Refine
Year of publication
Document Type
- Article (9)
- Doctoral Thesis (5)
Has Fulltext
- yes (14)
Is part of the Bibliography
- no (14)
Keywords
- mass spectrometry (14) (remove)
Institute
- Abteilung für Mikrobiologie und Molekularbiologie (6)
- Interfakultäres Institut für Genetik und Funktionelle Genomforschung (UMG) (2)
- Institut für Anatomie und Zellbiologie (1)
- Institut für Chemie und Biochemie (1)
- Institut für Immunologie u. Transfusionsmedizin - Abteilung Immunologie (1)
- Institut für Med. Biochemie u. Molekularbiologie (1)
- Institut für Mikrobiologie - Abteilung für Genetik & Biochemie (1)
- Institut für Physik (1)
- Klinik und Poliklinik für Chirurgie Abt. für Unfall- und Wiederherstellungschirurgie (1)
Publisher
- MDPI (8)
- Frontiers Media S.A. (1)
Posttranslationale Proteinmodifikationen beeinflussen Proteinaktivitäten und Signalwege innerhalb einer Zelle und haben somit vielfältige Auswirkungen auf den Stoffwechsel von Bakterien. Um die genauen Mechanismen besser verstehen zu können, wurde in dieser Arbeit das Phosphoproteom von Streptococcus pneumoniae D39 untersucht. Der Schwerpunkt lag dabei in der Entwicklung besserer Auswertestrategien und der damit einhergehenden verbesserten Identifizierung von Phosphoproteinen. Um dies zu bewerkstelligen, wurden die Proteinextrakte durch gelfreie und gelbasierte Methoden aufgetrennt. Die Auswertung der Experimente erfolgte zunächst durch klassische Proteinidentifizierung mit Hilfe von Proteindatenbanken. Zusätzlich wurden Spektrenbibliotheken von S. pneumoniae D39 aufgebaut und diese für eine bessere Proteinidentifizierung sowie Phosphoproteinidentifizierung genutzt. Anschließend wurden zur Quantifizierung des Phosphoproteoms dieses Pathogens verschiedene Quantifizierungsmethoden getestet und modifiziert. Hierbei wurde zum einen das Phosphoproteom einer Kinasedeletionsmutante von S. pneumoniae D39 über die Spotintensitäten von 2D Gelen mit dem Wildtyp verglichen. Zusätzlich wurden die Auswirkungen dieser Kinase auf das globale S. pneumoniae D39 Proteom mittels SILAC sowie der neu erstellten Spektrenbibliothek aufgezeigt. Eine weitere etablierte Quantifizierungsmethode für Phosphoproteine in der Arbeit war die Kombination von metabolischer Markierung und 2D Gelen. Die Veränderung des Phosphoproteoms wurde an dem industriell bedeutsamen Bakterium Bacillus pumilus anhand von oxidativem Stress aufgezeigt.
: Platelets are components of the blood that are highly reactive, and they quickly respond
to multiple physiological and pathophysiological processes. In the last decade, it became clear that
platelets are the key components of circulation, linking hemostasis, innate, and acquired immunity.
Protein composition, localization, and activity are crucial for platelet function and regulation. The
current state of mass spectrometry-based proteomics has tremendous potential to identify and quantify thousands of proteins from a minimal amount of material, unravel multiple post-translational
modifications, and monitor platelet activity during drug treatments. This review focuses on the role
of proteomics in understanding the molecular basics of the classical and newly emerging functions
of platelets. including the recently described role of platelets in immunology and the development
of COVID-19.The state-of-the-art proteomic technologies and their application in studying platelet
biogenesis, signaling, and storage are described, and the potential of newly appeared trapped ion
mobility spectrometry (TIMS) is highlighted. Additionally, implementing proteomic methods in
platelet transfusion medicine, and as a diagnostic and prognostic tool, is discussed.
Like eukaryotes, different bacterial species express one or more Ser/Thr kinases and phosphatases that operate in various signaling networks by catalyzing phosphorylation and dephosphorylation of proteins that can immediately regulate biochemical pathways by altering protein function. The human pathogen Streptococcus pneumoniae encodes a single Ser/Thr kinase-phosphatase couple known as StkP-PhpP, which has shown to be crucial in the regulation of cell wall synthesis and cell division. In this study, we applied proteomics to further understand the physiological role of pneumococcal PhpP and StkP with an emphasis on phosphorylation events on Ser and Thr residues. Therefore, the proteome of the non-encapsulated D39 strain (WT), a kinase (ΔstkP), and phosphatase mutant (ΔphpP) were compared in a mass spectrometry based label-free quantification experiment. Results show that a loss of function of PhpP causes an increased abundance of proteins in the phosphate uptake system Pst. Quantitative proteomic data demonstrated an effect of StkP and PhpP on the two-component systems ComDE, LiaRS, CiaRH, and VicRK. To obtain further information on the function, targets and target sites of PhpP and StkP we combined the advantages of phosphopeptide enrichment using titanium dioxide and spectral library based data evaluation for sensitive detection of changes in the phosphoproteome of the wild type and the mutant strains. According to the role of StkP in cell division we identified several proteins involved in cell wall synthesis and cell division that are apparently phosphorylated by StkP. Unlike StkP, the physiological function of the co-expressed PhpP is poorly understood. For the first time we were able to provide a list of previously unknown putative targets of PhpP. Under these new putative targets of PhpP are, among others, five proteins with direct involvement in cell division (DivIVA, GpsB) and peptidoglycan biosynthesis (MltG, MreC, MacP).
Proteomic Adaptation of Clostridioides difficile to Treatment with the Antimicrobial Peptide Nisin
(2021)
A method employing labeling of cell-surface proteins with Sulfo-NHS-SS-biotin and subsequent affinity enrichment with NeutrAvidin has been optimized in order to make cell-surface proteins from Gram-positive bacteria reliably accessible to quantitative mass spectrometric analyses. The optimized biotinylation approach was applied for analysis of the lipoproteome from S. aureus and S. pneumoniae on a global scale and the influence of mutations in the lipoprotein maturation pathway on the cell-surface and exoproteomes of both species was investigated. The biotinylation approach was integrated into a proteomic workflow that employs metabolic labeling with heavy nitrogen for relative protein quantification to investigate proteomic differences between S. aureus in a biofilm model and its free-floating, planktonic counterparts.
Deciphering the entire protein complement of a living cell together with the elucidation of dynamic processes on protein level are the main goals of proteomics as it is used today. To achieve this goal, namely the elucidation of dynamic processes of the entire bacterial cell, we have developed strategies and distinct workflows to cover the most proteins in different subcellular localizations in bacteria together with a stable isotopes labeling approach to follow temporal and spatial changes in different proteomic subfractions. In this work, it has been shown that the use of mass spectrometry based in vivo quantitation techniques and the application of subcellular and chromatographic fractionation has lead to a new level of qualitative and quantitative proteomics data. Emphasizing on the studies revealing the dynamics of the bacterial physiology on a time resolved base, both spatial and temporal processes can be monitored to obtain knowledge on physiological processes in a depth that has not been reached before in comparable global studies.
Insight into the Impact of Oxidative Stress on the Barrier Properties of Lipid Bilayer Models
(2022)
As a new field of oxidative stress-based therapy, cold physical plasma is a promising tool for several biomedical applications due to its potential to create a broad diversity of reactive oxygen and nitrogen species (RONS). Although proposed, the impact of plasma-derived RONS on the cell membrane lipids and properties is not fully understood. For this purpose, the changes in the lipid bilayer functionality under oxidative stress generated by an argon plasma jet (kINPen) were investigated by electrochemical techniques. In addition, liquid chromatography-tandem mass spectrometry was employed to analyze the plasma-induced modifications on the model lipids. Various asymmetric bilayers mimicking the structure and properties of the erythrocyte cell membrane were transferred onto a gold electrode surface by Langmuir-Blodgett/Langmuir-Schaefer deposition techniques. A strong impact of cholesterol on membrane permeabilization by plasma-derived species was revealed. Moreover, the maintenance of the barrier properties is influenced by the chemical composition of the head group. Mainly the head group size and its hydrogen bonding capacities are relevant, and phosphatidylcholines are significantly more susceptible than phosphatidylserines and other lipid classes, underlining the high relevance of this lipid class in membrane dynamics and cell physiology.
Niemann–Pick type C1 (NPC1) is a lysosomal storage disorder, inherited as an
autosomal-recessive trait. Mutations in the Npc1 gene result in malfunction of the NPC1 protein,
leading to an accumulation of unesterified cholesterol and glycosphingolipids. Beside visceral
symptoms like hepatosplenomegaly, severe neurological symptoms such as ataxia occur. Here,
we analyzed the sphingosine-1-phosphate (S1P)/S1P receptor (S1PR) axis in different brain regions
of Npc1−/− mice and evaluated specific effects of treatment with 2-hydroxypropyl-β-cyclodextrin
(HPβCD) together with the iminosugar miglustat. Using high-performance thin-layer chromatography
(HPTLC), mass spectrometry, quantitative real-time PCR (qRT-PCR) and western blot analyses, we
Int. J. Mol. Sci. 2020, 21, 4502; doi:10.3390/ijms21124502 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2020, 21, 4502 2 of 31
studied lipid metabolism in an NPC1 mouse model and human skin fibroblasts. Lipid analyses
showed disrupted S1P metabolism in Npc1−/− mice in all brain regions, together with distinct changes
in S1pr3/S1PR3 and S1pr5/S1PR5 expression. Brains of Npc1−/− mice showed only weak treatment
effects. However, side effects of the treatment were observed in Npc1+/+ mice. The S1P/S1PR axis
seems to be involved in NPC1 pathology, showing only weak treatment effects in mouse brain. S1pr
expression appears to be affected in human fibroblasts, induced pluripotent stem cells (iPSCs)-derived
neural progenitor and neuronal differentiated cells. Nevertheless, treatment-induced side effects
make examination of further treatment strategies indispensable
Tissue sections, which are widely used in research and diagnostic laboratories and have already been examined by immunohistochemistry (IHC), may subsequently provide a resource for proteomic studies, even though only small amount of protein is available. Therefore, we established a workflow for tandem mass spectrometry-based protein profiling of IHC specimens and characterized defined brain area sections. We investigated the CA1 region of the hippocampus dissected from brain slices of adult C57BL/6J mice. The workflow contains detailed information on sample preparation from brain slices, including removal of antibodies and cover matrices, dissection of region(s) of interest, protein extraction and digestion, mass spectrometry measurement, and data analysis. The Gene Ontology (GO) knowledge base was used for further annotation. Literature searches and Gene Ontology annotation of the detected proteins verify the applicability of this method for global protein profiling using formalin-fixed and embedded material and previously used IHC slides.