Project description:Combining data from multiple analytical platforms is essential for comprehensive study of the molecular phenotype (metabotype) of a given biological sample. The metabolite profiles generated are intrinsically dependent on the analytical platforms, each requiring optimization of instrumental parameters, separation conditions, and sample extraction to deliver maximal biological information. An in-depth evaluation of extraction protocols for characterizing the metabolome of the hepatobiliary fluke Fasciola hepatica , using ultra performance liquid chromatography and capillary electrophoresis coupled with mass spectroscopy is presented. The spectrometric methods were characterized by performance, and metrics of merit were established, including precision, mass accuracy, selectivity, sensitivity, and platform stability. Although a core group of molecules was common to all methods, each platform contributed a unique set, whereby 142 metabolites out of 14,724 features were identified. A mixture design revealed that the chloroform:methanol:water proportion of 15:59:26 was globally the best composition for metabolite extraction across UPLC-MS and CE-MS platforms accommodating different columns and ionization modes. Despite the general assumption of the necessity of platform-adapted protocols for achieving effective metabotype characterization, we show that an appropriately designed single extraction procedure is able to fit the requirements of all technologies. This may constitute a paradigm shift in developing efficient protocols for high-throughput metabolite profiling with more-general analytical applicability.
Project description:We report for the first time movement of Correia Repeat Enclosed Elements, through inversion of the element at its chromosomal location. Analysis of Ion Torrent generated genome sequence data from Neisseria gonorrhoeae strain NCCP11945 passaged for 8 weeks in the laboratory under standard conditions and stress conditions revealed a total of 37 inversions: 24 were exclusively seen in the stressed sample; 7 in the control sample; and the remaining 3 were seen in both samples. These inversions have the capability to alter gene expression in N. gonorrhoeae through the previously determined activities of the sequence features of these elements. In addition, the locations of predicted non-coding RNAs were investigated to identify potential associations with CREE. Associations varied between strains, as did the number of each element identified. The analysis indicates a role for CREE in disrupting ancestral regulatory networks, including non-coding RNAs. RNA-Seq was used to examine expression changes related to Correia repeats in the strain
Project description:Extracting histones from cells is a routine operation for studies that aim to characterize histones and their post-translational modifications (hPTMs). However, label-free quantitative mass spectrometry (MS) approaches, both data-dependent (DDA) and data-independent (DIA), require streamlined protocols that are highly reproducible even at the peptide level, to enable simultaneous accurate quantification of dozens to hundreds of these hPTMs. We present a step-by-step comparison of different histone extraction protocols based on literature and evaluate their suitability for label-free MS purposes using a nanoESI label-free MS1 intensity-based DDA MS experiment. We evaluate the data both using a targeted and an untargeted (Progenesis QI) approach.
Project description:In order to determine whether dis-regulation of a genetic pathway could explain the increased apoptosis of parp-2-/- double positive thymocytes, the gene expression profiles in double positive thymocytes derived from wild-type and parp-2-/- mice were analysed using Affymetrix oligonucleotide chips (mouse genome 430 2.0).
Project description:In the urinary metabolomics for finding biomarkers in urine, owing to high concentrations of urea, for chromatography-based metabolomic analysis, urea needed to be degraded by urease. This urease pretreatment has been the key step of sample preparation for standard urinary metabolomics until today even for mass spectrometry-based analysis. The urease pretreatment involving incubation of urine with urease contradicts the concept of metabolome sampling, which should immediately arrest metabolic reactions to prevent alterations of a metabolite profile. Nonetheless, the impact of urease pretreatment has not been clearly elucidated yet. We found that activities of urease and endogenous urinary enzymes and metabolite contaminants from the urease preparations introduce artefacts into metabolite profiles, thus leading to misinterpretation.
Project description:Ossification of the posterior longitudinal ligament (OPLL) is formed by heterogeneous ossification of posterior longitudinal ligament. The patho-mechanism of OPLL is still largely unknown. MicroRNAs are small nucleatides that function as regulators of gene expression in almost any biological process. However, few microRNAs are reported to have a role in the pathological process of OPLL. Therefore, we performed high-throughput microRNA sequencing and transcriptome sequencing of primary OPLL and PLL cells in order to decipher the interacting network of microRNAs in OPLL. MRNA and microRNA profiles were done using primary culture cells of human ossification of the posterior longitudinal ligament (OPLL) tissue and normal posterior longitudinal ligament (PLL) tissue.
Project description:Metabolomics is an essential discipline in omics technology that promotes research on the biology of microbial systems. Streptomyces albus J1074 is a model organism used in fundamental research and industrial microbiology. Nevertheless, a comprehensive and standardized method for analyzing the metabolome of S. albus J1074 is yet to be developed. Thus, we comprehensively evaluated and optimized the analytical procedure and sample preparation for profiling polar metabolites using hydrophilic interaction liquid chromatography (HILIC) coupled with high-resolution mass spectrometry (HRMS). We systematically examined the HILIC columns, quenching solutions, sample-to-quenching ratios, and extraction methods. Then, the optimal protocol was used to investigate the dynamic intracellular polar metabolite profile of the engineered S. albus J1074 strains during spinosad (spinosyn A and spinosyn D) fermentation. A total of 3648 compounds were detected, and 83 metabolites were matched to the standards. The intracellular metabolomic profiles of engineered S. albus J1074 strains (ADE-AP and OE3) were detected; furthermore, their metabolomes in different stages were analyzed to reveal the reasons for their differences in their spinosad production, as well as the current metabolic limitation of heterologous spinosad production in S. albus J1074. The HILIC-HRMS method is a valuable tool for investigating polar metabolomes, and provides a reference methodology to study other Streptomyces metabolomes.
Project description:Proteomics and metabolomics are essential in systems biology, and simultaneous proteo-metabolome liquid-liquid extraction (SPM-LLE) allows isolation of the metabolome and proteome from the same sample. Since the proteome is present as a pellet in SPM-LLE, it must be solubilized for quantitative proteomics. Solubilization and proteome extraction are critical factors in the information obtained at the proteome level. In this study, we investigated the performance of two surfactants (sodium deoxycholate (SDC), sodium dodecyl sulfate (SDS)) and urea in terms of proteome coverage and extraction efficiency of an interphase proteome pellet generated by methanol-chloroform based SPM-LLE. We also investigated how the performance differs when the proteome is extracted from the interphase pellet or by direct cell lysis. We quantified 12 lipids covering triglycerides and various phospholipid classes, and 25 polar metabolites covering central energy metabolism in chloroform and methanol extracts. Our study reveals that the proteome coverages between the two surfactants and urea for the SPM-LLE interphase pellet were similar, but the extraction efficiencies differed significantly. While SDS led to enrichment of basic proteins, which were mainly ribosomal and ribonuclear proteins, urea was the most efficient extraction agent for simultaneous proteo-metabolome analysis. The results of our study also show that the performance of surfactants for quantitative proteomics is better when the proteome is extracted through direct cell lysis rather than an interphase pellet. In contrast, the performance of urea for quantitative proteomics was significantly better when the proteome was extracted from an interphase pellet than by direct cell lysis. We demonstrated that urea is superior to surfactants for proteome extraction from SPM-LLE interphase pellets, with a particularly good performance for the extraction of proteins associated with metabolic pathways. Data are available via ProteomeXchange with identifier PXD027338.
Project description:Lipidomics is a rapidly developing field in modern biomedical research. While LC-MS systems are able to detect most of the known lipid classes in a biological matrix, there is no single technique able to extract all of them simultaneously. In comparison with two-phase extractions, one-phase extraction systems are of particular interest, since they decrease the complexity of the experimental procedure. By using an untargeted lipidomics approach, we explored the differences/similarities between the most commonly used two-phase extraction systems (Folch, Bligh and Dyer, and MTBE) and one of the more recently introduced one-phase extraction systems for lipid analysis based on the MMC solvent mixture (MeOH/MTBE/CHCl3). The four extraction methods were evaluated and thoroughly compared against a pooled extract that qualitatively and quantitatively represents the average of the combined extractions. Our results show that the lipid profile obtained with the MMC system displayed the highest similarity to the pooled extract, indicating that it was most representative of the lipidome in the original sample. Furthermore, it showed better extraction efficiencies for moderate and highly apolar lipid species in comparison with the Folch, Bligh and Dyer, and MTBE extraction systems. Finally, the technical simplicity of the MMC procedure makes this solvent system highly suitable for automated, untargeted lipidomics analysis.