title: Intact cell MALDI-TOF mass spectrometry on single bovine oocyte and follicular cells combined with top-down proteomics: A novel approach to characterise markers of oocyte maturation.
abstract: Intact cell MALDI-TOF mass spectrometry (ICM-MS) was adapted to bovine follicular cells from individual ovarian follicles to obtain the protein/peptide signatures (<17kDa) of single oocytes, cumulus cells (CC) and granulosa cells (GC), which shared a total of 439 peaks. By comparing the ICM-MS profiles of single oocytes and CC before and after in vitro maturation (IVM), 71 different peaks were characterised, and their relative abundance was found to vary depending on the stage of oocyte meiotic maturation. To identify these endogenous biomolecules, top-down workflow using high resolution MS/MS (TD HR-MS) was performed on the protein extracts from oocytes, CC and GC. The TD HR-MS proteomic approach allowed for: (1) identification of 386 peptide/proteoforms encoded by 194 genes; and (2) characterisation of proteolysis products likely resulting from the action of kallikreins and caspases. In total, 136 peaks observed by ICM-MS were annotated by TD HR-MS (ProteomeXchange PXD004892). Among these, 16 markers of maturation were identified, including IGF2 binding protein 3 and hemoglobin B in the oocyte, thymosins beta-4 and beta-10, histone H2B and ubiquitin in CC. The combination of ICM-MS and TD HR-MS proved to be a suitable strategy to identify non-invasive markers of oocyte quality using limited biological samples.
  
  
  
  Intact cell MALDI-TOF mass spectrometry on single oocytes and their surrounding cumulus cells, coupled to an optimised top-down HR-MS proteomic approach on ovarian follicular cells, was used to identify specific markers of oocyte meiotic maturation represented by whole low molecular weight proteins or products of degradation by specific proteases.
authors.affiliation: UMR PRC, INRA 85, CNRS, Université de Tours, IFCE, 37380 Nouzilly, France; INRA, Plateforme d'Analyse Intégrative des Biomolécules, Laboratoire de Spectrométrie de Masse, 37380 Nouzilly, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Labas
authors.affiliation: UMR ISP, INRA, Université de Tours, 37380 Nouzilly, France; INRA, Plateforme d'Analyse Intégrative des Biomolécules, Laboratoire de Spectrométrie de Masse, 37380 Nouzilly, France.
authors.forename: Ana-Paula
authors.initials: AP
authors.lastname: Teixeira-Gomes
authors.affiliation: UMR ISP, INRA, Université de Tours, 37380 Nouzilly, France; INRA, Plateforme d'Analyse Intégrative des Biomolécules, Laboratoire de Spectrométrie de Masse, 37380 Nouzilly, France.
authors.forename: Laura
authors.initials: L
authors.lastname: Bouguereau
authors.affiliation: UMR PRC, INRA 85, CNRS, Université de Tours, IFCE, 37380 Nouzilly, France; INRA, Plateforme d'Analyse Intégrative des Biomolécules, Laboratoire de Spectrométrie de Masse, 37380 Nouzilly, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Gargaros
authors.affiliation: INRA, Plateforme d'Analyse Intégrative des Biomolécules, Laboratoire de Spectrométrie de Masse, 37380 Nouzilly, France; INSA/CNRS 5504 - UMR INSA/INRA 792, Toulouse, France.
authors.forename: Lucie
authors.initials: L
authors.lastname: Spina
authors.affiliation: UMR PRC, INRA 85, CNRS, Université de Tours, IFCE, 37380 Nouzilly, France; INRA, Plateforme d'Analyse Intégrative des Biomolécules, Laboratoire de Spectrométrie de Masse, 37380 Nouzilly, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Marestaing
authors.affiliation: UMR PRC, INRA 85, CNRS, Université de Tours, IFCE, 37380 Nouzilly, France; INRA, Plateforme d'Analyse Intégrative des Biomolécules, Laboratoire de Spectrométrie de Masse, 37380 Nouzilly, France. Electronic address: svetlana.uzbekova@inra.fr.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Uzbekova
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002417:Cattle; D054885:Cumulus Cells / Q000737:chemistry; D005260:Female; D006107:Granulosa Cells / Q000737:chemistry; D008540:Meiosis; D009865:Oocytes / Q000737:chemistry / Q000166:cytology*; D006080:Ovarian Follicle / Q000737:chemistry / Q000166:cytology*; D040901:Proteomics / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D015415:Biomarkers
keywords: Bovine; Cumulus cells; MALDI-TOF mass spectrometry; Meiotic maturation; Oocyte; Top-down proteomics