Streamlined pentafluorophenylpropyl column liquid chromatography-tandem quadrupole mass spectrometry and global 13C-labeled internal standards improve performance for quantitative metabolomics in bacteria

摘要: Streamlined quantitative metabolomics in central metabolism of bacteria would be greatly facilitated by a high-efficiency liquid chromatography (LC) method conjunction with accurate quantitation. To achieve this goal, methodology for LC-tandem quadrupole mass spectrometry (LC-MS/MS) involving pentafluorophenylpropyl (PFPP) column and culture-derived global (13)C-labeled internal standards (I.Ss.) has been developed compared to hydrophilic interaction (HILIC)-MS/MS published combined two-dimensional gas LC methods. All 50 tested metabolite from 5 classes (amino acids, carboxylic nucleotides, acyl-CoAs sugar phosphates) displayed good chromatographic separation sensitivity on the PFPP column. In addition, many important critical pairs such as isomers/isobars (e.g. isoleucine/leucine, methylsuccinic acid/ethylmalonic acid malonyl-CoA/3-hydroxybutyryl-CoA) metabolites similar structure malate/fumarate) were resolved better than HILIC Compared only one I.S., addition I.Ss. improved linearity accuracy. PFPP-MS/MS allowed absolute quantitation 42 pool sizes Methylobacterium extorquens AM1. A comparison level changes previously ethylamine (C2) versus succinate (C4) cultures M. AM1 indicated consistency data obtained PFPP-MS/MS, suggesting single approach capability providing comprehensive profiling combination The more quantification forms fundamental basis flux measurements can used modeling future studies.