Multi-Protease Strategy Identifies Three PE2 Missing Proteins in Human Testis Tissue.

Although 5 years of the missing proteins (MPs) study have been completed, searching for MPs remains one of the core missions of the Chromosome-Centric Human Proteome Project (C-HPP). Following the next-50-MPs challenge of the C-HPP, we have focused on the testis-enriched MPs by various strategies since 2015. On the basis of the theoretical analysis of MPs (2017-01, neXtProt) using multiprotease digestion, we found that nonconventional proteases (e.g. LysargiNase, GluC) could improve the peptide diversity and sequence coverage compared with Trypsin. Therefore, a multiprotease strategy was used for searching more MPs in the same human testis tissues separated by 10% SDS-PAGE, followed by high resolution LC-MS/MS system (Q Exactive HF). A total of 7838 proteins were identified. Among them, three PE2 MPs in neXtProt 2017-01 have been identified: beta-defensin 123 ( Q8N688 , chr 20q), cancer/testis antigen family 45 member A10 ( P0DMU9 , chr Xq), and Histone H2A-Bbd type 2/3 ( P0C5Z0 , chr Xq). However, because only one unique peptide of ≥9 AA was identified in beta-defensin 123 and Histone H2A-Bbd type 2/3, respectively, further analysis indicates that each falls under the exceptions clause of the HPP Guidelines v2.1. After a spectrum quality check, isobaric PTM and single amino acid variant (SAAV) filtering, and verification with a synthesized peptide, and based on overlapping peptides from different proteases, these three MPs should be considered as exemplary examples of MPs found by exceptional criteria. Other MPs were considered as candidates but need further validation. All MS data sets have been deposited to the ProteomeXchange with identifier PXD006465.