Looking for Missing Proteins in the Proteome of Human Spermatozoa: An Update

The
Chromosome-Centric Human Proteome Project (C-HPP) aims to identify
“missing” proteins in the neXtProt knowledgebase. We
present an in-depth proteomics analysis of the human sperm proteome
to identify testis-enriched missing proteins. Using protein extraction
procedures and LC–MS/MS analysis, we detected 235 proteins
(PE2–PE4) for which no previous evidence of protein expression
was annotated. Through LC–MS/MS and LC–PRM analysis,
data mining, and immunohistochemistry, we confirmed the expression
of 206 missing proteins (PE2–PE4) in line with current HPP
guidelines (version 2.0). Parallel reaction monitoring acquisition
and sythetic heavy labeled peptides targeted 36 ≪one-hit wonder≫
candidates selected based on prior peptide spectrum match assessment.
24 were validated with additional predicted and specifically targeted
peptides. Evidence was found for 16 more missing proteins using immunohistochemistry
on human testis sections. The expression pattern for some of these
proteins was specific to the testis, and they could possibly be valuable
markers with fertility assessment applications. Strong evidence was
also found of four “uncertain” proteins (PE5); their
status should be re-examined. We show how using a range of sample
preparation techniques combined with MS-based analysis, expert knowledge,
and complementary antibody-based techniques can produce data of interest
to the community. All MS/MS data are available via ProteomeXchange
under identifier PXD003947. In addition to contributing to the C-HPP,
we hope these data will stimulate continued exploration of the sperm
proteome.