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 (22
PE2 and 1 PE4) were identified by MS, and 18 of them had translation
evidence; in addition, six PE5 proteins were identified by MS, three
with translation evidence. We showed that cytoplasmic DIPs were not
an enrichment of transmembrane proteins and were chromosome-, cell
type-, and tissue-specific. Furthermore, we demonstrated that DIPs
were distinct from DSPs in terms of structural and physical–chemical
features. In conclusion, we have found 23 missing proteins and 6 PE5
proteins from the cytoplasmic insoluble proteome that is biologically
and physical-chemically different from the soluble proteome, suggesting
that cytoplasmic DIPs carry comprehensive and valuable information
for finding PE of missing proteins. The mass spectrometry proteomics
data have been deposited to the ProteomeXchange Consortium with the
data set identifier PXD001694.