LC-MS based Metabolomics

Metabolomics, the qualitative and quantitative analysis of metabolites is a valuable approach for understanding the biochemical processes in the cells. Particularly important are the intracellular metabolites that supply the cell with energy, serve as building blocks and act as signaling molecules. The analytical tools applied for analysis of intracellular metabolites should be capable to cope with the large number of metabolites to be analyzed and the complex matrix in the samples. Therefore the combination of separation and detection techniques is commonly applied for analysis of intracellular metabolites with liquid chromatography mass spectrometry (LC-MS) as the most commonly used. The primary goal of this Ph.D. study was to develop an LC-MS method together with sample
preparation for analysis of intracellular metabolites such as nucleotides, sugar phosphates, organic acids, coenzymes etc.. In the studies conducted during this Ph.D. the developed method was used to understand how the genetic manipulations in various organisms, influence the levels of their intracellular metabolites. The method development was divided into three steps: i) optimization of the MS detection, ii) establishment and optimization of the chromatographic separation and iii) optimization of the sample preparation. A substantial part of the thesis was focused on the development of the LC-MS method. For quantitative targeted analysis of a group of defined metabolites, triple quadrupole (QqQ) MS was used. The optimization of the MS detection aimed to determine multiple reaction monitoring (MRM) transitions of the analytes and to increase the sensitivity by testing different ion-source parameters and collision energies. This resulted in optimized detection of more than 50
intracellular metabolites. During the optimization of the chromatographic separation, anion exchange (AEC), ion chromatography (IC) and ion-pair reversed phase (IP-RP) were tested with the ion-pair giving the best compromise between retention, separation and stability of the compounds during the chromatographic separation. By testing different types and concentrations of ion-pair reagent and different concentrations of acetic acid as a counter ion, it was found that a solution of 10 mM tributylamine (TBA) and 10 mM acetic acid gave the best compromise between chromatographic retention and separation. Establishment of proper sample preparation (quenching and extraction) procedures for intracellular metabolites was necessary in order to obtain meaningful metabolomics data. The main idea was to find a sample preparation method that will give the best compromise between an acceptable energy charge ratio (ECR, usually between 0.80-0.95) low leakage during the quenching and high recovery of the metabolites after the extraction. Quenching and extraction procedures for bacteria, yeast, mammalian cells and filamentous fungi were tested. Cold MeOH as a quenching method combined with boiling EtOH or MeOH/chloroform as extraction method showed to work well for Saccharomyces cerevisiae (S. cerevisiae) resulting in an ECR of 0.80-0.95 and less than 10 % leakage. Quenching of bacteria and fungi showed to be challenging task due to the high susceptibility of these organisms to leakage during quenching. Quenching using formic acid, where the cells were
not separated from the media, was shown to work well for Lactococcus lactis (L. lactis) but not for Streptomyces coelicolor (S. coelicolor) and Microbispora corallina (M. corallina). The reason for this was speculated to be due to the filamentous growth of these organisms. Other quenching procedures were tested for S. coelicolor and M. corallina with -40 °C MeOH/H2O (60/40, v/v) giving an acceptable ECR (in the range of 0.80-0.95). However leakage was observed for both organisms. For filamentous fungi, filtration in combination with cold methanol or 0 °C saline resulted in successful quenching. In the case of cold methanol quenching the concentration of AMP and ADP in the quenching supernatant was found to be 30 % of the total amount found in the biomass and the supernatant. Saline at 0 °C
showed to be a good quenching solution for mammalian cells as well and was combined with an extraction procedure based on the addition of cold MeOH and ACN/H2O (50/50, v/v). In another study conducted during this Ph.D. a novel approach for creating an authentic matrix that can be used for validation of analytical methods was established. This circumvents the problem with the absence of a matrix free of the analyte which is needed for preparation of the calibration curves for quantification of intracellular metabolites. The spiking matrix was produced by extracting biomass obtained from growing S. cerevisiae in a media that contained 13C labeled glucose/non-labeled glucose (50/50, w/w). The advantage of this matrix was that the pools of the compounds with only 12C or 13C carbons were very low or even not measurable and showed minimal or no interference to the spiked amount of nonlabeled standards and their stable isotope-labeled internal standards (SIL-IS). Finally the developed IP-RP LC-MS method was coupled to a quadrupole time of flight (QTOF) MS for multitargeted analysis, detection and identification of as many known unknown metabolites as possible in different biological matrices. The feasibility of using the Q-TOF MS was evaluated by analysis of extracts from three different organisms: S. cerevisiae, M. corallina and S. coelicolor. The screening concept in this study was based on two approaches: i) aggressive dereplication of the full scan high resolution MS (HR-MS) data using search lists of known compounds and ii) high resolution tandem MS (MS/HRMS) data searched in Metabolite Link (METLIN) library. The data presented here show that the methods developed during this Ph.D. study were successfully applied for targeted and multitargeted analysis of different classes of intracellular metabolites such as nucleotides, sugar phosphates, coeznymes and organic acids. In addition sample preparation methods were established for different microorganisms capable of extracting broad range of metabolites. Finally these methods have shown to be valuable addition to the ‘’omics’’ tools used to reveal key information regarding the
metabolism and the regulation in the biological systems.