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.