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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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Comparison of proteomic workflows for blastocoel analysis and development of targeted proteomic assays for single embryo protein quantificationNumber of peptides identified using different sample preparation methods (black: solvent-based, MonoPrep; gray: urea-based, standard) on cytosolic lysates of HeLa cells at different concentrations (100, 1,000 and 10,000 cells corresponding to an estimated protein amount of 8, 80 and 800 ng, respectively).Validation of ten SRM assays on samples of five blastocoels. Batched samples obtained from 5 blastocoels were spiked with isotopically labeled versions of the target peptides. SRM assays were manually validated on the basis of the co-elution and relative intensities of at least 4 transitions per assay. Transition traces are displayed using their relative intensities (with the intensity of the highest transition per assay being set to 1). Positive values are used for endogenous peptides, while negative values are used for reference (synthetic) peptides. Horizontal axis displays peptide retention time (rt) in minutes.Intensity calibration curve derived from 76 absolutely quantified (AQUA) peptides measured at different concentrations. The linear regression between log10-transformed maximum apex intensity (derived from the maximum of the most intense transition trace) and log10-transformed attomoles of peptide injected was used to transform SRM-derived peptide intensities for blastocoel proteins into absolute abundances.
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fig05ev: Comparison of proteomic workflows for blastocoel analysis and development of targeted proteomic assays for single embryo protein quantificationNumber of peptides identified using different sample preparation methods (black: solvent-based, MonoPrep; gray: urea-based, standard) on cytosolic lysates of HeLa cells at different concentrations (100, 1,000 and 10,000 cells corresponding to an estimated protein amount of 8, 80 and 800 ng, respectively).Validation of ten SRM assays on samples of five blastocoels. Batched samples obtained from 5 blastocoels were spiked with isotopically labeled versions of the target peptides. SRM assays were manually validated on the basis of the co-elution and relative intensities of at least 4 transitions per assay. Transition traces are displayed using their relative intensities (with the intensity of the highest transition per assay being set to 1). Positive values are used for endogenous peptides, while negative values are used for reference (synthetic) peptides. Horizontal axis displays peptide retention time (rt) in minutes.Intensity calibration curve derived from 76 absolutely quantified (AQUA) peptides measured at different concentrations. The linear regression between log10-transformed maximum apex intensity (derived from the maximum of the most intense transition trace) and log10-transformed attomoles of peptide injected was used to transform SRM-derived peptide intensities for blastocoel proteins into absolute abundances.

Mentions: Blastosol samples were collected using blastocentesis, allowing the collection of 4–6 nL from each blastocyst cavity (Materials and Methods) (Movie EV1). Samples were initially processed using a standard procedure involving urea for protein solubilization and peptide desalting following enzymatic digestion. However, this approach was only compatible with pooled samples containing at least 20 blastocoels. For smaller samples, the majority of the peptides were lost during the multiple steps of the procedure. Therefore, a previously described nano-scale protocol was adapted for the analysis of individual blastocoel samples (Wang et al, 2005). This methodology utilized volatile buffers and organic solvents and had no requirement for peptide desalting prior to MS analysis (referred to as MonoPrep). In preliminary experiments, the MonoPrep procedure allowed the detection of more than 2,000 peptides comprising as little as 80 ng of proteins (cytoplasmic extract obtained from ∼1,000 HeLa cells) (FigEV1A). We performed shotgun proteomics analysis on 80 blastocoels sampled from human embryos 5 or 6 days after fertilization. The samples were divided into four sets each composed of 20 pooled specimens. Two sets were processed using the standard urea-based procedure and two with MonoPrep. For both procedures, blank samples obtained from plated sterile PBS microdrops swiped with a microneedle of the same type used for blastosol collection were analyzed in parallel and treated as controls for the identification of contaminant proteins.


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)

Comparison of proteomic workflows for blastocoel analysis and development of targeted proteomic assays for single embryo protein quantificationNumber of peptides identified using different sample preparation methods (black: solvent-based, MonoPrep; gray: urea-based, standard) on cytosolic lysates of HeLa cells at different concentrations (100, 1,000 and 10,000 cells corresponding to an estimated protein amount of 8, 80 and 800 ng, respectively).Validation of ten SRM assays on samples of five blastocoels. Batched samples obtained from 5 blastocoels were spiked with isotopically labeled versions of the target peptides. SRM assays were manually validated on the basis of the co-elution and relative intensities of at least 4 transitions per assay. Transition traces are displayed using their relative intensities (with the intensity of the highest transition per assay being set to 1). Positive values are used for endogenous peptides, while negative values are used for reference (synthetic) peptides. Horizontal axis displays peptide retention time (rt) in minutes.Intensity calibration curve derived from 76 absolutely quantified (AQUA) peptides measured at different concentrations. The linear regression between log10-transformed maximum apex intensity (derived from the maximum of the most intense transition trace) and log10-transformed attomoles of peptide injected was used to transform SRM-derived peptide intensities for blastocoel proteins into absolute abundances.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4644378&req=5

fig05ev: Comparison of proteomic workflows for blastocoel analysis and development of targeted proteomic assays for single embryo protein quantificationNumber of peptides identified using different sample preparation methods (black: solvent-based, MonoPrep; gray: urea-based, standard) on cytosolic lysates of HeLa cells at different concentrations (100, 1,000 and 10,000 cells corresponding to an estimated protein amount of 8, 80 and 800 ng, respectively).Validation of ten SRM assays on samples of five blastocoels. Batched samples obtained from 5 blastocoels were spiked with isotopically labeled versions of the target peptides. SRM assays were manually validated on the basis of the co-elution and relative intensities of at least 4 transitions per assay. Transition traces are displayed using their relative intensities (with the intensity of the highest transition per assay being set to 1). Positive values are used for endogenous peptides, while negative values are used for reference (synthetic) peptides. Horizontal axis displays peptide retention time (rt) in minutes.Intensity calibration curve derived from 76 absolutely quantified (AQUA) peptides measured at different concentrations. The linear regression between log10-transformed maximum apex intensity (derived from the maximum of the most intense transition trace) and log10-transformed attomoles of peptide injected was used to transform SRM-derived peptide intensities for blastocoel proteins into absolute abundances.
Mentions: Blastosol samples were collected using blastocentesis, allowing the collection of 4–6 nL from each blastocyst cavity (Materials and Methods) (Movie EV1). Samples were initially processed using a standard procedure involving urea for protein solubilization and peptide desalting following enzymatic digestion. However, this approach was only compatible with pooled samples containing at least 20 blastocoels. For smaller samples, the majority of the peptides were lost during the multiple steps of the procedure. Therefore, a previously described nano-scale protocol was adapted for the analysis of individual blastocoel samples (Wang et al, 2005). This methodology utilized volatile buffers and organic solvents and had no requirement for peptide desalting prior to MS analysis (referred to as MonoPrep). In preliminary experiments, the MonoPrep procedure allowed the detection of more than 2,000 peptides comprising as little as 80 ng of proteins (cytoplasmic extract obtained from ∼1,000 HeLa cells) (FigEV1A). We performed shotgun proteomics analysis on 80 blastocoels sampled from human embryos 5 or 6 days after fertilization. The samples were divided into four sets each composed of 20 pooled specimens. Two sets were processed using the standard urea-based procedure and two with MonoPrep. For both procedures, blank samples obtained from plated sterile PBS microdrops swiped with a microneedle of the same type used for blastosol collection were analyzed in parallel and treated as controls for the identification of contaminant proteins.

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