Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this propert... more Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human...
The holistic nature of omics studies makes them ideally suited to generate hypotheses on health a... more The holistic nature of omics studies makes them ideally suited to generate hypotheses on health and disease. Sequencing-based genomics and mass spectrometry (MS)-based proteomics are linked through epigenetic regulation mechanisms. However, epigenomics is currently mainly focused on DNA methylation status using sequencing technologies, while studying histone posttranslational modifications (hPTMs) using MS is lagging, partly because reuse of raw data is impractical. Yet, targeting hPTMs using epidrugs is an established promising research avenue in cancer treatment. Therefore, we here present the most comprehensive MS-based preprocessed hPTM atlas to date, including 21 T-cell acute lymphoblastic leukemia (T-ALL) cell lines. We present the data in an intuitive and browsable single licensed Progenesis QIP project and provide all essential quality metrics, allowing users to assess the quality of the data, edit individual peptides, try novel annotation algorithms and export both peptide ...
INTRODUCTIONThe pandemic readiness toolbox needs to be extended, providing diagnostic tools that ... more INTRODUCTIONThe pandemic readiness toolbox needs to be extended, providing diagnostic tools that target different biomolecules, using orthogonal experimental setups and fit-for-purpose specification of detection. Here we build on a previous Cov-MS effort that used liquid chromatography-mass spectrometry (LC-MS) and describe a method that allows accurate, high throughput measurement of SARS-CoV-2 nucleocapsid (N) protein.MATERIALS and METHODSWe used Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) technology to enrich and quantify proteotypic peptides of the N protein from trypsin-digested samples from COVID-19 patients.RESULTSThe Cov2MS assay was shown to be compatible with a variety of sample matrices including nasopharyngeal swabs, saliva and blood plasma and increased the sensitivity into the attomole range, up to a 1000-fold increase compared to direct detection in matrix. In addition, a strong positive correlation was observed between the SISCAPA antige...
ICs were isolated from serum of RA patients (n=20) and healthy persons (n=6) by a sucrosegradient... more ICs were isolated from serum of RA patients (n=20) and healthy persons (n=6) by a sucrosegradient (20-65%) and ultracentrifugation. The sucrosegradient was fractionated and the different fractions were submitted to a spectrophotometric analysis at 280nm. The fractions with ICs underwent further purification by immunoprecipitation with immobilized protein G and anti-IgM. Analysis of the ICs was carried out by SDS-PAGE and Western Blotting. Possible citrullination of the proteins was investigated by AMC staining, and mass spectrometry was used to identify the proteins.
A collection of 215 specimens of different stadia of the African millipede Epibolus pulchripes al... more A collection of 215 specimens of different stadia of the African millipede Epibolus pulchripes allowed studying its post-embryonic development, with special attention drawn to (i) the number of segments and ocelli at each stadium and (ii) the development of the copulatory organs of males and females. Laboratory rearing of adult E. pulchripes from Kenya revealed some aspects of reproduction, from the structure of the adult reproductive organs to mating behaviour and oviposition, as well as the first stadia of post-embryonic development. Based on histological observations and scanning electron microscopy, the position of the copulatory organs during mating and the function of the different parts of the reproductive organs are hypothesized.
Background: Although the association of the MHC class I allele HLA-B27 with Spondyloarthropathy (... more Background: Although the association of the MHC class I allele HLA-B27 with Spondyloarthropathy (SpA) has been known for almost 35 years different hypotheses on its relation to disease mechanism still exist in parallel. Several lines of rats transgenic for HLA-B27 and human β2-microglobulin develop an inflammatory disease that strikingly resembles human SpA. It is hypothesized that disease in HLA-B27- transgenic rats arises as a consequence of interaction between antigen-presenting cells expressing high levels of HLA-B27 and peripheral T lymphocytes, and may result from a rupture of tolerance towards gut bacteria. Methods: We used 2D PAGE and iTRAQ to compare the protein expression profile of HLA-B27 dendritic cells (DCs) to that of healthy HLAB7 expressing and nontransgenic (NTG) rat DCs. MHC II surface expression and apoptotic sensitivity were quantified using flow cytometry. Results: Three protein sets from the proteome analysis were indicative for aberrant cellular processes. Fi...
Chikungunya virus (CHIKV), a positive stranded RNA alphavirus, recently reemerged causing multipl... more Chikungunya virus (CHIKV), a positive stranded RNA alphavirus, recently reemerged causing multiple outbreaks around the world. Generally, alphaviruses enter the cell via clathrin-mediated endocytosis. Entry is supported by the structural envelope proteins E2 and E1. Different experimental approaches have been applied previously to identify receptor(s) molecules responsible for CHIKV binding and entry in human and mosquito cells. However they cannot account for all CHIKV entry events in all susceptible cell types. We performed affinity purification coupled to mass spectrometry to identify entry factors of CHIKV in both human and mosquito cells. We transiently expressed the N-terminally Strep-tagged, full-length envelope gene in 293T and C6/36 cells. Affinity purifications were digested on-bead and analyzed by mass spectrometry. MiST analysis allowed the identification of 39 human proteins with a confidence score above 0.8. Twelve proteins were selected for validation with CRISPR/Cas9 knock-out cells. Three separate AP-MS experiments in C6/36 cells led to the identification of 31, 58 and 11 proteins with a MiST score higher than 0.7, of which 19 proteins were chosen for further analysis. Using knock-out experiments in 293T cells and a reporter CHIKV (ECSA strain) resulted in the identification of the CD147-CD98 protein complex on human cells as possible entry factor. Repetition of this knock-out experiment using an Asian CHIKV strain combined with E2 staining, confirmed these results. CD147 contains 2 immunoglobulin domains which is similar to MXRA8, a previously identified alphavirus entry factor. The interaction of CD147 with E2 was validated on Western Blot after affinity purification.
Data-independent acquisition (DIA) mass spectrometry (MS) has introduced deter-ministic, periodic... more Data-independent acquisition (DIA) mass spectrometry (MS) has introduced deter-ministic, periodic and simultaneous acquisition of all fragment ions. Despite the chimeric side-effects associated with this unprecedented data integrity, DIA data analysis approaches still use conventional spectra and extracted ion chromatograms (XICs) that represent individual precursors and fragments. Here, we introduce ion-networks, an alternative data format wherein nodes correspond to reproducible fragment ions from multiple runs and edges correspond to consistent co-elution. Each ion-network represents a complete experiment and computationally eliminates chimericy based on reproducibility without sacrificing data integrity.
Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on prot... more Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms, such as histone post-translational modifications (hPTMs). Due to substantial progress in the large-scale study of hPTMs, integration into the field of toxicology is promising and offers the opportunity to gain novel insights into toxicological phenomena. Moreover, there is a growing demand for high-throughput human-based in vitro assays for toxicity testing, especially for developmental toxicity. Consequently, we developed a mass spectrometry-based proof-of-concept to assess a histone code screening assay capable of simultaneously detecting multiple hPTM-changes in human embryonic stem cells. We first validated the untargeted workflow with valproic acid (VPA), a histone deacetylase inhibitor. These results demonstrate the capability of mapping the hPTM-dynamics, with a general increase in acetylations as an internal control. To il...
At the dawn of a new era in label-free quantitation on high-resolutionMS instruments, classical m... more At the dawn of a new era in label-free quantitation on high-resolutionMS instruments, classical methods such as iTRAQ continue to provide very useful insights in comparative proteomics. The potential to multiplex samples makes this reporter-based labeling technique highly suited for method optimization as demonstrated here by a set of standard series. Instead of studying ratios of annotated proteins, we propose an alternative method, based on the analysis of the average reporter ratios of all the spectra from a sample or a large distinct subset herein. This strategy circumvents the bias, associated with the annotation and iTRAQ quantitation, leading to increased adequacy inmeasuring yield differences betweenworkflows. As gel electrophoresis prior to MS analysis is highly beneficial, for example, as a fractionation step, the approach was applied to evaluate the influence of several parameters of the established in-gel digestion protocol. We quantified the negative effect of SYPRO Rub...
Histone clipping was first discovered in the 1960s and still is a lingering mystery. Considering ... more Histone clipping was first discovered in the 1960s and still is a lingering mystery. Considering the essential roles of histones in regulating eukaryotic transcription through the histone code, clipping is a post‐translational modification that appeals to the imagination. In this issue of EMBO Reports, Marruecos and colleagues investigate histone H4 clipping during intestinal development (Marruecos et al, 2021), and are providing crucial clues to finally elucidate the intricacies of this elusive modification.
Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this propert... more Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human...
The holistic nature of omics studies makes them ideally suited to generate hypotheses on health a... more The holistic nature of omics studies makes them ideally suited to generate hypotheses on health and disease. Sequencing-based genomics and mass spectrometry (MS)-based proteomics are linked through epigenetic regulation mechanisms. However, epigenomics is currently mainly focused on DNA methylation status using sequencing technologies, while studying histone posttranslational modifications (hPTMs) using MS is lagging, partly because reuse of raw data is impractical. Yet, targeting hPTMs using epidrugs is an established promising research avenue in cancer treatment. Therefore, we here present the most comprehensive MS-based preprocessed hPTM atlas to date, including 21 T-cell acute lymphoblastic leukemia (T-ALL) cell lines. We present the data in an intuitive and browsable single licensed Progenesis QIP project and provide all essential quality metrics, allowing users to assess the quality of the data, edit individual peptides, try novel annotation algorithms and export both peptide ...
INTRODUCTIONThe pandemic readiness toolbox needs to be extended, providing diagnostic tools that ... more INTRODUCTIONThe pandemic readiness toolbox needs to be extended, providing diagnostic tools that target different biomolecules, using orthogonal experimental setups and fit-for-purpose specification of detection. Here we build on a previous Cov-MS effort that used liquid chromatography-mass spectrometry (LC-MS) and describe a method that allows accurate, high throughput measurement of SARS-CoV-2 nucleocapsid (N) protein.MATERIALS and METHODSWe used Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) technology to enrich and quantify proteotypic peptides of the N protein from trypsin-digested samples from COVID-19 patients.RESULTSThe Cov2MS assay was shown to be compatible with a variety of sample matrices including nasopharyngeal swabs, saliva and blood plasma and increased the sensitivity into the attomole range, up to a 1000-fold increase compared to direct detection in matrix. In addition, a strong positive correlation was observed between the SISCAPA antige...
ICs were isolated from serum of RA patients (n=20) and healthy persons (n=6) by a sucrosegradient... more ICs were isolated from serum of RA patients (n=20) and healthy persons (n=6) by a sucrosegradient (20-65%) and ultracentrifugation. The sucrosegradient was fractionated and the different fractions were submitted to a spectrophotometric analysis at 280nm. The fractions with ICs underwent further purification by immunoprecipitation with immobilized protein G and anti-IgM. Analysis of the ICs was carried out by SDS-PAGE and Western Blotting. Possible citrullination of the proteins was investigated by AMC staining, and mass spectrometry was used to identify the proteins.
A collection of 215 specimens of different stadia of the African millipede Epibolus pulchripes al... more A collection of 215 specimens of different stadia of the African millipede Epibolus pulchripes allowed studying its post-embryonic development, with special attention drawn to (i) the number of segments and ocelli at each stadium and (ii) the development of the copulatory organs of males and females. Laboratory rearing of adult E. pulchripes from Kenya revealed some aspects of reproduction, from the structure of the adult reproductive organs to mating behaviour and oviposition, as well as the first stadia of post-embryonic development. Based on histological observations and scanning electron microscopy, the position of the copulatory organs during mating and the function of the different parts of the reproductive organs are hypothesized.
Background: Although the association of the MHC class I allele HLA-B27 with Spondyloarthropathy (... more Background: Although the association of the MHC class I allele HLA-B27 with Spondyloarthropathy (SpA) has been known for almost 35 years different hypotheses on its relation to disease mechanism still exist in parallel. Several lines of rats transgenic for HLA-B27 and human β2-microglobulin develop an inflammatory disease that strikingly resembles human SpA. It is hypothesized that disease in HLA-B27- transgenic rats arises as a consequence of interaction between antigen-presenting cells expressing high levels of HLA-B27 and peripheral T lymphocytes, and may result from a rupture of tolerance towards gut bacteria. Methods: We used 2D PAGE and iTRAQ to compare the protein expression profile of HLA-B27 dendritic cells (DCs) to that of healthy HLAB7 expressing and nontransgenic (NTG) rat DCs. MHC II surface expression and apoptotic sensitivity were quantified using flow cytometry. Results: Three protein sets from the proteome analysis were indicative for aberrant cellular processes. Fi...
Chikungunya virus (CHIKV), a positive stranded RNA alphavirus, recently reemerged causing multipl... more Chikungunya virus (CHIKV), a positive stranded RNA alphavirus, recently reemerged causing multiple outbreaks around the world. Generally, alphaviruses enter the cell via clathrin-mediated endocytosis. Entry is supported by the structural envelope proteins E2 and E1. Different experimental approaches have been applied previously to identify receptor(s) molecules responsible for CHIKV binding and entry in human and mosquito cells. However they cannot account for all CHIKV entry events in all susceptible cell types. We performed affinity purification coupled to mass spectrometry to identify entry factors of CHIKV in both human and mosquito cells. We transiently expressed the N-terminally Strep-tagged, full-length envelope gene in 293T and C6/36 cells. Affinity purifications were digested on-bead and analyzed by mass spectrometry. MiST analysis allowed the identification of 39 human proteins with a confidence score above 0.8. Twelve proteins were selected for validation with CRISPR/Cas9 knock-out cells. Three separate AP-MS experiments in C6/36 cells led to the identification of 31, 58 and 11 proteins with a MiST score higher than 0.7, of which 19 proteins were chosen for further analysis. Using knock-out experiments in 293T cells and a reporter CHIKV (ECSA strain) resulted in the identification of the CD147-CD98 protein complex on human cells as possible entry factor. Repetition of this knock-out experiment using an Asian CHIKV strain combined with E2 staining, confirmed these results. CD147 contains 2 immunoglobulin domains which is similar to MXRA8, a previously identified alphavirus entry factor. The interaction of CD147 with E2 was validated on Western Blot after affinity purification.
Data-independent acquisition (DIA) mass spectrometry (MS) has introduced deter-ministic, periodic... more Data-independent acquisition (DIA) mass spectrometry (MS) has introduced deter-ministic, periodic and simultaneous acquisition of all fragment ions. Despite the chimeric side-effects associated with this unprecedented data integrity, DIA data analysis approaches still use conventional spectra and extracted ion chromatograms (XICs) that represent individual precursors and fragments. Here, we introduce ion-networks, an alternative data format wherein nodes correspond to reproducible fragment ions from multiple runs and edges correspond to consistent co-elution. Each ion-network represents a complete experiment and computationally eliminates chimericy based on reproducibility without sacrificing data integrity.
Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on prot... more Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms, such as histone post-translational modifications (hPTMs). Due to substantial progress in the large-scale study of hPTMs, integration into the field of toxicology is promising and offers the opportunity to gain novel insights into toxicological phenomena. Moreover, there is a growing demand for high-throughput human-based in vitro assays for toxicity testing, especially for developmental toxicity. Consequently, we developed a mass spectrometry-based proof-of-concept to assess a histone code screening assay capable of simultaneously detecting multiple hPTM-changes in human embryonic stem cells. We first validated the untargeted workflow with valproic acid (VPA), a histone deacetylase inhibitor. These results demonstrate the capability of mapping the hPTM-dynamics, with a general increase in acetylations as an internal control. To il...
At the dawn of a new era in label-free quantitation on high-resolutionMS instruments, classical m... more At the dawn of a new era in label-free quantitation on high-resolutionMS instruments, classical methods such as iTRAQ continue to provide very useful insights in comparative proteomics. The potential to multiplex samples makes this reporter-based labeling technique highly suited for method optimization as demonstrated here by a set of standard series. Instead of studying ratios of annotated proteins, we propose an alternative method, based on the analysis of the average reporter ratios of all the spectra from a sample or a large distinct subset herein. This strategy circumvents the bias, associated with the annotation and iTRAQ quantitation, leading to increased adequacy inmeasuring yield differences betweenworkflows. As gel electrophoresis prior to MS analysis is highly beneficial, for example, as a fractionation step, the approach was applied to evaluate the influence of several parameters of the established in-gel digestion protocol. We quantified the negative effect of SYPRO Rub...
Histone clipping was first discovered in the 1960s and still is a lingering mystery. Considering ... more Histone clipping was first discovered in the 1960s and still is a lingering mystery. Considering the essential roles of histones in regulating eukaryotic transcription through the histone code, clipping is a post‐translational modification that appeals to the imagination. In this issue of EMBO Reports, Marruecos and colleagues investigate histone H4 clipping during intestinal development (Marruecos et al, 2021), and are providing crucial clues to finally elucidate the intricacies of this elusive modification.
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Papers by Maarten Dhaenens