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J Proteome Res. 2017 Dec 1;16(12):4340-4351. doi: 10.1021/acs.jproteome.7b00374. Epub 2017 Sep 26.

Validating Missing Proteins in Human Sperm Cells by Targeted Mass-Spectrometry- and Antibody-based Methods.

Author information

1
Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC, Université de Strasbourg, CNRS UMR7178 , 25 Rue Becquerel, Strasbourg F-67087, France.
2
CALIPHO Group, SIB-Swiss Institute of Bioinformatics, CMU , rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland.
3
H2P2 Core facility, UMS BioSit, University of Rennes 1 , Rennes F-35040, France.
4
Protim, Inserm U1085, Irset, Campus de Beaulieu , Rennes F-35042, France.
5
Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University , Uppsala, Sweden.
6
Service de Médecine de la Reproduction, CHU de Nantes , 38 boulevard Jean Monnet, Nantes F-44093, France.
7
Inserm UMR1064 , Nantes F-44093, France.
8
CEA, DRF, BIG, Laboratoire de Biologie à Grande Echelle, 17, rue des Martyrs, Grenoble F-38054, France.
9
Inserm U1038 , Grenoble F-38054, France.
10
Grenoble-Alpes University , Grenoble F-38054, France.
11
Department of Human Protein Sciences, Faculty of Medicine, University of Geneva , 1, rue Michel-Servet, 1211 Geneva 4, Switzerland.

Abstract

The present study is a contribution to the "neXt50 challenge", a coordinated effort across C-HPP teams to identify the 50 most tractable missing proteins (MPs) on each chromosome. We report the targeted search of 38 theoretically detectable MPs from chromosomes 2 and 14 in Triton X-100 soluble and insoluble sperm fractions from a total of 15 healthy donors. A targeted mass-spectrometry-based strategy consisting of the development of LC-PRM assays (with heavy labeled synthetic peptides) targeting 92 proteotypic peptides of the 38 selected MPs was used. Out of the 38 selected MPs, 12 were identified with two or more peptides and 3 with one peptide after extensive SDS-PAGE fractionation of the two samples and with overall low-intensity signals. The PRM data are available via ProteomeXchange in PASSEL (PASS01013). Further validation by immunohistochemistry on human testes sections and cytochemistry on sperm smears was performed for eight MPs with antibodies available from the Human Protein Atlas. Deep analysis of human sperm still allows the validation of MPs and therefore contributes to the C-HPP worldwide effort. We anticipate that our results will be of interest to the reproductive biology community because an in-depth analysis of these MPs may identify potential new candidates in the context of human idiopathic infertilities.

KEYWORDS:

bioinformatics; data mining; human proteome project; immunocytochemistry; immunohistochemistry; missing proteins; parallel reaction monitoring; spermatozoon; targeted proteomics

PMID:
28891297
DOI:
10.1021/acs.jproteome.7b00374
[Indexed for MEDLINE]
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