TY - CHAP A1 - Das, Hirakjyoti A1 - Zografakis, Alexandros A1 - Oeljeklaus, Silke A1 - Warscheid, Bettina T1 - Analysis of Yeast Peroxisomes via Spatial Proteomics T1 - Analyse von Hefeperoxisomen durch Spatial Proteomik T2 - Peroxisomes N2 - Peroxisomes are ubiquitous organelles with essential functions in numerous cellular processes such as lipid metabolism, detoxification of reactive oxygen species and signaling. Knowledge of the peroxisomal proteome including multi-localized proteins and, most importantly, changes of its composition induced by altering cellular conditions or impaired peroxisome biogenesis and function is of paramount importance for a holistic view on peroxisomes and their diverse functions in a cellular context. In this chapter, we provide a spatial proteomics protocol specifically tailored to the analysis of the peroxisomal proteome of baker's yeast that enables the definition of the peroxisomal proteome under distinct conditions and to monitor dynamic changes of the proteome including the relocation of individual proteins to a different cellular compartment. The protocol comprises subcellular fractionation by differential centrifugation followed by Nycodenz density gradient centrifugation of a crude peroxisomal fraction, quantitative mass spectrometric measurements of subcellular and density gradient fractions and advanced computational data analysis, resulting in the establishment of organellar maps on a global scale. KW - peroxisome purification KW - mass spectrometry KW - label-free quantification KW - protein localization KW - spatial proteomics KW - Saccharomyces cerevisiae KW - differential centrifugation KW - density gradient centrifugation KW - organellar mapping Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-327532 PB - Springer ET - accepted version ER - TY - JOUR A1 - Koderer, Corinna A1 - Schmitz, Werner A1 - Wünsch, Anna Chiara A1 - Balint, Julia A1 - El-Mesery, Mohamed A1 - Volland, Julian Manuel A1 - Hartmann, Stefan A1 - Linz, Christian A1 - Kübler, Alexander Christian A1 - Seher, Axel T1 - Low energy status under methionine restriction is essentially independent of proliferation or cell contact inhibition JF - Cells N2 - Nonlimited proliferation is one of the most striking features of neoplastic cells. The basis of cell division is the sufficient presence of mass (amino acids) and energy (ATP and NADH). A sophisticated intracellular network permanently measures the mass and energy levels. Thus, in vivo restrictions in the form of amino acid, protein, or caloric restrictions strongly affect absolute lifespan and age-associated diseases such as cancer. The induction of permanent low energy metabolism (LEM) is essential in this process. The murine cell line L929 responds to methionine restriction (MetR) for a short time period with LEM at the metabolic level defined by a characteristic fingerprint consisting of the molecules acetoacetate, creatine, spermidine, GSSG, UDP-glucose, pantothenate, and ATP. Here, we used mass spectrometry (LC/MS) to investigate the influence of proliferation and contact inhibition on the energy status of cells. Interestingly, the energy status was essentially independent of proliferation or contact inhibition. LC/MS analyses showed that in full medium, the cells maintain active and energetic metabolism for optional proliferation. In contrast, MetR induced LEM independently of proliferation or contact inhibition. These results are important for cell behaviour under MetR and for the optional application of restrictions in cancer therapy. KW - methionine restriction KW - caloric restriction KW - mass spectrometry KW - LC/MS KW - liquid chromatography/mass spectrometry KW - metabolomics KW - L929 KW - amino acid KW - proliferation KW - contact inhibition Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262329 SN - 2073-4409 VL - 11 IS - 3 ER - TY - JOUR A1 - Berger, Nathalie A1 - Demolombe, Vincent A1 - Hem, Sonia A1 - Rofidal, Valérie A1 - Steinmann, Laura A1 - Krouk, Gabriel A1 - Crabos, Amandine A1 - Nacry, Philippe A1 - Verdoucq, Lionel A1 - Santoni, Véronique T1 - Root membrane ubiquitinome under short-term osmotic stress JF - International Journal of Molecular Sciences N2 - Osmotic stress can be detrimental to plants, whose survival relies heavily on proteomic plasticity. Protein ubiquitination is a central post-translational modification in osmotic-mediated stress. In this study, we used the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry to compile a list of 719 ubiquitinated lysine (K-Ub) residues from 450 Arabidopsis root membrane proteins (58% of which are transmembrane proteins), thereby adding to the database of ubiquitinated substrates in plants. Although no ubiquitin (Ub) motifs could be identified, the presence of acidic residues close to K-Ub was revealed. Our ubiquitinome analysis pointed to a broad role of ubiquitination in the internalization and sorting of cargo proteins. Moreover, the simultaneous proteome and ubiquitinome quantification showed that ubiquitination is mostly not involved in membrane protein degradation in response to short osmotic treatment but that it is putatively involved in protein internalization, as described for the aquaporin PIP2;1. Our in silico analysis of ubiquitinated proteins shows that two E2 Ub-conjugating enzymes, UBC32 and UBC34, putatively target membrane proteins under osmotic stress. Finally, we revealed a positive role for UBC32 and UBC34 in primary root growth under osmotic stress. KW - aquaporin KW - mass spectrometry KW - osmotic stress KW - ubiquitination Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284003 SN - 1422-0067 VL - 23 IS - 4 ER - TY - JOUR A1 - Volland, Julian Manuel A1 - Kaupp, Johannes A1 - Schmitz, Werner A1 - Wünsch, Anna Chiara A1 - Balint, Julia A1 - Möllmann, Marc A1 - El-Mesery, Mohamed A1 - Frackmann, Kyra A1 - Peter, Leslie A1 - Hartmann, Stefan A1 - Kübler, Alexander Christian A1 - Seher, Axel T1 - Mass spectrometric metabolic fingerprinting of 2-Deoxy-D-Glucose (2-DG)-induced inhibition of glycolysis and comparative analysis of methionine restriction versus glucose restriction under perfusion culture in the murine L929 model system JF - International Journal of Molecular Sciences N2 - All forms of restriction, from caloric to amino acid to glucose restriction, have been established in recent years as therapeutic options for various diseases, including cancer. However, usually there is no direct comparison between the different restriction forms. Additionally, many cell culture experiments take place under static conditions. In this work, we used a closed perfusion culture in murine L929 cells over a period of 7 days to compare methionine restriction (MetR) and glucose restriction (LowCarb) in the same system and analysed the metabolome by liquid chromatography mass spectrometry (LC-MS). In addition, we analysed the inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) over a period of 72 h. 2-DG induced very fast a low-energy situation by a reduced glycolysis metabolite flow rate resulting in pyruvate, lactate, and ATP depletion. Under perfusion culture, both MetR and LowCarb were established on the metabolic level. Interestingly, over the period of 7 days, the metabolome of MetR and LowCarb showed more similarities than differences. This leads to the conclusion that the conditioned medium, in addition to the different restriction forms, substantially reprogramm the cells on the metabolic level. KW - amino acid restriction KW - glucose restriction KW - mass spectrometry KW - low carb KW - 2-deoxy-D-glucose KW - 2-DG KW - methionine KW - perfusion culture KW - energy restriction KW - caloric restriction Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286007 SN - 1422-0067 VL - 23 IS - 16 ER - TY - JOUR A1 - Schmitz, Werner A1 - Koderer, Corinna A1 - El-Mesery, Mohamed A1 - Gobik, Sebastian A1 - Sampers, Rene A1 - Straub, Anton A1 - Kübler, Alexander Christian A1 - Seher, Axel T1 - Metabolic fingerprinting of murine L929 fibroblasts as a cell-based tumour suppressor model system for methionine restriction JF - International Journal of Molecular Sciences N2 - Since Otto Warburg reported in 1924 that cancer cells address their increased energy requirement through a massive intake of glucose, the cellular energy level has offered a therapeutic anticancer strategy. Methionine restriction (MetR) is one of the most effective approaches for inducing low-energy metabolism (LEM) due to the central position in metabolism of this amino acid. However, no simple in vitro system for the rapid analysis of MetR is currently available, and this study establishes the murine cell line L929 as such a model system. L929 cells react rapidly and efficiently to MetR, and the analysis of more than 150 different metabolites belonging to different classes (amino acids, urea and tricarboxylic acid cycle (TCA) cycles, carbohydrates, etc.) by liquid chromatography/mass spectrometry (LC/MS) defines a metabolic fingerprint and enables the identification of specific metabolites representing normal or MetR conditions. The system facilitates the rapid and efficient testing of potential cancer therapeutic metabolic targets. To date, MS studies of MetR have been performed using organisms and yeast, and the current LC/MS analysis of the intra- and extracellular metabolites in the murine cell line L929 over a period of 5 days thus provides new insights into the effects of MetR at the cellular metabolic level. KW - methionine restriction KW - caloric restriction KW - mass spectrometry KW - LC/MS KW - liquid chromatography/mass spectrometry KW - metabolism KW - L929 KW - amino acid Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259198 SN - 1422-0067 VL - 22 IS - 6 ER - TY - JOUR A1 - Schmitz, Werner A1 - Ries, Elena A1 - Koderer, Corinna A1 - Völter, Maximilian Friedrich A1 - Wünsch, Anna Chiara A1 - El-Mesery, Mohamed A1 - Frackmann, Kyra A1 - Kübler, Alexander Christian A1 - Linz, Christian A1 - Seher, Axel T1 - Cysteine restriction in murine L929 fibroblasts as an alternative strategy to methionine restriction in cancer therapy JF - International Journal of Molecular Sciences N2 - Methionine restriction (MetR) is an efficient method of amino acid restriction (AR) in cells and organisms that induces low energy metabolism (LEM) similar to caloric restriction (CR). The implementation of MetR as a therapy for cancer or other diseases is not simple since the elimination of a single amino acid in the diet is difficult. However, the in vivo turnover rate of cysteine is usually higher than the rate of intake through food. For this reason, every cell can enzymatically synthesize cysteine from methionine, which enables the use of specific enzymatic inhibitors. In this work, we analysed the potential of cysteine restriction (CysR) in the murine cell line L929. This study determined metabolic fingerprints using mass spectrometry (LC/MS). The profiles were compared with profiles created in an earlier work under MetR. The study was supplemented by proliferation studies using D-amino acid analogues and inhibitors of intracellular cysteine synthesis. CysR showed a proliferation inhibition potential comparable to that of MetR. However, the metabolic footprints differed significantly and showed that CysR does not induce classic LEM at the metabolic level. Nevertheless, CysR offers great potential as an alternative for decisive interventions in general and tumour metabolism at the metabolic level. KW - methionine restriction KW - cysteine restriction KW - mass spectrometry KW - LC/MS KW - cancer therapy KW - caloric restriction KW - homocysteine KW - amino acid analogues KW - cysteine synthase inhibitor Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265486 SN - 1422-0067 VL - 22 IS - 21 ER - TY - THES A1 - Carstensen, Anne Carola T1 - Identification of novel N-MYC interacting proteins reveals N-MYC interaction with TFIIIC T1 - Identifizierung von neuen N-MYC interagierenden Proteinen offenbart N-MYC's Interaktion mit TFIIIC N2 - N-MYC is a member of the human MYC proto-oncogene family, which comprises three transcription factors (C-, N- and L-MYC) that function in multiple biological processes. Deregulated expression of MYC proteins is linked to tumour initiation, maintenance and progression. For example, a large fraction of neuroblastoma displays high N-MYC levels due to an amplification of the N-MYC encoding gene. MYCN-amplified neuroblastoma depend on high N-MYC protein levels, which are maintained by Aurora-A kinase. Aurora-A interaction with N-MYC interferes with degradation of N-MYC via the E3 ubiquitin ligase SCFFBXW7. However, the underlying mechanism of Aurora-A-mediated stabilisation of N-MYC remains to be elucidated. To identify novel N-MYC interacting proteins, which could be involved in N-MYC stabilisation by Aurora-A, a proteomic analysis of purified N-MYC protein complexes was conducted. Since two alanine mutations in MBI of N-MYC, T58A and S62A (N-MYC mut), disable Aurora-A-mediated stabilisation of N-MYC, N-MYC protein complexes from cells expressing either N-MYC wt or mut were analysed. Proteomic analysis revealed that N-MYC interacts with two deubiquitinating enzymes, USP7 and USP11, which catalyse the removal of ubiquitin chains from target proteins, preventing recognition by the proteasome and subsequent degradation. Although N-MYC interaction with USP7 and USP11 was confirmed in subsequent immunoprecipitation experiments, neither USP7, nor USP11 was shown to be involved in the regulation of N-MYC stability. Besides USP7/11, proteomic analyses identified numerous additional N-MYC interacting proteins that were not described to interact with MYC transcription factors previously. Interestingly, many of the identified N-MYC interaction partners displayed a preference for the interaction with N-MYC wt, suggesting a MBI-dependent interaction. Among these were several proteins, which are involved in three-dimensional organisation of chromatin domains and transcriptional elongation by POL II. Not only the interaction of N-MYC with proteins functioning in elongation, such as the DSIF component SPT5 and the PAF1C components CDC73 and CTR9, was validated in immunoprecipitation experiments, but also with the POL III transcription factor TFIIIC and topoisomerases TOP2A/B. ChIP-sequencing analysis of N-MYC and TFIIIC subunit 5 (TFIIIC5) revealed a large number of joint binding sites in POL II promoters and intergenic regions, which are characterised by the presence of a specific motif that is highly similar to the CTCF motif. Additionally, N-MYC was shown to interact with the ring-shaped cohesin complex that is known to bind to CTCF motifs and to assist the insulator protein CTCF. Importantly, individual ChIP experiments demonstrated that N-MYC, TFIIIC5 and cohesin subunit RAD21 occupy joint binding sites comprising a CTCF motif. Collectively, the results indicate that N-MYC functions in two biological processes that have not been linked to MYC biology previously. Furthermore, the identification of joint binding sites of N-MYC, TFIIIC and cohesin and the confirmation of their interaction with each other suggests a novel function of MYC transcription factors in three-dimensional organisation of chromatin. N2 - N-MYC ist ein Mitglied der humanen MYC proto-Onkogen Familie, welche drei Transkriptionsfaktoren umfasst (C-,N- und L-MYC), die in zahlreichen biologischen Prozessen fun-gieren. Deregulierte Expression der MYC Proteine ist mit Tumorinitiierung, -erhalt und -progression verbunden. Zum Beispiel zeigt ein großer Anteil an Neuroblastomen aufgrund einer Amplifizierung des N-MYC kodierenden Gens hohe N-MYC Level. MYCN-amplifizierte Neuroblastome hängen von den hohen N-MYC Protein Leveln ab, die durch die Aurora-A Kinase erhalten werden. Die Interaktion von Aurora-A mit N-MYC behindert den Abbau von N-MYC durch die E3 Ubiquitin Ligase SCFFBXW7. Allerdings muss der zugrunde liegende Mechanismus der Aurora-A vermittelten Stabilisierung von N-MYC noch aufgedeckt werden. Um neue N-MYC interagierende Proteine zu identifizieren, welche in der N-MYC Stabilisierung durch Aurora-A involviert sind, wurde eine Proteom Analyse der aufgereinigten N-MYC Proteinkomplexe durchgeführt. Da zwei Alanin-Mutationen in MBI von N-MYC, T58A und S62A (N-MYC mut), die Aurora-A vermittelte Stabilisierung von N-MYC verhindern, wurden N-MYC Protein-Komplexe von Zellen, die entweder N-MYC wt oder mut exprimieren analysiert. Die Proteom Analyse offenbarte, dass N-MYC mit zwei Deubiquitinierenden Enzymen, USP7 und USP11, interagiert, welche das Entfernen von Ubiquitinketten von Zielproteinen katalysieren und dadurch die Erkennung durch das Proteasom und den darauf folgenden Abbau verhindern. Obwohl die Interaktion von N-MYC mit USP7 und USP11 in darauf folgenden Immunpräzipitationsexperimenten bestätigt wurde, konnnte weder für USP7, noch für USP11 gezeigt werden, dass es in die Regulierung der Stabilität von N-MYC involviert ist. Neben USP7/11 wurden in der Proteom Analyse zusätzlich zahlreiche mit N-MYC interagierende Proteine identifiziert, die zuvor noch nicht beschrieben wurden mit MYC Transkriptionsfaktoren zu interagieren. Interessanterweise zeigten viele der identifizierten N-MYC Interaktionspartner eine Präferenz für die Interaktion mit N-MYC wt, was eine MBI-abhängige Interaktion suggeriert. Unter diesen waren einige Proteine, die in die drei-dimensionale Organisation von Chromatindomänen und transkriptioneller Elongation durch POL II involviert sind. Nicht nur die Interaktion von N-MYC mit Proteinen, die in der Elongation agieren, wie die DSIF Komponente SPT5 und die PAF1C Komponenten CDC73 und CTR9, wurden in Immunpräzipitationsexperimenten bestätigt, sondern auch mit dem POL III Transkriptionsfaktor TFIIIC und den Topoisomerasen TOP2A/B. Analyse von ChIP-Sequenzierungsexperimenten für N-MYC und TFIIIC Untereinheit 5 (TFIIIC5) offenbarte eine große Anzahl von gemeinsamen Bindungsstellen in POL II Promotoren und intergenen Regionen, welche durch das Vorkommen eines speziellen Motivs gekennzeichent waren, das dem CTCF Motiv sehr ähnlich ist. Zusätzlich wurde gezeigt, dass N-MYC mit dem ringförmigen Cohesin Komplex interagiert, der dafür bekannt ist an CTCF Motive zu binden und dem Insulator Protein CTCF zu assistieren. Entscheidender Weise zeigten individuelle ChIP Experimente, dass N-MYC, TFIIIC5 und die Cohesin Untereinheit RAD21 gemeinsame Bindungstellen haben, die ein CTCF Motiv enthalten. Zusammenfassend weisen die Ergebnisse darauf hin, dass N-MYC in zwei biologischen Prozessen fungiert, die zuvor nicht mit der Biologie von MYC verbunden wurden. Zudem suggeriert die Identifizierung von gemeinsamen Bindungstellen von N-MYC, TFIIIC und Cohesin und die Bestätigung der Interaktion untereinander eine neue Funktion von MYC Transkriptionsfaktoren in der drei-dimensionalen Organisation von Chromatin. KW - Biologie KW - Transkriptionsfaktor KW - Onkogen KW - N-MYC KW - neuroblastoma KW - TFIIIC KW - Aurora-A KW - mass spectrometry KW - cohesin Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143658 ER - TY - JOUR A1 - Sbirkov, Yordan A1 - Kwok, Colin A1 - Bhamra, Amandeep A1 - Thompson, Andrew J. A1 - Gil, Veronica A1 - Zelent, Arthur A1 - Petrie, Kevin T1 - Semi-quantitative mass spectrometry in AML cells identifies new non-genomic targets of the EZH2 methyltransferase JF - International Journal of Molecular Sciences N2 - Alterations to the gene encoding the EZH2 (KMT6A) methyltransferase, including both gain-of-function and loss-of-function, have been linked to a variety of haematological malignancies and solid tumours, suggesting a complex, context-dependent role of this methyltransferase. The successful implementation of molecularly targeted therapies against EZH2 requires a greater understanding of the potential mechanisms by which EZH2 contributes to cancer. One aspect of this effort is the mapping of EZH2 partner proteins and cellular targets. To this end we performed affinity-purification mass spectrometry in the FAB-M2 HL-60 acute myeloid leukaemia (AML) cell line before and after all-trans retinoic acid-induced differentiation. These studies identified new EZH2 interaction partners and potential non-histone substrates for EZH2-mediated methylation. Our results suggest that EZH2 is involved in the regulation of translation through interactions with a number of RNA binding proteins and by methylating key components of protein synthesis such as eEF1A1. Given that deregulated mRNA translation is a frequent feature of cancer and that eEF1A1 is highly expressed in many human tumours, these findings present new possibilities for the therapeutic targeting of EZH2 in AML. KW - acute myeloid leukaemia KW - EZH2 KW - mass spectrometry KW - methylation KW - eEF1A1 Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285541 SN - 1422-0067 VL - 18 IS - 7 ER - TY - JOUR A1 - Macintyre, Lynsey A1 - Zhang, Tong A1 - Viegelmann, Christina A1 - Martinez, Ignacio Juarez A1 - Cheng, Cheng A1 - Dowdells, Catherine A1 - Abdelmohsen, Usama Ramadan A1 - Gernert, Christine A1 - Hentschel, Ute A1 - Edrada-Ebel, RuAngelie T1 - Metabolomic Tools for Secondary Metabolite Discovery from Marine Microbial Symbionts JF - Marine Drugs N2 - Marine invertebrate-associated symbiotic bacteria produce a plethora of novel secondary metabolites which may be structurally unique with interesting pharmacological properties. Selection of strains usually relies on literature searching, genetic screening and bioactivity results, often without considering the chemical novelty and abundance of secondary metabolites being produced by the microorganism until the time-consuming bioassay-guided isolation stages. To fast track the selection process, metabolomic tools were used to aid strain selection by investigating differences in the chemical profiles of 77 bacterial extracts isolated from cold water marine invertebrates from Orkney, Scotland using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Following mass spectrometric analysis and dereplication using an Excel macro developed in-house, principal component analysis (PCA) was employed to differentiate the bacterial strains based on their chemical profiles. NMR H-1 and correlation spectroscopy (COSY) were also employed to obtain a chemical fingerprint of each bacterial strain and to confirm the presence of functional groups and spin systems. These results were then combined with taxonomic identification and bioassay screening data to identify three bacterial strains, namely Bacillus sp. 4117, Rhodococcus sp. ZS402 and Vibrio splendidus strain LGP32, to prioritize for scale-up based on their chemically interesting secondary metabolomes, established through dereplication and interesting bioactivities, determined from bioassay screening. KW - multivariate analysis KW - metabolic profiling KW - metabolomics KW - dereplication KW - symbiotic bacteria KW - mass spectrometry KW - NMR KW - sponge holicolona-simulans KW - bryozoan bugula-neritina KW - polyketide synthase gene Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116097 SN - 1660-3397 VL - 12 IS - 6 ER - TY - THES A1 - Jakob-Rodamer, Verena T1 - Development and validation of LC-MS/MS methods to determine PK/PD parameters of anti-infectives T1 - Entwicklung und Validierung von LC-MS/MS Methoden zur Bestimmung von PK/PD-Parametern von Antiinfektiva N2 - In the present thesis the development and validation of bioanalytical LC-MS/MS methods for the quantification of erythromycin A, erythromycin ethylsuccinate, roxithromycin, clarithromycin, 14 hydroxy clarithromycin, flucloxacillin, piperacillin and moxifloxacin in human plasma and human urine (piperacillin) is introduced. All methods were applied to analyze human plasma and urine samples from clinical trials and therefore, have been validated according to international guidelines. The methods were reliable in these studies and fulfilled all regulatory requirements known at the time of the study conduct. Moreover, the validation data of the macrolides were compared on three different mass spectrometers (API III Plus, API 3000™, API 5000™). The new innovations in the ion source (horizontal versus vertical electrospray), the ionpath (skimmer, QJet) and the diameter of the orifice resulted in better sensitivity and a larger linearity range for the majority of the analytes. Sensitivity was improved up to a factor of 12 (for clarithromycin) between API III Plus to API 3000™ and up to a factor of 8 (for erythromycin and roxithromycin) between API 3000™ and API 5000™, keeping the accuracy and precision data at about the same level. The high sensitivity was a benefit for example for the flucloxacillin study, because concentrations from all subject samples were detectable up to approximately eight half-lives, i.e. no concentrations needed to be reported below the quantification limit. Also the linearity range were extended from two orders of magnitude to up to four orders of magnitude, which increases the likelihood to allow to analyze all samples from a pharmacokinetic study in the same run. This is especially useful if a large concentration range needs to be analysed, for example, if the method shall be applied in an ascending dose study. Then, all low concentrations from the beginning of the study can be determined, as well as all high concentrations, without the need to dilute and analyse single samples repeatedly. The pharmacokinetic data were compared to previously reported literature data and correlated graphically with MIC values of popular microorganisms which might be a starting point for further PK/PD investigations. The PK/PD theory is a very helpful tool for prediction of the efficacy of given drugs against certain micro-organisms. Depending on the pharmacodynamic processes, e. g. the mode of action, three classes of drugs have been identified. In the same way this applies to adverse effects, which need to be minimised by reducing plasma concentrations. These coherences are not well-investigated, yet, and are not discussed further in this thesis. Still, a lot of research has to be done in this interdisciplinary field to minimise uncertainty in single values, like an AUC/MIC. These include: Improve accuracy and precision of bioanalytical methods determining total and free concentration data in biological matrices for calculation of AUC and Cmax These parameters are related to the MIC in pharmacodynamic considerations. Since the determination of the MIC often underlies significant variations and also differences between microbiological laboratories, the determination of concentrations of anti-infectives is particular important, being achievable by scientific exact techniques. Finally, from the volume of distribution of antibiotics can be used to derive information about intracellular concentrations and effectivity of antiinfectives. N2 - In der vorliegenden Arbeit wird die Entwicklung und Validierung von bioanalytischen LC-MS/MS-Methoden zur Quantifizierung von Erythromycin A, Erythromycin-ethylsuccinat, Roxithromycin, Clarithromycin, 14 Hydroxy-clarithromycin, Flucloxacillin, Piperacillin und Moxifloxacin in Humanplasma und Humanurin (Piperacillin) vorgestellt. Alle Methoden wurden für die Analyse von Plasma- und Urinproben aus klinischen Studien beim Menschen eingesetzt und deshalb nach international anerkannten Richtlinien validiert. In diesen Studien haben sich die Methoden bewährt und alle Anforderungen der zum Zeitpunkt der Studie bekannten behördlichen Ansprüche erfüllt. Die Validierungsdaten der Makrolid-Methoden konnten zudem an drei Massenspektrometern der selben Baureihe verglichen werden. Dabei zeigte sich, dass die Neuerungen an der Ionenquelle (horizontale versus vertikale ESI), dem Ionenpfad (Skimmer, QJet), sowie das immer größere Orifice in der Baureihe, nicht nur stetig verbesserte Sensitivität ermöglichen, sondern auch immer größere Linearitätsbereiche. So konnte ein Sensitivitätsgewinn bis zu Faktor 12 (Clarithromycin) von API III Plus auf API 3000™ und bis zu Faktor 8 (Erythromycin und Roxithromycin) von API 3000™ auf API 5000™ bei etwa gleichen Werten für die Präzision erreicht werden. Die hohe Sensitivität zeigte sich zum Beispiel bei der Flucloxacillin Studie von Vorteil, da alle Konzentrationen bis circa acht Halbwertszeiten nach Wirkstoffgabe bestimmt werden konnten, ohne dass einzelne Proben als „BLOQ“ (unter dem Quantifizierungslimit) berichtet werden mussten. Während früher noch Linearitätsbereiche um zwei Größenordnungen üblich waren, sind jetzt am API 5000™ Konzentrationsbereiche über bis zu vier Größenordnungen reproduzierbar linear. Das ist insbesondere von Nutzen, wenn bei einer Substanz ein größerer Konzentrationsbereich gemessen werden muss. Das ist beispielsweise dann der Fall, wenn die Methode für eine Dosissteigerungsstudie eingesetzt werden soll. Dann können sowohl alle kleinen Konzentrationen zu Beginn der Studie gemessen werden, als auch alle hohen Konzentrationen zum Ende der Studie, und zwar ohne, dass einzelne Proben gesondert verdünnt und wiederholt gemessen werden müssen. Die pharmakokinetischen Daten der Antibiotika wurden in den Kontext mit Literaturdaten gestellt und graphisch mit MHK-Werten für gängige Mikroorganismen verglichen, was den Ausgangspunkt für spätere PK/PD-Untersuchungen darstellt. Für die Vorhersage der Wirksamkeit einer verabreichten Substanz gegenüber bestimmten Mikroorganismen ist die PK/PD-Korrelation ein gutes Hilfsmittel. Anhand der pharmakodynamischen Parameter, wie der Wirkungsweise, können drei Antibiotikaklassen gebildet werden. In analoger Weise gilt für die Nebenwirkungen, dass die Plasma-Konzentrationen verringert werden müssen. Diese Zusammenhänge sind jedoch weniger gut untersucht und werden in dieser Dissertation nicht näher diskutiert. In diesem interdisziplinären Feld ist immer noch viel Forschung nötig um beispielsweise Unsicherheiten bei einzelnen Werten wie z.B. AUC/MIC zu minimieren. Diese beinhalten: Verbesserung der Genauigkeit und Präzision von bioanalytischen Methoden welche für die Bestimmung der Gesamtkonzentration und der freien Konzentration in biologischen Matrices verwendet werden um AUC und Cmax zu bestimmen. Diese Parameter werden in pharmakodynamischen Überlegungen in Beziehung zur MHK gesetzt. Da die MHK-Bestimmung oft erheblichen Schwankungen und auch Unterschieden zwischen verschiedenen mikrobiologischen Laboratorien unterliegt, kommt der Konzentrationsbestimmung der Antibiotika besondere Bedeutung zu, weil sie mit naturwissenschaftlich exakten Methoden möglich ist. Aus den Verteilungsvolumina von Antibiotika lassen sich schließlich auch noch Informationen über die intrazelluläre Konzentration und Wirksamkeit von Antibiotika ableiten. KW - Antimikrobieller Wirkstoff KW - Pharmakokinetik KW - Pharmakodynamik KW - LC-MS KW - LC-MS/MS KW - anti-infectives KW - Antiinfektiva KW - mass spectrometry KW - liquid chromatography KW - antibiotic KW - pharmacokinetic KW - clinical study Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-109215 ER -