Finding Missing Proteins from the Epigenetically Manipulated Human Cell with Stringent Quality Criteria

The chromosome-centric human proteome project (C-HPP) has made great progress of finding protein evidence (PE) for missing proteins (PE2–4 proteins defined by the neXtProt), which now becomes an increasingly challenging field. As a majority of samples tested in this field were from adult tissues/cells, the developmental stage specific or relevant proteins could be missed due to biological source availability. We posit that epigenetic interventions may help to partially bypass such a limitation by stimulating the expression of the “silenced” genes in adult cells, leading to the increased chance of finding missing proteins. In this study, we established <i>in vitro</i> human cell models to modify the histone acetylation, demethylation, and methylation with near physiological conditions. With mRNA-seq analysis, we found that histone modifications resulted in overall increases of expressed genes in an even distribution manner across different chromosomes. We identified 64 PE2–4 and six PE5 proteins by MaxQuant (FDR < 1% at both protein and peptide levels) and 44 PE2–4 and 7 PE5 proteins by Mascot (FDR < 1% at peptide level) searches, respectively. However, only 24 PE2–4 and five PE5 proteins in Mascot, and 12 PE2–4 and one PE5 proteins in MaxQuant searches could, respectively, pass our stringently manual spectrum inspections. Collectively, 27 PE2–4 and five PE5 proteins were identified from the epigenetically modified cells; among them, 19 PE2–4 and three PE5 proteins passed FDR < 1% at both peptide and protein levels. Gene ontology analyses revealed that the PE2–4 proteins were significantly involved in development and spermatogenesis, although their chemical–physical features had no statistical difference from the background. In addition, we presented an example of suspicious PE5 peptide spectrum matched with unusual AA substitutions related to post-translational modification. In conclusion, the epigenetically manipulated cell models should be a useful tool for finding missing proteins in C-HPP. The mass spectrometry data have been deposited to the iProx database (accession number: IPX00020200)