Background: There is a growing appreciation that many cardiomyopathies, such as myocardial infarction, dilated/hypertrophic cardiomyopathy (CM) and heart failure (HF) are associated with multiple and diverse cellular defects including protein aggregation, fibrosis, loss of membrane contact sites and metabolic disruption. Understanding these complex phenotypes (which likely involve multiple cellular pathways) and dissecting the underlying cellular and molecular mechanisms necessitates high-precision systems-biology and multi-omics approaches. For most cardiomyopathies, transgenic mouse lines and human induced-pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) represent the state-of-the-art model systems; however, both differ significantly from the native-human cardiac biology. Pig models offer the advantage of human-like physiology, as well as the possibility of conducting spatially-resolved proteomics studies; however, the relatively low-throughput necessitates highly reproducible and sensitive mass-spectrometry (MS)-based proteomics workflows. In addition, many critical cardiac functions, such as excitation-contraction coupling and SR-calcium handling are carried out by membrane proteins and protein complexes, for which reproducible and sensitive detection can sometimes be a challenge. Here, we highlight recent developments by our group aimed at improving proteomics analysis from small pig heart biopsies. Methods: We implemented an automated pressure-cycling technology (PCT) single-pot solid-phase-enhanced sample preparation (SP3) workflow for data-independent analysis (DIA)-MS. In brief: cardiac biopsies were obtained from live WT-control pigs and lysed by pressure-cycling techniques. Several detergents (8M urea, 1% SDS and 2% SDC) were trialed to improve reproducibility and sensitivity of membrane-protein extraction/detection. In place of manual fluid-handling and desalting workflows (which we hypothesize to be a significant source of inconsistency), an automated SP3 protocol with amine-coated magnetic beads was used for protein cleanup and trypsinization. Peptide mixtures were separated and analyzed using a hybrid ion mobility/quadrupole/time-of-flight mass spectrometer. Data analysis was performed using directDIA processing in Spectronaut v16. Overall recovery of protein groups, as well as recovery of specific cardiac proteins of interest (i.e., SERCA2a, RYR2, PLN) were assessed. Results: The PCT-SP3-DIA proteomics workflow enables quantitative analysis of approximately 6,000 protein groups from a 0.5 mm³ tissue volume, with a throughput of 12 samples per day. Reproducibility and protein recovery was significantly improved compared to legacy workflows. Lastly, 2% SDS was found to be the optimal detergent for the recovery of membrane proteins, increasing recovery by up to 90% for some cardiac proteins of interest such as PLN and SERCA2A. Conclusion: Our findings demonstrate the capabilities of the PCT-SP3-DIA workflow for high-throughput pig-cardiac proteome analysis in cardiac tissues from human biopsies, animal models, and engineered heart muscle. Notably, reproducible proteomics data could be obtained from biopsies as small as 0.5mm3, opening up the possibility of developing a spatially-resolved proteome “map” and performing spatially-resolved proteomics. We are currently in the process of developing biopsy techniques to facilitate spatially-resolved proteomics and are applying these techniques to a novel pig disease model.