@article{ArltBiehlTayloretal.2011, author = {Arlt, Wiebke and Biehl, Michael and Taylor, Angela E. and Hahner, Stefanie and Lib{\´e}, Rossella and Hughes, Beverly A. and Schneider, Petra and Smith, David J. and Stiekema, Han and Krone, Nils and Porfiri, Emilio and Opocher, Giuseppe and Bertherat, Jer{\^o}me and Mantero, Franco and Allolio, Bruno and Terzolo, Massimo and Nightingale, Peter and Shackleton, Cedric H. L. and Bertagna, Xavier and Fassnacht, Martin and Stewart, Paul M.}, title = {Urine Steroid Metabolomics as a Biomarker Tool for Detecting Malignancy in Adrenal Tumors}, series = {The Journal of Clinical Endocrinology \& Metabolism}, volume = {96}, journal = {The Journal of Clinical Endocrinology \& Metabolism}, number = {12}, doi = {10.1210/jc.2011-1565}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-154682}, pages = {3775 -- 3784}, year = {2011}, abstract = {Context: Adrenal tumors have a prevalence of around 2\% in the general population. Adrenocortical carcinoma (ACC) is rare but accounts for 2-11\% of incidentally discovered adrenal masses. Differentiating ACC from adrenocortical adenoma (ACA) represents a diagnostic challenge in patients with adrenal incidentalomas, with tumor size, imaging, and even histology all providing unsatisfactory predictive values. Objective: Here we developed a novel steroid metabolomic approach, mass spectrometry-based steroid profiling followed by machine learning analysis, and examined its diagnostic value for the detection of adrenal malignancy. Design: Quantification of 32 distinct adrenal derived steroids was carried out by gas chromatography/mass spectrometry in 24-h urine samples from 102 ACA patients (age range 19-84 yr) and 45 ACC patients (20-80 yr). Underlying diagnosis was ascertained by histology and metastasis in ACC and by clinical follow-up [median duration 52 (range 26-201) months] without evidence of metastasis in ACA. Steroid excretion data were subjected to generalized matrix learning vector quantization (GMLVQ) to identify the most discriminative steroids. Results: Steroid profiling revealed a pattern of predominantly immature, early-stage steroidogenesis in ACC. GMLVQ analysis identified a subset of nine steroids that performed best in differentiating ACA from ACC. Receiver-operating characteristics analysis of GMLVQ results demonstrated sensitivity = specificity = 90\% (area under the curve = 0.97) employing all 32 steroids and sensitivity = specificity = 88\% (area under the curve = 0.96) when using only the nine most differentiating markers. Conclusions: Urine steroid metabolomics is a novel, highly sensitive, and specific biomarker tool for discriminating benign from malignant adrenal tumors, with obvious promise for the diagnostic work-up of patients with adrenal incidentalomas.}, language = {en} } @article{BergerDemolombeHemetal.2022, author = {Berger, Nathalie and Demolombe, Vincent and Hem, Sonia and Rofidal, Val{\´e}rie and Steinmann, Laura and Krouk, Gabriel and Crabos, Amandine and Nacry, Philippe and Verdoucq, Lionel and Santoni, V{\´e}ronique}, title = {Root membrane ubiquitinome under short-term osmotic stress}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {4}, issn = {1422-0067}, doi = {10.3390/ijms23041956}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284003}, year = {2022}, abstract = {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.}, language = {en} } @article{BerghoffKonzerManketal.2013, author = {Berghoff, Bork A. and Konzer, Anne and Mank, Nils N. and Looso, Mario and Rische, Tom and F{\"o}rstner, Konrad U. and Kr{\"u}ger, Marcus and Klug, Gabriele}, title = {Integrative "Omics"-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses}, series = {PLOS Genetics}, volume = {9}, journal = {PLOS Genetics}, number = {6}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1003576}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127587}, pages = {e1003576}, year = {2013}, abstract = {Bacteria constantly face stress conditions and therefore mount specific responses to ensure adaptation and survival. Stress responses were believed to be predominantly regulated at the transcriptional level. In the phototrophic bacterium Rhodobacter sphaeroides the response to singlet oxygen is initiated by alternative sigma factors. Further adaptive mechanisms include post-transcriptional and post-translational events, which have to be considered to gain a deeper understanding of how sophisticated regulation networks operate. To address this issue, we integrated three layers of regulation: (1) total mRNA levels at different time-points revealed dynamics of the transcriptome, (2) mRNAs in polysome fractions reported on translational regulation (translatome), and (3) SILAC-based mass spectrometry was used to quantify protein abundances (proteome). The singlet oxygen stress response exhibited highly dynamic features regarding short-term effects and late adaptation, which could in part be assigned to the sigma factors RpoE and RpoH2 generating distinct expression kinetics of corresponding regulons. The occurrence of polar expression patterns of genes within stress-inducible operons pointed to an alternative of dynamic fine-tuning upon stress. In addition to transcriptional activation, we observed significant induction of genes at the post-transcriptional level (translatome), which identified new putative regulators and assigned genes of quorum sensing to the singlet oxygen stress response. Intriguingly, the SILAC approach explored the stress-dependent decline of photosynthetic proteins, but also identified 19 new open reading frames, which were partly validated by RNA-seq. We propose that comparative approaches as presented here will help to create multi-layered expression maps on the system level ("expressome"). Finally, intense mass spectrometry combined with RNA-seq might be the future tool of choice to re-annotate genomes in various organisms and will help to understand how they adapt to alternating conditions.}, language = {en} } @phdthesis{Boerner2010, author = {B{\"o}rner, Juliane}, title = {Charakterisierung der Phosphorylierungsstellen der Guanylyl Cyklase A, dem Rezeptor f{\"u}r das atriale natriuretische Peptid, mittels Massenspektrometrie}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-51914}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Das ANP/GC-A-System spielt durch die Produktion des sekund{\"a}ren Botenstoffs cGMP eine wichtige Rolle bei der Regulation des Blutdruckes und des Blutvolumens. Bei Patienten mit Herzhypertrophie oder Herzinsuffizienz sind die ANP-Plasmakonzentrationen erh{\"o}ht, aber die GC-A-vermittelten Effekte stark reduziert, was auf einen Defekt des Signalsystems hinweist. Studien an metabolisch markierten GC-A-{\"u}berexprimierenden HEK 293-Zellen zeigten, dass der GC-A-Rezeptor im basalen Zustand stark phosphoryliert und die homologe bzw. heterologe Desensitisierung wahrscheinlich mit einer Dephosphorylierung verbunden ist. Die Desensitisierung stellt einen Mechanismus dar, der in vivo zu einem Funktionsverlust des Rezeptors beitragen k{\"o}nnte. Im Rahmen dieser Arbeit konnten mittels Massenspektrometrie sieben Phosphorylierungsstellen in der Kinasehomologen Dom{\"a}ne aus FLAG-GC-A exprimierenden HEK 293-Zellen detektiert werden: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 und Thr513. Die massenspektrometrische relative Quantifizierung basierend auf der Multiple-Reaction-Monitoring (MRM)-Methode zeigte bei ANP-induzierter, homologer Desensitisierung eine Dephosphorylierung der Phosphorylierungsstellen Ser497, Thr500, Ser502, Ser506, Ser510 und Thr513, was mit bereits publizierten Daten {\"u}bereinstimmt, und einen starken Anstieg der Phosphorylierung an Ser487. Nach Inkubation mit Angiotensin II, welches eine heterologe Desensitisierung hervorruft, wurde eine Reduzierung aller Phosphorylierungen verzeichnet, die zudem st{\"a}rker ausgepr{\"a}gt war als bei der ANP-abh{\"a}ngigen Desensitisierung. Die Funktion der neu identifizierten Phosphorylierung an Ser487 wurde mittels Mutagenese analysiert. Die Substitution des Serins durch Alanin, welche den unphosphorylierten Zustand nachstellt, resultierte in einer Rezeptoraktivit{\"a}t und desensitisierung vergleichbar zum GC-A Wildtyp-Rezeptor. Wurde hingegen Serin gegen Glutamat getauscht, um den phosphorylierten Zustand zu imitieren, konnte der Rezeptor weder aktiviert noch desensitisiert werden. Diese Ergebnisse best{\"a}tigen vorherige Studien, dass die GC-A-Rezeptorantwort auf ANP durch die Phosphorylierungen reguliert wird. Allerdings scheint bei der homologen Desensitisierung die Phosphorylierung an der Position Ser487 eine Rolle zu spielen, da sie die Aktivit{\"a}t des Rezeptors inhibiert. Die Identifizierung und Charakterisierung dieser Phosphorylierungsstelle tr{\"a}gt zum Verst{\"a}ndnis des Mechanismus der homologen Desensitierung bei. Zus{\"a}tzlich konnten einige der beschriebenen Phosphorylierungen in Zellsystemen detektiert werden, die die GC-A endogen exprimieren. Dadurch sind unter physiologischen Bedingungen Analysen der Mechanismen m{\"o}glich, die bei der Aktivierung und Deaktivierung der GC-A involviert sind und somit wichtige pathophysiologische Konsequenzen haben k{\"o}nnen.}, subject = {Guanylatcyclase}, language = {de} } @phdthesis{Carstensen2018, author = {Carstensen, Anne Carola}, title = {Identification of novel N-MYC interacting proteins reveals N-MYC interaction with TFIIIC}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143658}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {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.}, subject = {Biologie}, language = {en} } @incollection{DasZografakisOeljeklausetal.2023, author = {Das, Hirakjyoti and Zografakis, Alexandros and Oeljeklaus, Silke and Warscheid, Bettina}, title = {Analysis of Yeast Peroxisomes via Spatial Proteomics}, series = {Peroxisomes}, booktitle = {Peroxisomes}, edition = {accepted version}, publisher = {Springer}, doi = {10.1007/978-1-0716-3048-8_2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-327532}, publisher = {Universit{\"a}t W{\"u}rzburg}, pages = {13-31}, year = {2023}, abstract = {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.}, language = {en} } @phdthesis{Deubel2006, author = {Deubel, Alexandra}, title = {Charakterisierung des Verunreinigungsprofils von Erythromycin mittels chromatographischer Methoden und massenspektrometrischer Detektion}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-20160}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Im Rahmen der vorliegenden Arbeit wurden analytische Methoden zur Bestimmung des Verunreinigungsprofils von Erythromycin entwickelt, die der bestehenden Ph.Eur.-Methode {\"u}berlegen sind. Die neue HPLC-Methode ist in der Lage, alle verwandten Verbindungen mit angemessener Pr{\"a}zision nachzuweisen und zu quantifizieren. Mit Hilfe der Massenspektrometrie konnten alle Hauptkomponenten und verwandten Verbindungen der Base, ihrer Ester und Salze eindeutig identifiziert und quantifiziert werden. Zudem konnten zwei neue verwandte Verbindungen von Erythromycin gefunden und als N,N-Didemethylerythromycin A und Anhydroerythromycin F identifiziert werden.}, subject = {Erythromycin}, language = {de} } @phdthesis{JakobRodamer2014, author = {Jakob-Rodamer, Verena}, title = {Development and validation of LC-MS/MS methods to determine PK/PD parameters of anti-infectives}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-109215}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {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.}, subject = {Antimikrobieller Wirkstoff}, language = {en} } @article{KodererSchmitzWuenschetal.2022, author = {Koderer, Corinna and Schmitz, Werner and W{\"u}nsch, Anna Chiara and Balint, Julia and El-Mesery, Mohamed and Volland, Julian Manuel and Hartmann, Stefan and Linz, Christian and K{\"u}bler, Alexander Christian and Seher, Axel}, title = {Low energy status under methionine restriction is essentially independent of proliferation or cell contact inhibition}, series = {Cells}, volume = {11}, journal = {Cells}, number = {3}, issn = {2073-4409}, doi = {10.3390/cells11030551}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-262329}, year = {2022}, abstract = {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.}, language = {en} } @article{MacintyreZhangViegelmannetal.2014, author = {Macintyre, Lynsey and Zhang, Tong and Viegelmann, Christina and Martinez, Ignacio Juarez and Cheng, Cheng and Dowdells, Catherine and Abdelmohsen, Usama Ramadan and Gernert, Christine and Hentschel, Ute and Edrada-Ebel, RuAngelie}, title = {Metabolomic Tools for Secondary Metabolite Discovery from Marine Microbial Symbionts}, series = {Marine Drugs}, volume = {12}, journal = {Marine Drugs}, number = {6}, issn = {1660-3397}, doi = {10.3390/md12063416}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116097}, pages = {3416-3448}, year = {2014}, abstract = {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.}, language = {en} }