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

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Download Hi-Res ImageDownload to MS-PowerPointCite This:J. Proteome Res. 2017, 16, 12, 4340-4351
    Article

    Validating Missing Proteins in Human Sperm Cells by Targeted Mass-Spectrometry- and Antibody-based Methods
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    † Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC, Université de Strasbourg, CNRS UMR7178, 25 Rue Becquerel, Strasbourg F-67087, France
    ‡ CALIPHO Group, SIB-Swiss Institute of Bioinformatics, CMU, rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland
    § H2P2 Core facility, UMS BioSit, University of Rennes 1, Rennes F-35040, France
    ∥ Protim, Inserm U1085, Irset, Campus de Beaulieu, Rennes F-35042, France
    ⊥ Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden
    # Service de Médecine de la Reproduction, CHU de Nantes, 38 boulevard Jean Monnet, Nantes F-44093, France
    ▽ Inserm UMR1064, Nantes F-44093, France
    ○ CEA, DRF, BIG, Laboratoire de Biologie à Grande Echelle, 17, rue des Martyrs, Grenoble F-38054, France
    ◆ Inserm U1038, Grenoble F-38054, France
    Grenoble-Alpes University, Grenoble F-38054, France
    + Department of Human Protein Sciences, Faculty of Medicine, University of Geneva, 1, rue Michel-Servet, 1211 Geneva 4, Switzerland
    *C.C.: E-mail: [email protected]. Tel: +33 (0)3 68 85 27 30.
    *C.P.: E-mail: [email protected]. Tel: +33 (0)2 23 23 52 79.
    Other Access OptionsSupporting Information (4)

    Journal of Proteome Research

    Cite this: J. Proteome Res. 2017, 16, 12, 4340–4351
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.jproteome.7b00374
    Published September 11, 2017
    Copyright © 2017 American Chemical Society
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    Abstract

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    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.

    Copyright © 2017 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.7b00374.

    • Analytical details of the acquisition of reference MS/MS spectra for the 92 proteotypic peptides corresponding to the 38 targeted MPs and on the targeted LC–PRM time-scheduled method development. (PDF)

    • Supplementary Figure 1: LC–PRM data of validated peptides. (PDF)

    • Supplementary Table 1: Targeted LC–PRM inclusion list. List of the 212 targets followed during LC–PRM analyses: precursor m/z ratio, charge state, polarity, scheduling start and end times, normalized collision energy (CE), amino acid sequence including modifications and precursor type (light form and heavy labeled form), are provided for each target. (XLSX)

    • Supplementary Table 2: List of MPs for which immunohistochemistry or immunocytochemistry was successful, HPA antibody names, and dilutions used. (XLSX)

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    Cited By

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    This article is cited by 22 publications.

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    Journal of Proteome Research

    Cite this: J. Proteome Res. 2017, 16, 12, 4340–4351
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.jproteome.7b00374
    Published September 11, 2017
    Copyright © 2017 American Chemical Society
    Request reuse permissions

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