LAUSR

Pluripotent stem cells (PSC) endocrine differentiation at a large scale allows sampling of transcriptome and proteome with phosphoproteome (proteoform) at specific time points. We describe the dynamic time course of changes in cells undergoing directed beta‐cell differentiation and show target proteins or previously unknown phosphorylation of critical proteins in pancreas development, NKX6‐1, and Chromogranin A (CHGA). We describe fluctuations in the correlation between gene expression, protein abundance, and phosphorylation, following differentiation protocol perturbations at all stages to identify proteoform profiles. Our modeling recognizes outliers on a phenomic landscape of endocrine differentiation, and we describe new biological pathways involved. We have validated our proteomic data by analyzing independent single‐cell RNAseq datasets for in‐vitro pancreatic islet production and corroborated our findings for several proteins suggestive as targets for future research. The single‐cell analysis combined with proteoform data places new protein targets within the specific time point and at the specific pancreatic lineage of differentiating stem cells. We suggest that non‐correlating proteins abundances or new phosphorylation motifs of NKX6.1 and CHGA point to new signaling pathways that may play an essential role in beta‐cell development. We present our findings for the research community's use to improve endocrine differentiation protocols and developmental studies.