Multiproteases Combined with High-pH Reverse-Phase Separation Strategy Verified Fourteen Missing Proteins in Human Testis Tissue

Subsequent to conducting the Chromosome-Centric Human Proteome Project, we have focused on human testis-enriched missing proteins (MPs) since 2015. For protein coverage to be enhanced, a multiprotease strategy was used for separation of samples by 10% SDS-PAGE. For the separating efficiency to be improved, a high-pH reverse phase (RP) separation strategy was applied to fractionate complex samples in this study. A total of 11,558 proteins was identified, which is the largest proteome data set for single human tissue sample so far. On the basis of this large-scale data set, we verified 14 MPs (PE2) in neXtProt (2018-01) after spectrum quality analysis, isobaric post-translational modification, and single amino acid variant filtering, and synthesized peptide matching. Tissue expression analysis showed that 3 of 14 MPs were testis-specific proteins. Functional analysis showed that 10 of 14 MPs were closely related to liver tumor, liver carcinoma, and hepatocellular carcinoma. Another 100 MPs were listed as candidates but required additional verification information. All MS data sets have been deposited into the ProteomeXchange with the identifier PXD009737.