Several protocols now support efficient differentiation of human pluripotent stem cells to cardiomyocytes (hPSC-CMs) but these still indicate line-to-line variability. As the number of studies implementing this technology expands, accurate assessment of cell identity is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity has not yet been established. Therefore, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in day 25 hPSC-CMs and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges to interpreting data generated by published methods and inform the development of a robust protocol for routine assessment of hPSC-CMs. The data, workflow for antibody evaluation, and standardized protocol described here should benefit investigators new to this field and those with expertise in hPSC-CM differentiation.

Day 25 hPSC-CM cell lysate and recombinant human TNNI3(ProSpec, PRO-324) were digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry using an Orbitrap Fusion Lumos (Thermo). In initial discovery studies, data-dependent acquisition was used for selection of peptides that had favorable characteristics (i.e., well-defined chromatographic peak, easily ionized). Subsequently, stable isotopically labeled synthetic peptides were obtained for the three best-performing TNNI3 peptides and used as internal standards. A targeted mass spectrometry assay was developed using PRM (Peterson et al., 2012) to selectively detect three peptides from TNNI3 and four peptides from TNNT2 at their observed mass and time of elution from preliminary experiments of recombinant protein and cell lysate, respectively. This assay was applied to day 25 hPSC-CMs, and hPSC and cardiac tissue samples (collected with Institutional Review Board approval) were included as negative and positive controls, respectively.

Human cardiac tissue, day 25 hPSC-CM cell lysate, recombinant human TNNI3 tryptic peptides all with spiked in SIL peptides of TNNI3.