Separation, identification and quantitation of metabolites in biological systems by liquid chromatography and tandem mass spectrometry

Die Trennung, Identifizierung und Quantifizierung von Metaboliten in biologischen Systemen stellt eine große Herausforderung für analytisch chemische Methoden dar. Generell, macht die Analytik von Metaboliten und Biomolekülen in biologischen Proben meist eine Reduktion der hohen Probenkomplexizität nötig. Darüber hinaus bedingt die Identifizierung von Analyten im Spurenbereich auch die Einführung hoch selektiver und sensitiver Messtechniken. Hochleistungsflüssigkeitschromatographie (HPLC) ist unter zu Hilfenahme einer breiten Anzahl an verfügbaren Selektivitätsprinzipien eine sehr vielseitige Methode zur Stofftrennung. Die Kopplung von HPLC und Massenspektrometrie (MS) stellt eine Kombination orthogonaler Selektivitäten mit sehr hoher Detektionsempfindlichkeit dar. Zielgerichtete Derivatisierungsstrategien können synergistisch eine weitere Steigerung sowohl der Selektivität, als auch der Empfindlichkeit der so erstellten Methoden zur Folge haben. Aminosäuren stellen die zentralen Analyten dieser Arbeit dar und sie wurden mit Hinblick auf eine große Breite ihrer biologischen Relevanz untersucht. Cystein wurde in seiner Funktion als Acetaldehyd-Fänger betrachtet und lieferte dabei wertvolle Informationen zum Alkoholstoffwechsel im menschlichen Körper, weiters wurde es als Biomarker für kurzfristigen Alkoholkonsum erforscht. In einem fundamentalwissenschaftlichen Ansatz wurden enantioselektive Zwitterionenaustauscher als stationäre Phasen für die HPLC untersucht. Sie wurden dabei auf präparative Trennungen von derivatisierten Aminosäureenantiomeren angewandt. Durch die Anwendung diverser LC-MS Methoden in Kombination mit selektivitäts-fördernden Deriviatisierungsstrategien konnten Spuren von D – Aminosäuren in biologischen Flüssigkeiten nachgewiesen werden. Zu diesem Zweck wurden zwei verschiedene Labelling Methoden angewendet. Die Einführung eines Metalloferrocen – Tags erlaubte die Trennung fast aller chiralen, proteinogenen Aminosäuren und erlaubte eine sehr empfindliche massenspektrometrische Detektion. In einem Ansatz diese Eigenschaften noch weiter zu verbessern, führte die Derivatisierung von Aminosäuren mit Methoxychinolin zur Trennung aller chiralen, proteinogenen Aminosäuren und einer weiteren Steigerung der MS-Empfindlichkeit, darüber hinaus wurde so eine fluoreszierende Funktionalität eingeführt, die zusätzlich HPLC mit hochempfindlicher Fluoreszenzdetektion erlaubte. Zuletzt wurde Strukturinformation über Proteine und Proteinkomplexe durch chemisches Quervernetzen gewonnen. Bei dieser Methode wurden räumlich nahe Lysin Seitenketten interagierender Proteine kovalent aneinander gebunden. In einem weiteren Schritt wurden die Proteine proteolytisch verdaut und die so gewonnen quervernetzten Peptide mit Größenausschlusschromatographie angereichert. Die nachfolgende nano-HPLC Kopplung mit hochauflösender MS stellte die ultimative Herausforderung für die Leistungfähigkeit sowohl der Chromatographie, als auch der Massenspektrometrie dar. Letztlich lieferten drei Faktoren komplementäre und essenzielle Beiträge zur Einführung von Selektivität und somit zum erfolgreichen Abschluss der Experimente: chemische Derivatisierung, chromatographische Trennung und tandem-massenspektrometrische Detektion.The separation, identification and quantitation of metabolites in biological systems poses an important, yet challenging field of application in analytical chemistry. Generally, monitoring metabolites and biomolecules in biological samples demands for a reduction of sometimes very high sample complexity. Furthermore, the identification of trace amounts of these analytical targets necessitates the introduction of highly selective and highly sensitive analytical techniques. High performance liquid chromatography (HPLC) is a powerful and versatile method for compound separations utilizing a broad range of selectivity principles. The coupling of HPLC with mass spectrometric (MS) detection combines orthogonal selectivities with very high detection sensitivity. Dedicated analyte derivatization procedures can be synergistically employed to introduce a final advance in both detection selectivity and sensitivity. Amino acids pose the central analytical targets in the present thesis and they were analysed according to a wide spectrum of their biological roles. Cysteine was investigated as an acetaldehyde scavenger to deliver valuable information on the metabolic pathways of alcohol in humans; moreover it was evaluated as a biomarker for recent ethanol consumption. In a fundamental scientific approach, new enantioselective zwitter ion exchanger chiral stationary phases for liquid chromatography were studied with respect to their ability of preparatively separating amino acid derivatives. The detection of trace amounts of D – amino acids in biological fluids was established by different HPLC-MS strategies in combination with selectivity enhancing derivatization protocols. For this reason two different labelling strategies were developed. The introduction of a metalloferrocene tag to amino acids facilitated enantioselective separation of most proteinogenic amino acids and enabled very sensitive mass spectrometric detection. In an attempt to further improve these selectivity and sensitivity promoting properties, a methoxyquinolinoyl tag was employed that allowed for the enantioseparation of all chiral proteinogenic amino acids, delivered supreme mass spectrometric detectability and moreover introduced fluorescence activity. Thereby this labelling approach additionally offered the applicability to highly sensitive LC methods with fluorescence detection. Finally, structural information of protein complexes was gathered in approaches involving the covalent crosslinking between the lysine side chains of protein regions in close proximity and consecutive proteolytic digest of the proteins. Due to a very high number of chemically similar matrix constituents, protein crosslinking experiments demanded for ultimate separation capability of liquid chromatography and pushed the performance of MS detection towards its limits. Thus, enrichment of the generated cross linked peptides via size exclusion fractionation and nano – HPLC – high resolution mass spectrometric detection were employed to deliver this analytical peak performance. Essentially, the successful outcome of the experiments was generally associated with the complementary contribution of chemical derivatization, chromatographic separation and tandem mass spectrometric detection to overall method selectivity and sensitivity

Copper oxide nanoparticle toxicity profiling using untargeted metabolomics

BackgroundThe rapidly increasing number of engineered nanoparticles (NPs), and products containing NPs, raises concerns for human exposure and safety. With this increasing, and ever changing, catalogue of NPs it is becoming more difficult to adequately assess the toxic potential of new materials in a timely fashion. It is therefore important to develop methods which can provide high-throughput screening of biological responses. The use of omics technologies, including metabolomics, can play a vital role in this process by providing relatively fast, comprehensive, and cost-effective assessment of cellular responses. These techniques thus provide the opportunity to identify specific toxicity pathways and to generate hypotheses on how to reduce or abolish toxicity.ResultsWe have used untargeted metabolome analysis to determine differentially expressed metabolites in human lung epithelial cells (A549) exposed to copper oxide nanoparticles (CuO NPs). Toxicity hypotheses were then generated based on the affected pathways, and critically tested using more conventional biochemical and cellular assays. CuO NPs induced regulation of metabolites involved in oxidative stress, hypertonic stress, and apoptosis. The involvement of oxidative stress was clarified more easily than apoptosis, which involved control experiments to confirm specific metabolites that could be used as standard markers for apoptosis; based on this we tentatively propose methylnicotinamide as a generic metabolic marker for apoptosis.ConclusionsOur findings are well aligned with the current literature on CuO NP toxicity. We thus believe that untargeted metabolomics profiling is a suitable tool for NP toxicity screening and hypothesis generation

Frontiers in Plant Science / Phosphoglucomutase is not the target for galactose toxicity in plants

Plants synthesize a number of different oligomeric or polymeric sugars containing galactose. During growth and development some of these carbohydrates are metabolized or remodeled releasing galactose as a breakdown product. All plants have established recycling pathways for such sugars, for which they seem to have a limited capacity to cope with. Exceeding these limits results in sugar toxicity, which is observed already at concentrations as low as 1 mmol·l1 for galactose. The mechanism of galactose toxicity is poorly understood but it seems plausible that the enzymes involved in carbohydrate metabolism also might be the targets responsible for the adverse effects. Data from yeast and bacteria suggests that the enzyme phosphoglucomutase (PGM) is inhibited by galactose-1-phosphate. To test this hypothesis for plants we expressed recombinant cytosolic PGM3 from Arabidopsis in E. coli. Intriguingly, the enzyme was not inhibited by galactose-1-phosphate at physiological concentrations. Furthermore, PGM3 did not convert galactose-1-phosphate to galactose-6-phosphate, which was suggested as the inhibitory mode of action in yeast. In addition, metabolite levels in Arabidopsis roots were analyzed for their galactose-1-phosphate concentration by means of GCMS. Seedlings grown on MS-media with sucrose contained less than 10 nmol·g FW1 of galactose-1-phosphate. However, seedlings from plates, in which the sucrose was replaced by galactose, showed a strong increase of Gal-1-P to levels of up to 200 nmol·g FW1.(VLID)491972

Selectivity issues in targeted metabolomics: Separation of phosphorylated carbohydrate isomers by mixed-mode hydrophilic interaction/weak anion exchange chromatography

Phosphorylated carbohydrates are important intracellular metabolites and thus of prime interest in metabolomics research. Complications in their analysis arise from the existence of structural isomers that do have similar fragmentation patterns in MS/MS and are hard to resolve chromatographically. Herein, we present selective methods for the liquid chromatographic separation of sugar phosphates, such as hexose and pentose phosphates, 2- and 3-phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, as well as glucosamine 1- and 6-phosphate utilizing mixed-mode chromatography with reversed-phase/weak anion-exchangers and a charged aerosol detector. The best results were obtained when the reversed-phase/weak anion-exchanger column was operated under hydrophilic interaction liquid chromatography elution conditions. The effects of various chromatographic parameters were examined and are discussed on the basis of a simple stoichiometric displacement model for explaining ion-exchange processes. Employed acidic conditions have led to the complete separation of α- and β-anomers of glucose 6-phosphate at low temperature. The anomers coeluted in a single peak at elevated temperatures (>40°C) (peak coalescence), while at intermediate temperatures on-column interconversion with a plateau in-between resolved anomer peaks was observed with apparent reaction rate constants between 0.1 and 27.8×10(-4) s(-1). Dynamic HPLC under specified conditions enabled to investigate mutarotation of phosphorylated carbohydrates, their interconversion kinetics, and energy barriers for interconversion. A complex mixture of six hexose phosphate structural isomers could be resolved almost completely

Imaging Peptide and Protein Chirality via Amino Acid Analysis by Chiral × Chiral Two-Dimensional Correlation Liquid Chromatography

The present contribution illustrates the utilization of a chiral × chiral two-dimensional liquid chromatography (2DLC) setup with <i>tert</i>-butylcarbamoyl quinine chiral stationary phase (CSP) in the first dimension (¹D) and <i>tert</i>-butylcarbamoyl quinidine CSP in the second dimension (²D) to analyze FMOC-derivatized d and l amino acids from peptide hydrolysates. Hereby, in the ¹D and ²D chiral separation dimensions factors such as selector and immobilization chemistry of the CSPs, mobile phase, temperature, column hardware dimensions, stationary phase supports, particle type and packing were identical. Orthogonality between ¹D and ²D CSPs was solely based on their stereochemistry, i.e. their opposite configurations in two chiral centers of the selector molecules, which results in inversion of enantiomer elution orders in the two dimensions. Using Coreshell CSPs for fast chromatography allowed ²D-flow rates which were 60 times faster than the ¹D-flow rates to enable online comprehensive two-dimensional chromatography (LC × LC). Due to very similar chemoselectivity, yet opposite elution orders of corresponding enantiomers in ¹D and ²D, characteristic 2D-elution patterns for achiral and chiral components can be generated. Peaks of achiral components and impurities are lined up on the diagonal line in the 2D separation space (contour plot) and thereby removed from the chromatographic space of the target enantiomers avoiding overlaps with potential interferences. Corresponding enantiomers provide cross peaks on the 2D chromatogram. Moreover, enantioselectivity of both single CSPs is combined to result in an enhanced overall 2D enantioselectivity. The concept is illustrated for the therapeutic peptides gramicidin and bacitracin. Since all amino acids give a consistent elution order as FMOC-derivatives, all enantiomers of the same configuration are either above or below the diagonal line allowing straightforward imaging of the configuration of the amino acids in peptides by the 2D chromatogram

Fatal anthrax infection in a heroin user from southern Germany, June 2012

Blood cultures from a heroin user who died in June 2012, a few hours after hospital admission, due to acute septic disease, revealed the presence of Bacillus anthracis. This report describes the extended diagnosis by MALDI-TOF and real-time PCR and rapid confirmation of the anthrax infection through reference laboratories. Physicians and diagnostic laboratories were informed and alerted efficiently through the reporting channels of German public health institutions, which is essential for the prevention of further cases

Injektionsmilzbrand – neu aufgetretene Fälle bei Heroinabhängigen

Hintergrund: Injektionsmilzbrand ist eine seltene Erkrankung bei Heroinkonsumierenden und wird durch Bacillus anthracis verursacht. Im Jahr 2012 sind vier Fälle in Deutschland aufgetreten, wovon ein Fall tödlich verlief, sowie vereinzelt Fälle in verschiedenen Ländern Europas wie Dänemark, Frankreich und Großbritannien. Bereits 2009/2010 gab es drei mit Drogen assoziierte Injektionsmilzbrandfälle in Deutschland. Der Fokus des damaligen Ausbruchs lag mit 119 Erkrankten in Schottland. Falldarstellung und Verlauf: Es werden drei Fälle von Injektionsmilzbrand vorgestellt, wovon zwei Patienten in Regensburg und einer in Berlin behandelt wurden. Ein Patient starb am Tag der Aufnahme infolge eines Multiorganversagens. Die anderen Patienten erhielten Antibiotika, und es wurde ein chirurgisches Wunddebridement durchgeführt. Die Labordiagnose beruhte auf dem Erregernachweis vorwiegend mittels kultureller Anzucht und/oder Polymerasekettenreaktion direkt aus der Patientenprobe und wurde durch einen spezifischen Antikörpernachweis unterstützt. Schlussfolgerung: Injektionsmilzbrand kann als eigene oder als Sonderform des Hautmilzbrands mit massiver Ödembildung sowie häufig nekrotisierender Fasciitis und einer Letalität von circa 30 % betrachtet werden. Die Erkrankung ist in den letzten Jahren in verschiedenen Ländern Europas unter Heroinkonsumierenden aufgetreten. Bei entsprechendem klinischen Bild verbunden mit Heroinkonsum muss frühzeitig eine Anthrax-Infektion in Betracht gezogen und labordiagnostisch untersucht werden. Eine frühe adäquate Therapie dieser schweren Erkrankung kann lebensrettend sein, daher sollten die Ärzteschaft und die Risikogruppe hierüber unterrichtet sein.Background: Injection anthrax is a rare disease that affects heroin users and is caused by Bacillus anthracis. In 2012, there were four cases in Germany, one of which was fatal, as well as a small number of cases in other European countries, including Denmark, France, and the United Kingdom. Three cases among drug users occurred in Germany in 2009/2010, in the setting of a larger outbreak centered on Scotland, where there were 119 cases. Case presentation and clinical course: We present three cases of injection anthrax, two of which were treated in Regensburg and one in Berlin. One patient died of multi-organ-system failure on the day of admission to the hospital. The others were treated with antibiotics, one of them also with surgical wound debridement. The laboratory diagnosis of injection anthrax is based on the demonstration of the pathogen, generally by culture and/or by polymerase chain reaction, in material removed directly from the patient’s wound. The diagnosis is additionally supported by the detection of specific antibodies. Conclusion: Injection anthrax may be viewed either as an independent disease entity or as a special type of cutaneous anthrax with massive edema, necrotizing fasciitis in many cases, and about 30% mortality. It has appeared in recent years among heroin users in various European countries. In patients with suggestive clinical presentation and a history of heroin use, anthrax infection must be suspected early, so that the appropriate diagnostic tests can be performed without delay. Timely treatment can be life-saving. It is therefore important that physicians—and the individuals at risk—should be well-informed about this disease