Metabolome in transitional phases during differentiation of multipotent cells
Description
Data includes three metabolome profiles of multipotent cells in their steady-states and after inducing differentiation. Metabolomes of Myoblast (MBs) undergoing differentiation into myofibers, Mesenchymal Stem Cells (MSCs) undergoing differentiation into chondrocytes, and Neural Stem Cells (NSCs) undergoing differentiation into astrocytes. Data is aimed to characterize the metabolomic status of cells undergoing a transitional phase between the original identity and their next commitment. Biological samples were generated at Salk Institute (San Diego, CA) and the Metabolomics was performed by the company Metabolon, Inc. (Morrisville, NC).
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Following receipt samples at Metabolon Inc, company inventoried (LIMSsystem) and stored at -80C. Samples were prepared using the automated MicroLabSTAR system (Hamilton). To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2min followed by centrifugation. Resulting extract was divided into 5fractions: 2 for analysis by 2 separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization, 1 for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap to remove the organic solvent. Sample extracts were stored overnight under nitrogen before analysis. UPLC-MS/MS: Waters ACQUITYultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution, were used. Sample extract was dried then reconstituted in solvents compatible to each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. Extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1x100mm, 1.7µm) using water+methanol, containing 0.05%PFPA+0.1%FA. Another aliquot was analyzed using acidic positive ion conditions; however, it was chromatographically optimized for more hydrophobic compounds. Extract was gradient eluted from the same afore mentioned C18 column using methanol, acetonitrile, water, 0.05%PFPA+0.01%FA and was operated at an overall higher organic content. Another aliquot was analyzed using basic negative ion optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water, however with 6.5mM Ammonium Bicarbonate at pH8. The 4th aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150mm, 1.7µm) using a gradient consisting of water and acetonitrile with 10mM Ammonium Formate, pH 10.8. MSanalysis alternated between MS and data-dependent MSn scans using dynamic exclusion. Scan range varied slighted between methods but covered 70-1000m/z. Raw data archived and extracted using Metabolon’s hardware and software. Following normalization to Bradford protein concentration, log transformation and imputation of missing values, if any, with the minimum observed value for each compound, ANOVA contrasts and Welch’s two-sample t-test was used to identify biochemicals that differed.