22 research outputs found

    A phosphoproteomics view at human pluripotent stem cells

    No full text
    Reversible protein phosphorylation has been one of the most investigated post-translational modifications (PTM) in the decade, made possible by advances in enrichment technologies and MS-based proteomics. PTMs are generally involved in the regulation of many biological processes, including processes that regulate pluripotency and self-renewal of stem cells. In recent years, several enrichment methods have been introduced that helped to improve the detection and quantitation of phosphorylated peptides by MS enabling the study of phosphorylation dynamics in cells and tissues. The work described in this thesis is focused on the application of a selection of such proteomic strategies, for the investigation of phosphorylation events in pluripotent stem cells

    Finding the same needles in the haystack? A comparison of phosphotyrosine peptides enriched by immuno-affinity precipitation and metal-based affinity chromatography

    No full text
    Analysis of tyrosine (Tyr) phosphorylation by mass spectrometry (MS)-based proteomics remains challenging, due to the low occurrence of this post-translational modification compared to serine and threonine phosphorylation events in mammalian systems. Conventional metal-based affinity chromatography methods used to enrich phosphopeptides can nowadays isolate over 10,000 phosphopeptides. However, these approaches are not particularly suitable for the selective enrichment of low abundant Tyr phosphorylated peptides as the higher abundant co-enriched serine (Ser) and threonine (Thr) phosphorylated peptides typically obscure their detection. Therefore, a more targeted approach based on immuno-affinity precipitation at the peptide level has been introduced for the specific analysis of Tyr phosphorylated species. This method typically leads to the detection of a few hundreds of phosphopeptides, albeit typically over 70% of those are Tyr phosphorylated. Here, we evaluated and compared phosphotyrosine peptides enriched by a phospho-Tyr immuno-affinity enrichment (employing pY99 antibodies) and a multidimensional approach consisting of metal–affinity based enrichment (Ti4 +-IMAC) followed by hydrophilic interaction liquid chromatography (HILIC) fractionation. Our aim was to assess differences and similarities in the set of Tyr phosphorylated peptides detected by each approach. Our data suggest that both strategies are not redundant but complementary and should ideally be combined for a more comprehensive view at phosphotyrosine signaling

    Finding the same needles in the haystack? A comparison of phosphotyrosine peptides enriched by immuno-affinity precipitation and metal-based affinity chromatography

    No full text
    Analysis of tyrosine (Tyr) phosphorylation by mass spectrometry (MS)-based proteomics remains challenging, due to the low occurrence of this post-translational modification compared to serine and threonine phosphorylation events in mammalian systems. Conventional metal-based affinity chromatography methods used to enrich phosphopeptides can nowadays isolate over 10,000 phosphopeptides. However, these approaches are not particularly suitable for the selective enrichment of low abundant Tyr phosphorylated peptides as the higher abundant co-enriched serine (Ser) and threonine (Thr) phosphorylated peptides typically obscure their detection. Therefore, a more targeted approach based on immuno-affinity precipitation at the peptide level has been introduced for the specific analysis of Tyr phosphorylated species. This method typically leads to the detection of a few hundreds of phosphopeptides, albeit typically over 70% of those are Tyr phosphorylated. Here, we evaluated and compared phosphotyrosine peptides enriched by a phospho-Tyr immuno-affinity enrichment (employing pY99 antibodies) and a multidimensional approach consisting of metal–affinity based enrichment (Ti4 +-IMAC) followed by hydrophilic interaction liquid chromatography (HILIC) fractionation. Our aim was to assess differences and similarities in the set of Tyr phosphorylated peptides detected by each approach. Our data suggest that both strategies are not redundant but complementary and should ideally be combined for a more comprehensive view at phosphotyrosine signaling

    Bacterial homologs of innate eukaryotic antiviral defenses with anti-phage activity highlight shared evolutionary roots of viral defenses

    No full text
    Prokaryotes have evolved a multitude of defense systems to protect against phage predation. Some of these resemble eukaryotic genes involved in antiviral responses. Here, we set out to systematically project the current knowledge of eukaryotic-like antiviral defense systems onto prokaryotic genomes, using Pseudomonas aeruginosa as a model organism. Searching for phage defense systems related to innate antiviral genes from vertebrates and plants, we uncovered over 450 candidates. We validated six of these phage defense systems, including factors preventing viral attachment, R-loop-acting enzymes, the inflammasome, ubiquitin pathway, and pathogen recognition signaling. Collectively, these defense systems support the concept of deep evolutionary links and shared antiviral mechanisms between prokaryotes and eukaryotes
    corecore