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J Proteome Res. 2016 Nov 4;15(11):3998-4019. Epub 2016 Aug 23.

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

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CEA, DRF, BIG, Laboratoire de Biologie à Grande Echelle, 17 rue des martyrs, Grenoble F-38054, France.
Inserm U1038 , 17, rue des Martyrs, Grenoble F-38054, France.
Université de Grenoble , Grenoble F-38054, France.
Department of Human Protein Sciences, Faculty of Medicine, University of Geneva , 1, rue Michel-Servet, 1211 Geneva 4, Switzerland.
CALIPHO Group, SIB-Swiss Institute of Bioinformatics, CMU , rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland.
Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC, Université de Strasbourg, CNRS UMR7178, 25 Rue Becquerel, 67087 Strasbourg, France.
Protim, Inserm U1085, Irset, Campus de Beaulieu, Rennes 35042, France.
Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, 31062 Toulouse, France.
Proteome Informatics Group, Centre Universitaire d'Informatique , Route de Drize 7, 1227 Carouge, CH, Switzerland.
Service de Médecine de la Reproduction, CHU de Nantes , 38 boulevard Jean Monnet, 44093 Nantes cedex, France.
INSERM UMR1064 , Nantes 44093, France.


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.


bioinformatics; cilia; data mining; human proteome project; immunohistochemistry; mass spectrometry proteomics; missing proteins; spermatozoon

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