Project description:Due to its antimicrobial activity, silver nanoparticles (Ag-NPs) are among the most used NPs worldwide, yet little information is available regarding their effects, particularly in soil dwelling organisms. Enchytraeids (Oligochaeta) are important members of the soil fauna which actively contribute to the acceleration of organic matter decomposition and nutrient recycling processes. Hence, for hazard and risk assessment it is important to provide toxicity data for these organisms and to understand more in regard to the mode of action of Ag-NPs within organism. To study this we conducted toxicity experiments using the OECD standard guideline, testing Ag-NPs and AgNO3, having assessed survival, reproduction and differential gene expression. Population toxicity responses were assessed showing higher toxicity for the AgNO3. In an attempt to understand the mode of action we performed transcription profiling using the microarray. Gene expression profile of Enchytraeus albidus was analysed after 2 days of exposure to 100 and 200 mg/kg of two silver forms (nanoparticles and salt_silver nitrate) in OECD soil. Three biological replicates per test treatment and control (clean OECD soil) were used.
Project description:Due to its antimicrobial activity, silver nanoparticles (Ag-NPs) are among the most used NPs worldwide, yet little information is available regarding their effects, particularly in soil dwelling organisms. Enchytraeids (Oligochaeta) are important members of the soil fauna which actively contribute to the acceleration of organic matter decomposition and nutrient recycling processes. Hence, for hazard and risk assessment it is important to provide toxicity data for these organisms and to understand more in regard to the mode of action of Ag-NPs within organism. To study this we conducted toxicity experiments using the OECD standard guideline, testing Ag-NPs and AgNO3, having assessed survival, reproduction and differential gene expression. Population toxicity responses were assessed showing higher toxicity for the AgNO3. In an attempt to understand the mode of action we performed transcription profiling using the microarray.
Project description:Data independent acquisition (DIA) has become a well-established method in LC-MS driven proteomics. Nonetheless, there are still a lot of possibilities at the data analysis level. By benchmarking different DIA analysis workflows through a ground truth sample mimicking real differential abundance samples, consisting of a differential spike-in of UPS2 in a constant yeast background, we provide a roadmap for DIA data analysis of shotgun samples based on whether sensitivity, precision or accuracy is of the essence. Three different commonly used DIA software tools (DIA-NN, EncyclopeDIA and SpectronautTM) were tested in both spectral library mode and spectral library free mode. In spectral library mode we used the independent spectral library prediction tools Prosit and MS2PIP together with DeepLC, next to the classical DDA-based spectral libraries. In total we benchmarked 12 DIA workflows. DIA-NN in library free mode or using in silico predicted libraries shows the highest sensitivity maintaining a high reproducibility and accuracy.
Project description:This set of submissions contains the mass spectrometry files acquired using Data-Dependent Acquisition mode (DDA), which were paired with Data-Independent Acquisition mode (DIA) files (MSV000081414) for the manuscript by Ji-Young Youn et al. that describes the high-density proximity mapping of RNA bodies. These DDA files were used to generate spectral library in MSPLIT analysis of both DDA and DIA files. The paired SAINT 3090 set entails APMS experiments of stress granule baits treated or untreated briefly with sodium arsenite and these were were performed from Flp-In T-REx HEK293 cells and MS files were acquired on AbSciex Triple TOF 6600 instrument in data-independent acquisition mode.
Project description:Data independent acquisition (DIA or DIA/SWATH ) mass spectrometry has emerged as a primary measurement strategy in the field of quantitative proteomics. diaPASEF is a recent adaptation that leverages trapped ion mobility spectrometry (TIMS) to improve selectivity and increase sensitivity. The complex fragmentation spectra generated by co-isolation of peptides in DIA mode are most typically analyzed with reference to prior knowledge in the form of spectral libraries. The best established method for generating libraries uses data dependent acquisition (DDA) mode, or DIA mode if appropriately deconvoluted, often including offline fractionation to increase depth of coverage,to create spectral libraries. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow window DIA methods designed to cover different slices of the precursor space, have been introduced and performed comparably to deep offline fractionation-based libraries for DIA data analysis. Here, we investigated whether an analogous GPF-based library building approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data and can remove the need for offline fractionation. To enable a rapid library development approach for diaPASEF we designed a GPF acquisition scheme covering the majority of multiply charged precursors in the m/z vs 1/K0 space requiring 7 injections of a representative sample and compared this with libraries generated by direct deconvolution-based analysis of diaPASEF data or by deep offline fractionation and ddaPASEF. . We found that the GPF based library outperformed library generation by direct deconvolution of the diaPASEF data, and performed comparably to deep offline fractionation libraries, when analysing diaPASEF data acquired from 200ng of commercial HeLa digest. With the ion mobility integrated GPF scheme we establish a pragmatic approach to rapid and comprehensive library generation for the analysis of diaPASEF data.
Project description:Data independent acquisition (DIA or DIA/SWATH ) mass spectrometry has emerged as a primary measurement strategy in the field of quantitative proteomics. diaPASEF is a recent adaptation that leverages trapped ion mobility spectrometry (TIMS) to improve selectivity and increase sensitivity. The complex fragmentation spectra generated by co-isolation of peptides in DIA mode are most typically analyzed with reference to prior knowledge in the form of spectral libraries. The best established method for generating libraries uses data dependent acquisition (DDA) mode, or DIA mode if appropriately deconvoluted, often including offline fractionation to increase depth of coverage,to create spectral libraries. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow window DIA methods designed to cover different slices of the precursor space, have been introduced and performed comparably to deep offline fractionation-based libraries for DIA data analysis. Here, we investigated whether an analogous GPF-based library building approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data and can remove the need for offline fractionation. To enable a rapid library development approach for diaPASEF we designed a GPF acquisition scheme covering the majority of multiply charged precursors in the m/z vs 1/K0 space requiring 7 injections of a representative sample and compared this with libraries generated by direct deconvolution-based analysis of diaPASEF data or by deep offline fractionation and ddaPASEF. . We found that the GPF based library outperformed library generation by direct deconvolution of the diaPASEF data, and performed comparably to deep offline fractionation libraries, when analysing diaPASEF data acquired from 200ng of commercial HeLa digest. With the ion mobility integrated GPF scheme we establish a pragmatic approach to rapid and comprehensive library generation for the analysis of diaPASEF data.
Project description:This set of submissions contains the mass spectrometry files acquired using Data-Dependent Acquisition mode (DDA), which were paired with Data-Independent Acquisition mode (DIA) files (MSV000081419) for the manuscript by Ji-Young Youn et al. that describes the high-density proximity mapping of RNA bodies. These DDA files were used to generate spectral library in MSPLIT analysis of both DDA and DIA files. The paired SAINT 3108 set entails BioID experiments of stress granule and Processing-body baits expressed in Flp-In T-REx HEK293 cells, treated or untreated with sodium arsenite during labeling period.
Project description:Toxicometabolomics, essentially applying metabolomics to toxicology of endogenous compounds such as drugs of abuse or new psychoactive substances (NPS), can be investigated by using different in vitro models and dedicated metabolomics techniques to enhance the number of relevant findings. The present study aimed to study the toxicometabolomics of the two NPS α-pyrrolidinobutiophenone (1-phenyl-2-(pyrrolidin-1-yl)butan-1-one, α-PBP) and α-pyrrolidinoheptaphenone (1-phenyl-2-(pyrrolidin-1-yl)heptan-1-one, α-PEP, PV8) in HepaRG cell line incubates. Evaluation was performed using reversed-phase and normal-phase liquid chromatography coupled with high-resolution mass spectrometry in positive and negative ionization mode, respectively, to analyze cells and cell media. Statistical evaluation was performed using one-way ANOVA, principal component discriminant function analysis, as well as hierarchical clustering. In general, the analysis of cells did not mainly reveal any features, but the parent compounds of the drugs of abuse. For α-PBP an increase in N-methylnicotinamide was found, which may indicate hepatotoxic potential of the substance. After analysis of cell media, significant features led to the identification of several metabolites of both compounds. Amino acid adducts with glycine and alanine were found, and these have not been described in any study before and are likely to appear in vivo. Additionally, significant changes in the metabolism of cholesterol were revealed after incubation with α-PEP. In summary, the application of metabolomics techniques after HepaRG cells exposure to NPS did not lead to an increased number of identified drug metabolites compared to previously published studies, but gave a wider perspective on the physiological effect of the investigated compounds on human liver cells.