Project description:Identification of unknown peaks in gas chromatography/mass spectrometry (GC/MS)-based discovery metabolomics is challenging, and remains necessary to permit discovery of novel or unexpected metabolites that may elucidate disease processes and/or further our understanding of how genotypes relate to phenotypes. Here, we introduce two new technologies and an analytical workflow that can facilitate the identification of unknown peaks. First, we report on a GC/Quadrupole-Orbitrap mass spectrometer that provides high mass accuracy, high resolution, and high sensitivity analyte detection. Second, with an "intelligent" data-dependent algorithm, termed molecular-ion directed acquisition (MIDA), we maximize the information content generated from unsupervised tandem MS (MS/MS) and selected ion monitoring (SIM) by directing the MS to target the ions of greatest information content, that is, the most-intact ionic species. We combine these technologies with (13)C- and (15)N-metabolic labeling, multiple derivatization and ionization types, and heuristic filtering of candidate elemental compositions to achieve (1) MS/MS spectra of nearly all intact ion species for structural elucidation, (2) knowledge of carbon and nitrogen atom content for every ion in MS and MS/MS spectra, (3) relative quantification between alternatively labeled samples, and (4) unambiguous annotation of elemental composition.
Project description:Fatty acids (FAs) play critical roles in health and disease. The detection of FA imbalances through metabolomics can provide an overview of an individual's health status, particularly as regards chronic inflammatory disorders. In this study, we aimed to establish sensitive reference value ranges for targeted plasma FAs in a well‑defined population of healthy adults. Plasma samples were collected from 159 participants admitted as outpatients. A total of 24 FAs were analyzed using gas chromatography‑mass spectrometry, and physiological values and 95% reference intervals were calculated using an approximate method of analysis. The differences among the age groups for the relative levels of stearic acid (P=0.005), the omega‑6/omega‑3 ratio (P=0.027), the arachidonic acid/eicosapentaenoic acid ratio (P<0.001) and the linoleic acid‑produced dihomo‑gamma‑linolenic acid (P=0.046) were statistically significant. The majority of relative FA levels were higher in males than in females. The levels of myristic acid (P=0.0170) and docosahexaenoic acid (P=0.033) were significantly different between the sexes. The reference values for the FAs examined in this study represent a baseline for further studies examining the reproducibility of this methodology and sensitivities for nutrient deficiency detection and investigating the biochemical background of pathological conditions. The application of these values to clinical practice will allow for the discrimination between health and disease and contribute to early prevention and treatment.
Project description:To better understand proteostasis in health and disease, determination of protein half-lives is essential. We improved the precision and accuracy of peptide-ion intensity based quantification in order to enable accurate determination of protein turnover in non-dividing cells using dynamic-SILAC. This enabled precise and accurate protein half-life determination ranging from 10 to more than 1000 hours. We achieve good proteomic coverage ranging from four to six thousand proteins in several types of non-dividing cells, corresponding to a total of 9699 unique proteins over the entire dataset. Good agreement was observed in half-lives between B-cells, natural killer cells and monocytes, while hepatocytes and mouse embryonic neurons showed substantial differences. Our comprehensive dataset enabled extension and statistical validation of the previous observation that subunits of protein complexes tend to have coherent turnover. Furthermore, we observed complex architecture dependent turnover within complexes of the proteasome and the nuclear pore complex. Our method is broadly applicable and might be used to investigate protein turnover in various cell types.
Project description:The mouse spot test, an in vivo mutation assay, has been used to assess a number of chemicals. It is at present the only in vivo mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484). It is however rather insensitive, animal consuming and expensive type of test. More recently several assays using transgenic animals have been developed. From data in the literature, the present study compares the results of in vivo testing of over twenty chemicals using the mouse spot test and compares them with results from the two transgenic mouse models with the best data base available, the lacI model (commercially available as the Big Blue(R) mouse), and the lacZ model (commercially available as the Mutatrade mark Mouse). There was agreement in the results from the majority of substances. No differences were found in the predictability of the transgenic animal assays and the mouse spot test for carcinogenicity. However, from the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo.
Project description:Purpose: The aim of this study is to investigate the translational regulation of skeletal muscle during acute endurance exercise. Methods: We used mRNA-Seq and ribosome profiling to examine transcriptional and translational regulation, respectively. Result: There were clear distinctions between the profiles of transcription and translation even at a basal condition. TOP-motif genes were translationally suppressed immediately after the exercise. Other genes, such as Slc25a25 was significantly translationally up-regulated presumably in a mTOR-independent manner. Conclusion: There were diverse regulation between transcription and translation. Although many focused on overall protein synthesis to understand the effect of exercise, translational regulation of individual genes are required. Transcriptional and translational profiles of mouse gastrocnemius with or without acute endurance exercise were generated using Ion PGM sequencer.
Project description:Hypertension is caused by polygenic inheritance and the interaction of various environmental factors. Abnormal function of the renin-angiotensin-aldosterone system (RAAS) is closely associated with changes in blood pressure. As an essential factor in the RAAS, angiotensin II (Ang II) contributes to vasoconstriction and inflammatory responses. However, the effects of overproduction of Ang II on the whole body-metabolism have been unclear. In this study, we established a hypertensive mouse model by micro-osmotic pump perfusion of Ang II, and the maximum systolic blood pressure reached 140 mmHg after 2 weeks. By ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, the metabolites in the serum of hypertensive model and control mice were analyzed. Partial least squares discriminant analysis (PLS-DA) in both positive and negative ionization modes showed clear separation of the two groups. Perfusion of Ang II induced perturbations of multiple metabolic pathways in mice, such as steroid hormone biosynthesis and galactose metabolism. Tandem mass spectrometry revealed 40 metabolite markers with potential diagnostic value for hypertension. Our data indicate that non-targeted metabolomics can reveal biochemical pathways associated with Ang II-induced hypertension. Although researches about the clinical use of these metabolites as potential biomarkers in hypertension is still needed, the current study improves the understanding of systemic metabolic response to sustained release of Ang II in hypertensive mice, providing a new panel of biomarkers that may be used to predict blood pressure fluctuations in the early stages of hypertension.