with a handheld homogenizer (OMNI International). Samples were placed on ice for 1 minute to allow cooling down. Repeat for a total of 3 rounds of homogenization. Homogenized tissues were sonicated with a handheld sonicator (Fisher Model 120), at 40% amplitude for 1 s, pause for 5 s, for 15 cycles. The samples were then centrifuged at 5,000 × g for 1 min at 4°C, and vortex for 10 s, and repeated for three rounds of sonication and centrifugation. The extracted protein samples were further centrifuged at 14,000 × g for 15 min at 4°C to collect supernatants and measure protein concentration with the BCA protein assay kit following manufacturer instructions. The samples were then reduced, alkylated, and digested with sequencing-grade trypsin. The digested peptides were labeled with TMT 10-plex isobaric tags (Thermo), and fractionated using high-pH reversed-phase columns (Pierce) following manufacturer instructions. Mass spectrometry was performed on a Q-Exactive HF orbitrap high-resolution mass spectrometer (Thermo) coupled to a Thermo Easy-nLC UPLC liquid chromatography system. The LC gradient was as follows: 0 – 105 minutes, 0 – 30% B; 105 – 110 minutes, 30 – 70% B, 110 – 115 minutes: 70 – 100% B; 115 – 120 minutes: hold; at 300 nL/min. Mass spectrometry data were acquired in data dependent acquisition (DDA) mode using typical settings including 60,000 MS1 and MS2 resolution, 3e6 MS1 AGC; 2e5 MS2 AGC; 110 ms maximum IT; 15 TopN; isolation window 0.7 m/z; normalized CE 28, 30, 32; and 30 seconds dynamic exclusion. Mass spectrometry data was searched using Sage v.0.14.5 aarch64-apple-darwin build to identify proteins and to measure peptide MS2 TMT and MS1 LFQ (label-free quantification) intensity, using typical settings including: missed_cleavages: 2, precursor_tol: –20 to +20 ppm; fragment_tol: –20 to +20 ppm; isotope: false; static_mods: C 57.0215 K 229.1629; variable mods: M 15.9949 ^ 229.1629 S 229.1629; generate_decoys: true; isotope_errors: 0 – 3; quant: tmt: Tmt10; quant: lfq: true, The database used was retrieved on 2023-09-15 from UniProt to contain 25,530 Swiss-Prot Mus musculus reviewed canonical and isoform entries. A peptide-level multiple testing adjusted q value cutoff of 0.01 was used for confident identification.