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Characterization and quantification of proteins secreted by single human embryos prior to implantation.

Poli M, Ori A, Child T, Jaroudi S, Spath K, Beck M, Wells D - EMBO Mol Med (2015)

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.

View Article: PubMed Central - PubMed

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.

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Targeted proteomics analysis of single human blastocoelsProtein abundance profile of 20 pooled blastocoels. SRM assays were developed for the proteins shown in red.Summary of estimated protein abundances from measurements of 9 targets in 21 single blastocoel fluids. Absolute protein amounts were estimated from peptide intensities upon calibration using a set of absolutely quantified (AQUA) peptides (FigEV1B, see Materials and Methods for details). Blue dots correspond to individual data points. Black vertical bars indicate median values. Grey boxes show inter quartile ranges (IQR, Q3–Q1). Whiskers show Q1–1.5×IQR and Q3+1.5×IQR ranges.GAPDH abundances per single blastocoel grouped based on embryo chromosomal status. P-values were calculated with Mann–Whitney U-test. Black bar: mean value; gray bars: SEM.Measurements of GAPDH and detection of H2A family proteins in samples derived from 14 single blastocoels and chromosomal status of the embryo. The presence or absence of embryo aneuploidy was assessed by aCGH.
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fig03: Targeted proteomics analysis of single human blastocoelsProtein abundance profile of 20 pooled blastocoels. SRM assays were developed for the proteins shown in red.Summary of estimated protein abundances from measurements of 9 targets in 21 single blastocoel fluids. Absolute protein amounts were estimated from peptide intensities upon calibration using a set of absolutely quantified (AQUA) peptides (FigEV1B, see Materials and Methods for details). Blue dots correspond to individual data points. Black vertical bars indicate median values. Grey boxes show inter quartile ranges (IQR, Q3–Q1). Whiskers show Q1–1.5×IQR and Q3+1.5×IQR ranges.GAPDH abundances per single blastocoel grouped based on embryo chromosomal status. P-values were calculated with Mann–Whitney U-test. Black bar: mean value; gray bars: SEM.Measurements of GAPDH and detection of H2A family proteins in samples derived from 14 single blastocoels and chromosomal status of the embryo. The presence or absence of embryo aneuploidy was assessed by aCGH.

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).


Characterization and quantification of proteins secreted by single human embryos prior to implantation.

Poli M, Ori A, Child T, Jaroudi S, Spath K, Beck M, Wells D - EMBO Mol Med (2015)

Targeted proteomics analysis of single human blastocoelsProtein abundance profile of 20 pooled blastocoels. SRM assays were developed for the proteins shown in red.Summary of estimated protein abundances from measurements of 9 targets in 21 single blastocoel fluids. Absolute protein amounts were estimated from peptide intensities upon calibration using a set of absolutely quantified (AQUA) peptides (FigEV1B, see Materials and Methods for details). Blue dots correspond to individual data points. Black vertical bars indicate median values. Grey boxes show inter quartile ranges (IQR, Q3–Q1). Whiskers show Q1–1.5×IQR and Q3+1.5×IQR ranges.GAPDH abundances per single blastocoel grouped based on embryo chromosomal status. P-values were calculated with Mann–Whitney U-test. Black bar: mean value; gray bars: SEM.Measurements of GAPDH and detection of H2A family proteins in samples derived from 14 single blastocoels and chromosomal status of the embryo. The presence or absence of embryo aneuploidy was assessed by aCGH.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4644378&req=5

fig03: Targeted proteomics analysis of single human blastocoelsProtein abundance profile of 20 pooled blastocoels. SRM assays were developed for the proteins shown in red.Summary of estimated protein abundances from measurements of 9 targets in 21 single blastocoel fluids. Absolute protein amounts were estimated from peptide intensities upon calibration using a set of absolutely quantified (AQUA) peptides (FigEV1B, see Materials and Methods for details). Blue dots correspond to individual data points. Black vertical bars indicate median values. Grey boxes show inter quartile ranges (IQR, Q3–Q1). Whiskers show Q1–1.5×IQR and Q3+1.5×IQR ranges.GAPDH abundances per single blastocoel grouped based on embryo chromosomal status. P-values were calculated with Mann–Whitney U-test. Black bar: mean value; gray bars: SEM.Measurements of GAPDH and detection of H2A family proteins in samples derived from 14 single blastocoels and chromosomal status of the embryo. The presence or absence of embryo aneuploidy was assessed by aCGH.
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).

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus