ABSTRACT: This set of profiles was utilized to construct, validate and evaluate a gene-expression based classifier of outcome of neuroblastoma patients
Project description:This set of 320 profiles was utilized to construct, validate and evaluate a gene-expression based classifier of outcome of neuroblastoma patients
Project description:This set of profiles was utilized to construct, validate and evaluate a gene-expression based classifier of outcome of neuroblastoma patients
Project description:Phenotypic plasticity, the ability of one genotype to express different phenotypes in response to changing environmental conditions, is one of the most common phenomena characterising the living world and is not only relevant for the ecology but also for the evolution of species. Daphnia, the waterflea, is a textbook example for predator induced phenotypic plastic defences including changes in life-history, behaviour and morphology. However, the analysis of molecular mechanisms underlying these inducible defences is still in its early stages.<br><br>We exposed Daphnia magna to chemical cues of the predator Triops cancriformis to identify key processes underlying plastic defensive trait formation. D. magna is known to develop an array of morphological changes in the presence of T. cancriformis including changes of carapace morphology and cuticle hardening. To get a more comprehensive idea of this phenomenon, we studied four different genotypes originating from habitats with different predation history, reaching from predator-free to temporary habitats containing T. cancriformis.<br><br>We analysed the morphologies as well as proteomes of predator-exposed and control animals. Three genotypes showed morphological changes when the predator was present. Using a high-throughput proteomics approach, we found 294 proteins which were significantly altered in their abundance after predator exposure in a general or genotype dependant manner. Proteins connected to genotype dependant responses were related to the cuticle, protein synthesis and calcium binding whereas the yolk protein vitellogenin increased in abundance in all genotypes, indicating their involvement in a more general response. Furthermore, genotype dependant responses at the proteome level correlated well with local adaptation to Triops predation.<br><br>Altogether, our study provides new insights concerning genotype dependant and general molecular processes involved in predator-induced phenotypic plasticity in D. magna.
Project description:Single-cell proteomics (SCP) holds the promise of decoding cellular heterogeneity at the functional level, yet achieving deep and reproducible proteome coverage from individual cells has remained a formidable challenge. Here, we establish a high-sensitivity, label-free SCP platform that extends current depth and reproducibility. By integrating optimized low-input sample processing, refined liquid chromatography, and the Orbitrap Astral Zoom mass spectrometer, our approach routinely quantifies up to 7,000 proteins per individual HeLa cell at over 100 samples-per-day (SPD), capturing thousands of low-abundance proteins that eluded prior SCP studies. Applied to very small human peripheral blood mononuclear cells (PBMCs), we identify up to 4,000 proteins per cell, including key markers that distinguish monocytes, T cells, and activated lymphocytes within heterogeneous populations, underscoring that single-cell proteomics can now directly elucidate clinically relevant primary samples with both depth and precision.
Project description:Single-cell proteomics (SCP) holds the promise of decoding cellular heterogeneity at the functional level, yet achieving deep and reproducible proteome coverage from individual cells has remained a formidable challenge. Here, we establish a high-sensitivity, label-free SCP platform that extends current depth and reproducibility. By integrating optimized low-input sample processing, refined liquid chromatography, and the Orbitrap Astral Zoom mass spectrometer, our approach routinely quantifies up to 7,000 proteins per individual HeLa cell at over 100 samples-per-day (SPD), capturing thousands of low-abundance proteins that eluded prior SCP studies. Applied to very small human peripheral blood mononuclear cells (PBMCs), we identify up to 4,000 proteins per cell, including key markers that distinguish monocytes, T cells, and activated lymphocytes within heterogeneous populations, underscoring that single-cell proteomics can now directly elucidate clinically relevant primary samples with both depth and precision.
Project description:ADP-ribosylation (ADPr) is a regulatory post-translational modification targeting nine amino acid residues, but glutamate/aspartate-linked ADPr (Glu/Asp-ADPr) is labile and remains challenging to detect using conventional mass spectrometry (MS)-based workflows. Using synthetic peptides, we show that ester-linked Glu/Asp-ADPr is lost under alkaline conditions, elevated temperatures, and by hydrolysis via wildtype Af1521. We developed an acidic enrichment workflow incorporating an Af1521 mutant that preserves Glu/Asp-ADPr, enabling site-specific, system-wide MS analysis. In cytokine-stimulated A549 and HeLa cells, we identified >600 Glu/Asp- and >200 Cys-ADPr sites. Glu/Asp-ADPr marks cytoplasmic, immune-related protein networks, contrasting with nuclear Ser-ADPr. Quantitative profiling revealed reproducible, cell type- and treatment-specific patterns. PARP10-mediated Glu/Asp ADPr of ubiquitin indicates direct crosstalk with ubiquitin signaling pathways. Interferon treatments revealed conserved antiviral PARP networks extensively modified on Glu/Asp residues. Together, our work establishes a robust MS-based workflow and provides a resource of site-specific ADPr events, revealing residue-specific ADPr in innate immune signaling.
Project description:Single-cell proteomics (SCP) holds the promise of decoding cellular heterogeneity at the functional level, yet achieving deep and reproducible proteome coverage from individual cells has remained a formidable challenge. Here, we establish a high-sensitivity, label-free SCP platform that extends current depth and reproducibility. By integrating optimized low-input sample processing, refined liquid chromatography, and the Orbitrap Astral Zoom mass spectrometer, our approach routinely quantifies up to 7,000 proteins per individual HeLa cell at over 100 samples-per-day (SPD), capturing thousands of low-abundance proteins that eluded prior SCP studies. Applied to very small human peripheral blood mononuclear cells (PBMCs), we identify up to 4,000 proteins per cell, including key markers that distinguish monocytes, T cells, and activated lymphocytes within heterogeneous populations, underscoring that single-cell proteomics can now directly elucidate clinically relevant primary samples with both depth and precision.
Project description:ADP-ribosylation (ADPr) is a regulatory post-translational modification targeting nine amino acid residues, but glutamate/aspartate-linked ADPr (Glu/Asp-ADPr) is labile and remains challenging to detect using conventional mass spectrometry (MS)-based workflows. Using synthetic peptides, we show that ester-linked Glu/Asp-ADPr is lost under alkaline conditions, elevated temperatures, and by hydrolysis via wildtype Af1521. We developed an acidic enrichment workflow incorporating an Af1521 mutant that preserves Glu/Asp-ADPr, enabling site-specific, system-wide MS analysis. In cytokine-stimulated A549 and HeLa cells, we identified >600 Glu/Asp- and >200 Cys-ADPr sites. Glu/Asp-ADPr marks cytoplasmic, immune-related protein networks, contrasting with nuclear Ser-ADPr. Quantitative profiling revealed reproducible, cell type- and treatment-specific patterns. PARP10-mediated Glu/Asp ADPr of ubiquitin indicates direct crosstalk with ubiquitin signaling pathways. Interferon treatments revealed conserved antiviral PARP networks extensively modified on Glu/Asp residues. Together, our work establishes a robust MS-based workflow and provides a resource of site-specific ADPr events, revealing residue-specific ADPr in innate immune signaling.