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

Abstract

The Chromosome-Centric Human Proteome Project aims to identify proteins classed as « missing » in the neXtProt knowledgebase. In this article, we present an in-depth proteomics analysis of the human sperm proteome to identify testis-enriched missing proteins. Using a range of protein extraction procedures and LC-MS/MS analysis, we detected a total of 235 proteins (PE2-PE4) for which no previous evidence of protein expression was annotated. Through a combination of LC-MS/MS and LC-PRM analysis, data mining and immunohistochemistry, we were able to confirm the expression of 206 missing proteins (PE2-4) in line with current HPP guidelines (version 2.0). Parallel Reaction Monitoring (PRM) acquisition combined with synthetic heavy labeled peptides was used to target 36 « one-hit wonder » candidates selected on the basis of prior PSM assessment. Of this subset of candidates, 24 were validated with additional predicted and specifically targeted peptides. Evidence was found for a further 16 missing proteins using immunohistochemistry on human testis sections. The expression pattern for some of these proteins was specific to the testis, and they could potentially be valuable markers with applications in fertility assessment. Strong evidence was also found for the existence of 4 proteins labeled as "uncertain" (PE5); the status of these proteins should therefore be re-examined.Our results show how the use of 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.

Novel homozygousCFAP69mutations in humans and mice cause severe asthenoteratospermia with multiple morphological abnormalities of the sperm flagella

“…Furthermore, the CFAP69 protein was also identified as a testis-enriched protein using an in-depth proteomics analysis in human spermatozoa 22. The orthologue of CFAP69 in Chlamydomonas reinhardtii is FAP69 (flagellar protein 69), a 135 kDa protein containing two armadillo repeat domains 23 24.…”

Toward Completion of the Human Proteome Parts List: Progress Uncovering Proteins That Are Missing or Have Unknown Function and Developing Analytical Methods

“…Hundreds of PE2,3,4 MPs have been detected in sperm and testis, led by the Chromosome 2/14 consortium in Switzerland and France 12,13 and the Chromosome 1 team in China. 14,15 Sun et al 16 identified 11 558 proteins in testis using SDS-PAGE fractionation, digestion with a combination of the proteases trypsin, LysC, and GluC, and high-pH reverse-phase fractionation; this total is 3700 more proteins than the previous year.…”

Progress in the Chromosome-Centric Human Proteome Project as Highlighted in the Annual Special Issue IV

“…4 This also emphasizes the difficulty in closing the human proteome gap with high-quality unambiguous MS data and the necessity for further targeted orthogonal studies and strategies to reveal shy proteins. Vandenbrouck et al 15 started with 235 missing protein candidates in spermatozoa, of which 206 met the HPP guidelines v2.1. Some were confirmed with immunohistochemistry, such as orphan proteins CXorf58 and C19orf81.…”

“…As noted above, SRMs were used very effectively by Vandenbrouck et al 15 to confirm missing protein identifications in testis, and they are at the heart of the targeted approach for detecting the 40 missing proteins proposed by Duek et al 10 SRMs were also used by Zhao et al 16 to confirm missing proteins identified in the membrane enrichment studies. Poverennaya et al 21 from the Chromosome 18 team used stable isotope-labeled standards for SRM (SRM/SIS) analyses, as well as shotgun LC–MS/MS, of normal liver and HepG2 liver cells.…”