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.