Identification of Missing Proteins Defined by Chromosome-Centric Proteome Project in the Cytoplasmic Detergent-Insoluble Proteins.

Finding protein evidence (PE) for protein coding genes is a primary task of the Phase I Chromosome-Centric Human Proteome Project (C-HPP). Currently, there are 2948 PE level 2–4 coding genes per neXtProt, which are deemed missing proteins in the human proteome. As most samples prepared and analyzed in the C-HPP framework were focusing on detergent soluble proteins, we posit that as a natural composition the cytoplasmic detergent-insoluble proteins (DIPs) represent a source of finding missing proteins. We optimized a workflow and separated cytoplasmic DIPs from three human lung and three human hepatoma cell lines via differential speed centrifugation. We verified that the detergent-soluble proteins (DSPs) could be sufficiently depleted and the cytoplasmic DIP isolation was partially reproducible with Spearman r > 0.70 according to two independent SILAC MS experiments. Through label-free MS, we identified 4524 and 4156 DIPs from lung and liver cells, respectively. Among them, a total of 23 missing proteins ...