title: Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes.
abstract: Oocytes undergo a range of complex processes via oogenesis, maturation, fertilization, and early embryonic development, eventually giving rise to a fully functioning organism. To understand proteome composition and diversity during maturation of human oocytes, here we have addressed crucial aspects of oocyte collection and proteome analysis, resulting in the first proteome and secretome maps of human oocytes. Starting from 100 oocytes collected via a novel serum-free hanging drop culture system, we identified 2,154 proteins, whose function indicate that oocytes are largely resting cells with a proteome that is tailored for homeostasis, cellular attachment, and interaction with its environment via secretory factors. In addition, we have identified 158 oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)(1) not observed in high-coverage proteomics studies of other human cell lines or tissues. Exploiting SP3, a novel technology for proteomic sample preparation using magnetic beads, we scaled down proteome analysis to single cells. Despite the low protein content of only ∼100 ng per cell, we consistently identified ∼450 proteins from individual oocytes. When comparing individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage, we found that the Tudor and KH domain-containing protein (TDRKH) is preferentially expressed in immature oocytes, while Wee2, PCNA, and DNMT1 were enriched in mature cells, collectively indicating that maintenance of genome integrity is crucial during oocyte maturation. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and preimplantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types. Data associated with this study are available via ProteomeXchange with identifier PXD004142.
authors.affiliation: From the ‡Reproductive Unit, Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, Slajmerjeva 3, 1000 Ljubljana, Slovenia;
authors.forename: Irma
authors.initials: I
authors.lastname: Virant-Klun
authors.affiliation: §European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany;
authors.forename: Stefan
authors.initials: S
authors.lastname: Leicht
authors.affiliation: ¶British Columbia Cancer Research Agency, 675 West 10th Avenue, Vancouver, Canada;
authors.forename: Christopher
authors.initials: C
authors.lastname: Hughes
authors.affiliation: §European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany; ‖German Cancer Research Center and Heidelberg University, Im Neuenheimer Feld 581, 69120, Heidelberg, Germany irma.virant@kclj.si j.krijgsveld@dkfz.de.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Krijgsveld
mesh_terms: D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D009928:Organ Specificity; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*; D053719:Tandem Mass Spectrometry