Characterization and quantification of proteins secreted by single human embryos prior to implantation.
Bottom Line: By using targeted proteomics, we demonstrate the feasibility of quantifying multiple proteins in samples derived from single blastocoels and that such measurements correlate with aspects of embryo viability, such as chromosomal (ploidy) status.This study illustrates the potential of high-sensitivity proteomics to measure clinically relevant biomarkers in minute samples and, more specifically, suggests that key aspects of embryo competence could be measured using a proteomic-based strategy, with negligible risk of harm to the living embryo.Our work paves the way for the development of "next-generation" embryo competence assessment strategies, based on functional proteomics.
Affiliation: Nuffield Department of Obstetrics and Gynaecology, Institute of Reproductive Sciences University of Oxford, Oxford, UK Oxford Fertility Unit, Institute of Reproductive Sciences, Oxford, UK Reprogenetics UK, Institute of Reproductive Sciences, Oxford, UK.Show MeSH
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Mentions: In order to enable single blastocoel analysis, we developed selected reaction monitoring (SRM) assays for 10 peptides (corresponding to 9 protein groups) that were selected from among the most abundant peptides identified in the shotgun experiments (Fig3A). For assay development, spectral libraries generated from the discovery phase were used and employed a procedure previously described for peptide selection and assay development (Ori et al, 2014) (see Materials and Methods). Additional SRM assays aimed at identifying nuclear (H2A histone family) and cytoskeletal (ACTA) control targets were also developed. First, SRM assays using isotopically labeled synthetic peptides that were spiked in pools of blastosols obtained from 5 embryos were tested and successfully validated (Fig EV1B and Table EV5). Using these assays, 21 single blastosol samples were screened and the 9 tested proteins were detected, thus demonstrating the ability of targeted proteomics to measure multiple protein species deriving from a single embryo blastocoel (Tables EV6 and EV7). In order to estimate the concentration of the detected peptides in the blastosol, we first calibrated the signal intensity deriving from the mass spectrometer using a pool of absolutely quantified (AQUA) peptides derived from human proteins (Ori et al, 2013) that were injected in different known amounts (FigEV1C). The concentration of embryo-derived peptides was then estimated in the blastocoel fluid by linear regression using the fitted equation deriving from AQUA peptides. The concentration of the detected target proteins spans approximately one order of magnitude, ranging from 13 ng/μl (GAPDH) to 0.8 ng/μl (ECAT1) (Fig3B). For 16 blastocoels, multiple peptides were successfully detected (3.3 proteins per sample on average).
Affiliation: Nuffield Department of Obstetrics and Gynaecology, Institute of Reproductive Sciences University of Oxford, Oxford, UK Oxford Fertility Unit, Institute of Reproductive Sciences, Oxford, UK Reprogenetics UK, Institute of Reproductive Sciences, Oxford, UK.