Magdenoska, Olivera

Metabolomics, den kvalitative og kvantitative analyse af metabolitter er en værdifuld metode til forståelsen af de biokemiske processer i cellerne. Særligt vigtige er de intracellulære metabolitter, der supplerer cellen med energi, bruges som byggeklodser og fungerer som signalmolekyler. De analytiske redskaber anvendt til måling af intracellulære metabolitter bør være i stand til at håndtere den store mængde af metabolitter, der skal analyseres og den komplekse matrix i prøverne. Derfor anvender man normalt kombinationen af separation og detektion, til analysen af intracellulære metabolitter med flydende kromatografi og masse spektrometri (LC-MS) som den mest almindeligt anvendte. Hovedformålet med dette Ph.D. studium var at udvikle en LC-MS metode sammen medprøve forberedelse til analyse af intracellulære metabolitter såsom nukleotider, sukker fosfater, organiske syre, co-enzymer etc.. I studierne udført under denne Ph.D., blev den udviklede metode brugt til at forstå hvordan den genetiske manipulering i forskellige organismer påvirker niveauet af deres intracellulære metabolitter. Metode udviklingen blev inddelt i tre trin: i) optimering af MS-detektionen, ii) etablering og optimering af den kromatografiske separation og iii) optimering af prøve forberedelse. En betydelig del af afhandlingen fokuserede på udviklingen af denne LC-MS metode. Triple quadrupole (QqQ) MS blev anvendt til kvantitativ målrettet analyse af en gruppe af definerede metabolitter. Optimeringen af MS detektionen stilede imod at bestemme multiple reaction monitoring (MRM) i.f.t. overgange af analytterne og mod at forøge følsomheden ved at teste forskellige ion-kilde parametre samt kollisions energier. Dette resulterede i optimeret detektion af af mere end 50 intracellulære metabolitter. Undervejs i optimeringen af kromatografisk adskillelse blev anion bytning (AEC), ion kromatografi (IC) og ion-par omvendt fase (IP-RP) testet med det ion-par der gav det bedste kompromis mellem retention, adskillelse og stabilitet af stofferne igennem den kromatografiske adskillelse. Ved testning af forskellige typer og koncentrationer af ion-par reagent og forskellige koncentrationer af eddikesyre som en modbalance ion blev det vist at en opløsning af 10 mM tributylamine og 10 mM eddikesyre gav det bedste kompromis mellem kromatografisk retention og adskillelse. Etableringen af ordentlige prøve forberedelse (quenching og ekstraktion) procedure til intracellulære metabolitter var nødvendig for at kunne opnå betydningsfulde data. Hovedformålet var at finde en metode, der ville give det bedste kompromis mellem et acceptabelt energi ladnings forhold (ECR, normal mellem 0.80-0.95), lav lækage under quenchingen og høj gendannelse af metabolitterne efter ekstraktionen. Quenching og ekstraktions procedure for bakterier, gær, mammale celler og filamentiøse svampe blev analyseret. Quenchingen af bakterier og svampe viste sig at være en udfordrende opgave på grund af den høje modtagelighed af disse organismer overfor lækage under quenchingen. Quenching med myresyre, hvor cellerne ikke blev adskilt fra mediet, blev påvist at virke godt for Lactococcus lactis (L. lactis), men ikke for Streptomyces coelicolor (S. coelicolor) eller Microbispora corallina (M. corallina). Årsagen til dette blev antaget at være på grund aforganismernes filamentiøse vækst. Andre quenching procedurer blev testet for S. coelicolor og M. corallina med -40 °C MeOH/H2O (60/40, v/v), resulterende i en acceptabel ECR (i området 0.80-0.90). Dog blev lækage observeret for begge organismer. For filamentiøse svampe blev filtrering i kombination med kold metanol brugt til quenching. Men koncentrationerne af AMP og ADP i quenching supernatanten var omkring 30 % af den totale mængde fundet i biomassen og supernatanten. Kold MeOH som en quenching metode i kombination med EtOH eller MeOH/kloroform som ekstraktions metode viste sig at fungere godt for Saccharomyces cerevisiae (S. cerevisiae). Saltvand ved 0 °C viste sig, som en god quenchings opløsning for mammale celler imens ekstraktions proceduren var baseret på tilsætning af kold MeOH og ACN/H2O (50/50, v/v). I et andet studie udført under denne Ph.D. blev en ny tilgang etableret til dannelsen af en kunstig matrix som kan bruges til validering af analytiske metoder. Med dette omgås problemet med mangel af en matrix fri for analytterne, som er nødvendig til forberedelsen af kalibreringskurverne til kvantificering af intracellulære metabolitter. Spiking matricen blev produceret ved at ekstrahere biomasse opnået fra voksende S. cerevisiae i et medie, som indeholdt 13C mærket glukose/umærket glukose (50/50, w/w). Fordelen ved at bruge denne matrix var, at puljerne af stoffer med kun 12C eller 13C kulstoffer var meget lave eller næsten ikke målbare og viste minimal eller ingen interferens af den spikede mængde af umærkede standarder og deres stabile isotopiske mærkede interne standarder (SIL-IS). Til slut blev den udviklede IR-RP LC-MS metode koblet til en quadropol time of flight (QTOF) MS til multimålrettet analyse, detektion og identifikation af så mange kendte ukendte metabolitter, som muligt i forskellige biologiske matricer. Gennemførligheden ved brugen af Q-TOF MS blev evalueret ved at analysere ekstrakter fra tre forskellige organismer: S. cerevisiae, M. corallina og S. coelicolor. Screenings konceptet i dette studie, var baseret på to tilgange: i) aggresiv dereplikation af fuldt scannet høj opløsning MS (HR-MS) data ved brug af søgningslister med kendte stoffer og ii) høj opløsning tandem MS (MS/HRMS) data gennemsøgt i Metabolite Link (METLIN) biblioteket. Den data, som er blevet præsenteret her viser at metoderne udviklet under dette Ph.D. studium blev succesfuldt anvendt til målrettet og multimålrettet analyse af forskellige klasser af intracellulære metabolitter såsom nukleotider, sukker fosfater, coenzymer og organiske syre. Endvidere blev prøve forberedelses metoder etableret til forskellige mikroorganismer der let kan ekstrahere et bredt udvalg af metabolitter med god gendannelse. Slutteligt blev disse metoder påvist at være en værdifuld tilføjelse til ”omics” værktøjerne brugt til at afslører nøgle informationer angående metabolismen og reguleringen i biologiske systemer.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 samplepreparation 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 50intracellular 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 werenot 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 °Cshowed 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 themetabolism and the regulation in the biological systems

Online Research Database In Technology

LC-MS based Metabolomics

http://orbit.dtu.dk/ws/files/127980671/LC_MS_based_Metabolomics.pdf

LC-MS based Metabolomics

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Online Research Database In Technology