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 tes...