| Metadata |
| datasetIdentifier | PASS01195 |
| datasetType | SRM |
| submitter | Yang Ni <yang.ni@kuleuven.vib.be> |
| submitter_organization | Mass Spectrometry Facility, Max Planck Institute for Molecular Genetics; Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin |
| lab_head_full_name | David Meierhofer |
| lab_head_email | Meierhof@molgen.mpg.de |
| lab_head_organization | Mass Spectrometry Facility, Max Planck Institute for Molecular Genetics |
| lab_head_country | Germany |
| datasetTag | NDUFS1MTND5CI |
| datasetTitle | Targeted metabolome profiling of cultured skin fibroblasts from patients carrying missense mutations in NDUFS1 and MT-ND5, which encode two core subunits of mitochondrial complex I in human |
| publicReleaseDate | 2019-09-26 00:00:00 |
| finalizedDate | 2019-09-25 12:18:04 |
| summary | Mitochondrial diseases occur at an estimated prevalence of 1 in 5000 live births, and are collectively the most common inborn error of metabolism. Complex I (CI) deficiency is the most frequent mitochondrial disorder among inborn errors of metabolism. The aim of this project is to dissect disease mechanisms of CI mutation. We applied an integrative proteome and metabolome profiling approach to investigate the molecular and cellular consequences of pathogenic mutations in two core subunits of mitochondrial CI. |
| contributors | Yang Ni; David Meierhofer |
| publication | Ni, Y, et al, Mutations in NDUFS1 Cause Metabolic Reprogramming and Disruption of the Electron Transfer, Cells, published (https://doi.org/10.3390/cells8101149) |
| growth | Human-derived primary skin fibroblast cells (patients and controls) were cultivated in high glucose DMEM and supplemented with 10% FBS, 1% Penicillin-Streptomycin-Neomycin antibiotic mixture at 37 °C in a normoxic incubator with a humidified atmosphere of 5% CO2. Cells were grown to 90% confluency in one T300 polystyrene flask (TPP, Switzerland) as one biological replicate. Cells were harvested in biological triplicates for targeted metabolome profiling. |
| treatment | Cells were untreated. |
| extraction | Metabolite extraction was done as reported previously with a minor modification for cell culture samples (Matyash, V., et al. 2008). In brief, T300 flasks with fibroblasts between the passages of 11–15 at 90% confluence were harvested in triplicates for each experiment. Twenty-four hours before harvest, the cell culture was replenished with fresh medium. In order to keep the original metabolic state of the cell and minimize metabolite degradation, cells were harvested within 2 minutes. Culture medium was aspirated and the cells were rinsed quickly twice with ice-chilled 1× phosphate-buffered saline, pH 7.4. Then, 1 ml water was added into the flask, which was immediately shock-frozen in liquid nitrogen. The flask was kept on ice, and cell lysates were collected with a cell scraper (TPP) and transferred into a 15 ml tube for three thaw-and-freeze cycles to extract the metabolites. Metabolites were extracted with methyl tert-butyl ether (MTBE), methanol, and water. The remaining protein pellet was used in the bicinchoninic acid (BCA) protein assay for normalization among samples. Extracts were aliquoted equally into three tubes for later reconstitution in water, acetonitrile, and 50% methanol in acetonitrile, respectively. Additionally, an internal standard mixture, containing chloramphenicol and C13-labeled L-glutamine, L-arginine, L-proline, L-valine, and uracil was added to each sample (10 µM final concentration). A SpeedVac was used to dry the aliquots. Dry residuals were dissolved in three different solvents (1) 100 µL 50% acetonitrile in MeOH with 0.1% formic acid, (2) 100 µL MeOH with 0.1% formic acid for analysis by hydrophilic interaction liquid chromatography (HILIC) column, or (3) 100 µL water with 0.1% formic acid for C18 column mode. The supernatants were transferred to micro-volume inserts. Then, 20 µL per run was injected for subsequent LC-MS analysis. |
| separation | Two different LC columns have been used for metabolite separation: a Reprosil-PUR C18-AQ (1.9 µm, 120 Å, 150 × 2 mm ID; Dr. Maisch, Ammerbuch, Germany) column, and a zicHILIC (3.5 µm, 100 Å, 150 × 2.1 mm ID; Merck). The settings of the LC-MS instrument, 1290 series ultra high pressure liquid chromatography (UHPLC) (Agilent) online coupled to a QTRAP 6500 (Sciex, Foster City, CA) were reported previously (Gielisch, I., et al. 2015). The buffer conditions were A1—10 mM ammonium acetate, pH 3.5 (adjusted with acetic acid); B1—99.9% acetonitrile with 0.1% formic acid; A2—10 mM ammonium acetate, pH 7.5 (adjusted with ammonia solution); and B2—99.9% methanol with 0.1% formic acid. All buffers were prepared in LC-MS grade water and organic solvents. |
| digestion | No enzymatic digestion of samples |
| acquisition | All raw data files (.wiff and .wiff.scan) and q.session files are included in this data set. |
| informatics | The import and processing of the raw data (.wiff) were performed using MultiQuant software v.2.1.1. Peak integrations of each metabolite were reviewed manually. The q.session files after the peak integration were exported from MultiQuant to Excel for further analysis. Retention time and MRM ion ratios of each metabolite were checked. Peak intensities were normalized, first against the internal standards, and subsequently against protein abundancies obtained from the BCA assay. The first transition per metabolite was used for relative quantification between samples and controls. Perseus software (v.1.6.0.2, Cox, J., et al. 2016) was used for further statistics analysis of the data. |
| instruments | Sciex QTRAP 6500 |
| species | Human |
| massModifications | none |
