PMC:4351692 / 37821-40468 JSONTXT

{"project":"2_test","denotations":[{"id":"25889900-17376459-14869138","span":{"begin":564,"end":566},"obj":"17376459"},{"id":"25889900-21134484-14869139","span":{"begin":1147,"end":1149},"obj":"21134484"},{"id":"25889900-20885382-14869140","span":{"begin":1436,"end":1438},"obj":"20885382"},{"id":"25889900-14705791-14869141","span":{"begin":1439,"end":1441},"obj":"14705791"},{"id":"25889900-21388201-14869142","span":{"begin":1618,"end":1620},"obj":"21388201"}],"text":"Sample and evaluation of mass isotopomer datasets\nMetabolite extracts were analysed by an LC-MS/MS method and a GC-MS/MS method to generate mass isotopomer distribution datasets for the P. fluorescens SBW25 wild-type and the mucA-ΔalgC strains. Prior to LC-MS/MS analysis, samples were reconstituted in 500 μL 60% (v/v) methanol. Analysis was performed on an Agilent 1200 series LC connected via an electrospray ion source to an Agilent 6410 triple-quadrupole MS using an adaptation of the reverse phase tributylamine ion exchange method introduced by Luo et al. [36]. The same LC-MS/MS method was used in the metabolome study of P. fluorescens [9]. A total of eight central carbon metabolism metabolites were analysed for mass isotopomer distributions by LC-MS/MS and used in the study. For each compound, the number of precursor-to-fragment transitions (multiple reaction monitoring (MRM)-transitions) was expanded to account for all mass isotopomers of the compound, i.e., (n-m + 1)(m + 1) MRM-transitions for each compound, where n is the number of carbons in the precursor ion and m is the number of carbons in the product ion, respectively [37]. A list of all MRM-transitions and accompanying MS-settings for the LC-MS/MS method can be found in Additional file 4.\nSamples for GC-MS/MS analysis were derivatised prior to analysis as described previously [9] using an adaptation of the methyl chloroformate derivatisation protocol [38,39]. Analysis was performed on an Agilent 7890A GC – 7000 triple-quadrupole MS interfaced with a chemical ionisation (CI) ion source based on a previously described MS/MS method [40]. The GC method was as described previously [9], with the exception that the injected volume was increased from 1 μL to 3 μL. The MS method used positive CI with methane as the reagent gas and nitrogen as the collision gas. MRM-transitions to account for all mass isotopomers of the 14 detected compounds (central carbon metabolism metabolites and amino acids) were included in the fluxomics models. A list of all MRM-transitions and accompanying GC-MS/MS-settings can be found in Additional file 4.\nThe resulting mass isotopomer fractions are listed in Additional file 5. Average relative standard deviations (STD) for the mass isotopomers are calculated from the biological replicates for the estimation of intracellular fluxes. The obtained LC-MS/MS STD values were 13% for the wild-type experimental design cultivation, and 10% and 8% for the main experiment cultivations on wild type and mucA-ΔalgC, respectively. For mass isotopomers detected by the GC-MS/MS method, these STD values were 10%, 12% and 15%, respectively."}