Functionally Integrated Top-Down Proteomics for Standardized Assessment of Human Induced Pluripotent Stem Cell-Derived Engineered Cardiac Tissues.

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The ability to perform functional and molecular assessments using the same hiPSC-ECT construct would allow for more reliable correlation between observed functional performance and underlying molecular events, and thus is critically needed. Herein, for the first time, we have established an integrated method that permits sequential assessment of functional properties and top-down proteomics from the same single hiPSC-ECT construct. We quantitatively determined the differences in isometric twitch force and the sarcomeric proteoforms between two groups of hiPSC-ECTs that differed in the duration of time of 3D-ECT culture. Importantly, by using this integrated method we discovered a new and strong correlation between the measured contractile parameters and the phosphorylation levels of alpha-tropomyosin between the two groups of hiPSC-ECTs. The integration of functional assessments together with molecular characterization by top-down proteomics in the same hiPSC-ECT construct enables a holistic analysis of hiPSC-ECTs to accelerate their applications in disease modeling, cardiotoxicity, and drug discovery. Data are available via ProteomeXchange with identifier PXD022814.", "claims": [{"id": "claim-method", "statement": "Statistical analysis of twitch force amplitude, time from pacing stimulus to twitch force peak (CT100), time from twitch force peak to 50% twitch force decay (RT50), and time from 50% to 90% twitch force decay (RT50–90), relative phosphorylation, and isoform ratios between the different groups of hiPSC-ECTs were determined by a Student’s two-tailed t test assuming equal variances. ns indicated a p > 0.05, * indicated a p < 0.05, and ** indicated a p < 0.01 by Student’s two-tailed t test with equal variances. Standard error of the mean was used for calculating the variation between the two groups of hiPSC-ECTs. Phosphorylation and contractile parameter values were fit to a multivariate linear model with age as the independent variable.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:33395532", "locator": "sections[12] \"Statistical Analysis\" paragraph 2", "quote": "Statistical analysis of twitch force amplitude, time from pacing stimulus to twitch force peak (CT100), time from twitch force peak to 50% twitch force decay (RT50), and time from 50% to 90% twitch force decay (RT50–90), relative phosphorylation, and isoform ratios between the different groups of hiPSC-ECTs were determined by a Student’s two-tailed t test assuming equal variances. ns indicated a p > 0.05, * indicated a p < 0.05, and ** indicated a p < 0.01 by Student’s two-tailed t test with equal variances. Standard error of the mean was used for calculating the variation between the two groups of hiPSC-ECTs. Phosphorylation and contractile parameter values were fit to a multivariate linear model with age as the independent variable."}], "state": "complete"}, {"id": "claim-data-access", "statement": "Data are available via ProteomeXchange with identifier PXD022814.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:33395532", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Data are available via ProteomeXchange with identifier PXD022814."}], "state": "complete"}, {"id": "claim-target-result", "statement": "Sequential functional assessment and top-down proteomics of the same hiPSC-ECT construct revealed a strong correlation between alpha-tropomyosin phosphorylation and contractile performance.", "kind": "quantitative", "target": {"value": 2.0, "unit": "correlation coefficient", "tolerance": null, "source": "Strikingly, we found that there was an extremely high correlation (R2 = 0.9666) between the CT100 and the phosphorylation levels of α-Tpm between the two groups of hiPSC-ECTs (Figure 4A)."}, "evidence": [{"asset": "source:pmid:33395532", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Importantly, by using this integrated method we discovered a new and strong correlation between the measured contractile parameters and the phosphorylation levels of alpha-tropomyosin between the two groups of hiPSC-ECTs."}, {"asset": "source:pmid:33395532", "locator": "sections[16] \"Integration of Functional Assessments and Top-Down Proteomics Data\" paragraph 2", "quote": "Strikingly, we found that there was an extremely high correlation (R2 = 0.9666) between the CT100 and the phosphorylation levels of α-Tpm between the two groups of hiPSC-ECTs (Figure 4A)."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "Sequential functional assessment and top-down proteomics of the same hiPSC-ECT construct revealed a strong correlation between alpha-tropomyosin phosphorylation and contractile performance.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:33395532"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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