Entwicklung und Etablierung von Massenspektrometrie-basierten relativen und absoluten Quantifizierungsmethoden zur physiologischen Proteomanalyse Gram positiver Bakterien

Massenspektrometrie hat sich zur Methode der Wahl für die globale relative und absolute Proteinquantifizierung entwickelt. Da das vorhandene Methodenspektrum in der Anzahl der zu analysierenden Proben limitiert ist und bei der Vermeidung von Vorfraktionierungstechniken keine globale Analyse erlaubt, war es das Ziel dieser Dissertation das Methodenspektrum anhand von anschaulichen Beispielen zur physiologischen Proteomanalyse Gram positiver Bakterien zu erweitern. Dazu erstreckt sich diese Arbeit von der Erweiterung der Anwendungsmöglichkeiten der Isotopen markierten relativen Quantifizierungsmethode, über die Entwicklung eines globalen markierungsfreien relativen Quantifizierungsansatzes bis zur globalen absoluten Quantifizierung und weiter im speziellen der Stöchiometrie-Aufklärung eines Proteinkomplexes. Die Kombination aus 14N/15N metabolischer Markierung mit der GeLC-MS Technik erlaubt eine robuste relative Quantifizierung auf globaler Ebene. Durch die Verwendung eines internen 15N-markierten Referenzextraktes wurde eine bisher nicht erreichte zeitliche Auflösung von zehn Zeitpunkten bei der Untersuchung eines Nährstoffwechsels zwischen den bevorzugten Kohlenstoffquellen, Glukose und Malat, des Gram positiven Modellorganismus Bacillus subtilis erreicht. Dieses Experiment zeigte klar, dass die Anpassung an Malat als zweite Kohlenstoffquelle sehr schnell passiert. Im Gegensatz dazu findet die Anpassung an Glukose als zusätzliche Kohlenstoffquelle mit einer zeitlichen Verschiebung von ca. 45 Min. statt. Diese Ergebnisse legen den Schluss nahe, dass die Anpassung an Malat hauptsächlich auf post-transkriptioneller Ebene geschieht und die Anpassung an Glukose auf transkriptioneller Ebene stattfindet. Die geringe Reproduzierbarkeit von Vorfraktionierungstechniken beschränkt ihre Anwendung während einer markierungsfreien Quantifizierung. Die eingeschränkte Kombinationsmöglichkeit mit Vorfraktionierungstechniken führt zu einer geringeren Anzahl an identifizierten und quantifizierten Proteinen, was durch den Einsatz von Ausschlusslisten mit optimierten Messparametern in wiederholten Messungen mit einer eindimensionalen Chromatographie ausgeglichen wurde. Im Vergleich zu einer einfachen Wiederholung der Messung konnte die Anzahl an identifizierten Peptiden um 32 % gesteigert werden. Der Ausschlusslistenansatz konnte anschließend erfolgreich für eine markierungsfreie globale Proteinquantifizierung der Stickstoffmonoxid (NO) Stressantwort des humanpathogenen Stapylococcus aureus eingesetzt werden. Die Ergebnisse wurden mittels paralleler Quantifizierung mit 14N/15N metabolischen Markierung verifiziert. Mit dem Ansatz wurden fast 50 % des gesamten Proteoms identifiziert und 70 % davon konnten mit einem zu dem Markierungsexperiment vergleichbaren Ergebnis quantifiziert werden. Die Proteomsignatur der NO-Stressantwort zeigte eine hohe Ähnlichkeit zu der von Antibiotika, die wie NO zu DNA-Strangbrüchen führen. Auch bei der absoluten Proteinquantifizierung kann nicht ohne Weiteres eine Vorfraktionierung eingesetzt werden. Durch die Verwendung einer „multiplexed LC-MS“ (LC-MSE) Methode wurde fast die Hälfte aller zytosolischen Proteine von B. subtilis mit einer hohen durchschnittlichen Sequenzabdeckung von 40 % identifiziert. Die Hi3-Methode ermögliche zusätzlich die absolute Quantifizierung fast aller identifizierten Proteine, die über fast vier Größenordnungen nachgewiesen werden konnten. Die Zuverlässigkeit des Ansatzes wurde für sechs Proteine mit der gut etablierten AQUA-Technik bestätigt. Mit der Hi3-Methode wurden zum einen absolute Proteomsignaturen für unterschiedliche Nährstoffsituationen erstellt, was auch Einblicke in die Regulation der Expression von Aminosäure-Biosynthese und –abbau-Enzyme ermöglichte. Zum anderen konnte gezeigt werden, dass die intrazelluläre Konzentration von ribosomalen und weiteren Wachstumsraten-abhängig benötigten Proteinen sich bei niedrigen Wachstumsraten nicht unterscheidet und erst ab einer Wachstumsrate von 0,8 Std.-1 linear ansteigt. Die vergleichsweise hohe Standardabweichung der Hi3-Methode (~30 %) erschwert ihre Anwendung bei der Bestimmung von nicht gradzahligen Protein-Komplex-Stöchiometrien. Deswegen wurde zur Analyse des RNA-Polymerase-Komplexes von B. subtilis der AQUA-Ansatz gewählt, der sich durch eine sehr geringe Standardabweichung auszeichnet (< 10 %). Dazu wurde ein Protokoll entwickelt, welches auf einer mTRAQ-Markierung der Referenzpeptide und des verdauten Komplexes beruhte. Es war so möglich die bekannte Stöchiometrie des Kernkomplexes RpoA:RpoB:RpoC 2:1:1 zu bestätigen und zusätzlich die zwei ω-Unterheiten und die σ-Faktoren σA und σB absolut zu bestimmen. Die Menge an σB im Komplex nahm nach Glukose-Hunger und Ethanol-Stress auf bis zu 5 % zu und es konnte gezeigt werden, dass sich die Menge einer ω-Unterheit (YloH) sich im gleichen Maße im Komplex ändert, wie die Menge an σA.Mass spectrometry has been developed as method of choice for comprehensive relative and absolute proteome analysis. Because the established quantification methods are limited in the number of samples for analysis and cannot be combined in each case with a pre fractionation, there is specific need for the development of new methods for mass spectrometry based proteome analysis. These methods should be established for Gram-positive bacteria as proof of principle. This thesis aimed at the expansion of the range of methods for demonstrative examples for physiological proteome analysis of Gram positive bacteria compiling the extension of label based relative protein quantification, the establishment of a global label free quantification approach, the development of a comprehensive absolute quantification method and furthermore the determination of the stoichiometry of a protein complex. The combination of 14N/15N metabolic labeling and GeLC-MS allows robust relative protein quantification on a global scale. The application of an internal 15N labeled reference extract enables never before achieved time resolved protein quantification (ten time points) within a nutritional shifts between the two preferred carbon sources (glucose and malate). Therefore Bacillus subtilis was selected as Gram positive model organism. In the result of this a very fast adaptation to malate as second carbon source has been observed, whereas the adaptation to glucose as second carbon is delayed by approx. 45 min. This finding revealed that the adaptation to malate is mainly post transcriptionally regulated and the adaptation to glucose in a transcriptional manner. The limited reproducibility of common proteomic pre fractionation techniques confines their application in combination with label free quantification methods leading to lower number of quantified proteins. To circumvent this drawback exclusion lists with iteratively improved instrument settings were introduced to LC-MS/MS analysis based on one dimensional chromatographic separation. This exclusion list based methods enabled to increase the number of identified peptides by 32 % compared to simple replicate measurements. By application of this workflow to nitric oxide stress (NO) response of human pathogen Staphylococcus aureus almost half of the entire proteome could be identified. Subsequent reliable label free quantification for 70 % of the identified proteins could be achieved. In comparison to the metabolic labeling approach combined with GeLC-MS a comparable result in terms of number of quantified proteins and fold changes was obtained. The quantitative evaluation of S. aureus stressed by NO adaptations illustrated the bacterial adaptation to the stress by higher expression of detoxifying enzyme, NO insensitive lactate dehydrogenase, DNA repair enzymes, biotin biosynthesis enzymes and phage L54a related proteins. Therefore this proteomic signature was similar to resistance mechanisms to antibiotics, also causing DNA strand breaks. The capabilities of pre fractionation techniques are limited for absolute quantification approaches as well. By application of the multiplexed LC-MS (LC-MSE) technique almost half of the predicted proteome of B subtilis with high average sequence coverage of 40 % could be identified. Absolute quantification for nearly all identified proteins over four orders of magnitude was achieved by application of the Hi3 approach. Comparative analysis with AQUA approach proved the high reliability of the global quantification results. The Hi3 approach enabled to set up absolute proteome signatures for different nutrient situation of B. subtilis and revealed new insights in the regulation of the expression of the amino acids biosynthesis and – degradation enzymes. Additionally the results showed that the intracellular proteins concentrations of ribosomal and other probably growth rate dependent regulated protein groups stay constant at low growth rates and are increasing linearly at a growth rate higher than 0.8 h-1. After development of reliable techniques for the purification of protein complexes the determination of the stoichiometric composition of multi-protein complexes becomes more and more important. The comparably high standard deviation of the Hi3 method confines the application of this approach for the exact determination of protein complex. Therefore the stoichiometry of RNA polymerase complex of B. subtilis was elucidated under different growth conditions by AQUA approach based on mTRAQ labeled synthetic peptides. The known stoichiometry of the core enzyme RpoA:RpoB:RpoC 2:1:1 could be shown. Under chosen growth conditions a stoichiometry of 1:1 relative to the core enzyme for the ω-subunit YkzG was determined. The concentration of the other ω subunit YloH was under all experimental conditions in the same range of σA (20 – 35 %). After ethanol stress and glucose starvation σB was found to be bound up to 5 % to the polymerase

FLEXIQuant-LF to quantify protein modification extent in label-free proteomics data

Improvements in LC-MS/MS methods and technology have enabled the identification of thousands of modified peptides in a single experiment. However, protein regulation by post-translational modifications (PTMs) is not binary, making methods to quantify the modification extent crucial to understanding the role of PTMs. Here, we introduce FLEXIQuant-LF, a software tool for large-scale identification of differentially modified peptides and quantification of their modification extent without knowledge of the types of modifications involved. We developed FLEXIQuant-LF using label-free quantification of unmodified peptides and robust linear regression to quantify the modification extent of peptides. As proof of concept, we applied FLEXIQuant-LF to data-independent-acquisition (DIA) data of the anaphase promoting complex/cyclosome (APC/C) during mitosis. The unbiased FLEXIQuant-LF approach to assess the modification extent in quantitative proteomics data provides a better understanding of the function and regulation of PTMs. The software is available at https://github.com/SteenOmicsLab/FLEXIQuantLF.Peer Reviewe

Costs of life - Dynamics of the protein inventory of Staphylococcus aureus during anaerobiosis

Absolute protein quantification was applied to follow the dynamics of the cytoplasmic proteome of Staphylococcus aureus in response to long-term oxygen starvation. For 1,168 proteins, the majority of all expressed proteins, molecule numbers per cell have been determined to monitor the cellular investments in single branches of bacterial life for the first time. In the presence of glucose the anaerobic protein pattern is characterized by increased amounts of glycolytic and fermentative enzymes such as Eno, GapA1, Ldh1, and PflB. Interestingly, the ferritin-like protein FtnA belongs to the most abundant proteins during anaerobic growth. Depletion of glucose finally leads to an accumulation of different enzymes such as ArcB1, ArcB2, and ArcC2 involved in arginine deiminase pathway. Concentrations of 29 exo- and 78 endometabolites were comparatively assessed and have been integrated to the metabolic networks. Here we provide an almost complete picture on the response to oxygen starvation, from signal transduction pathways to gene expression pattern, from metabolic reorganization after oxygen depletion to beginning cell death and lysis after glucose exhaustion. This experimental approach can be considered as a proof of principle how to combine cell physiology with quantitative proteomics for a new dimension in understanding simple life processes as an entity

Comprehensive absolute quantification of the cytosolic proteome of <em>Bacillus</em> <em>subtilis</em> by data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MSE)

International audienceIn the growing field of systems biology, the knowledge of protein concentrations is highly required to truly understand metabolic and adaptational networks within the cells. Therefore we established a workflow relying on long chromatographic separation and mass spectrometric analysis by data independent, parallel fragmentation of all precursor ions at the same time (LC/MSE). By prevention of discrimination of co-eluting low and high abundant peptides a high average sequence coverage of 40% could be achieved, resulting in identification of almost half of the predicted cytosolic proteome of the Gram-positive model organism Bacillus subtilis (>1,050 proteins). Absolute quantification was achieved by correlation of average MS signal intensities of the three most intense peptides of a protein to the signal intensity of a spiked standard protein digest. Comparative analysis with heavily labeled peptides (AQUA approach) showed the use of only one standard digest is sufficient for global quantification. The quantification results covered almost four orders of magnitude, ranging roughly from 10 to 150,000 copies per cell. To prove this method for its biological relevance selected physiological aspects of B. subtilis cells grown under conditions requiring either amino acid synthesis or alternatively amino acid degradation were analyzed. This allowed both in particular the validation of the adjustment of protein levels by known regulatory events and in general a perspective of new insights into bacterial physiology. Within new findings the analysis of “protein costs” of cellular processes is extremely important. Such a comprehensive and detailed characterization of cellular protein concentrations based on data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MSE) data has been performed for the first time and should pave the way for future comprehensive quantitative characterization of microorganisms as physiological entities

The dynamic protein partnership of RNA polymerase in Bacillus subtilis

In prokaryotes, transcription results from the activity of a 400 kDa RNA polymerase (RNAP) protein complex composed of at least five subunits (2 alpha, beta, beta', omega). To ensure adequate responses to changing environmental cues, RNAP activity is tightly controlled by means of interacting regulatory proteins. Here, we report the affinity-purification of the Bacillus subtilis RNAP complexes from cells in different growth states and stress conditions, and the quantitative assessment by mass spectrometry of the dynamic changes in the composition of the RNAP complex. The stoichiometry of RNA polymerase was determined by a comparison of two mass spectrometry-based quantification methods: a label-based and a label-free method. The validated label-free method was then used to quantify the proteins associated with RNAP. The levels of sigma factors bound to RNAP varied during growth and exposure to stress. Elongation factors, helicases such as HelD and PcrA, and novel unknown proteins were also associated with RNAP complexes. The content in 6S RNAs of purified RNAP complexes increased at the onset of the stationary phase. These quantitative variations in the protein and RNA composition of the RNAP complexes well correlate with the known physiology of B. subtilis cells under different conditions

Global, in situ analysis of the structural proteome in individuals with Parkinson's disease to identify a new class of biomarker

Parkinson's disease (PD) is a prevalent neurodegenerative disease for which robust biomarkers are needed. Because protein structure reflects function, we tested whether global, in situ analysis of protein structural changes provides insight into PD pathophysiology and could inform a new concept of structural disease biomarkers. Using limited proteolysis-mass spectrometry (LiP-MS), we identified 76 structurally altered proteins in cerebrospinal fluid (CSF) of individuals with PD relative to healthy donors. These proteins were enriched in processes misregulated in PD, and some proteins also showed structural changes in PD brain samples. CSF protein structural information outperformed abundance information in discriminating between healthy participants and those with PD and improved the discriminatory performance of CSF measures of the hallmark PD protein alpha-synuclein. We also present the first analysis of inter-individual variability of a structural proteome in healthy individuals, identifying biophysical features of variable protein regions. Although independent validation is needed, our data suggest that global analyses of the human structural proteome will guide the development of novel structural biomarkers of disease and enable hypothesis generation about underlying disease processes.ISSN:1545-9993ISSN:1545-998

FLEXITau: Quantifying Post-translational Modifications of Tau Protein in Vitro and in Human Disease.

Tauopathies, including Alzheimer's disease (AD), are associated with the aggregation of modified microtubule associated protein tau. This pathological state of tau is often referred to as "hyperphosphorylated". Due to limitations in technology, an accurate quantitative description of this state is lacking. Here, a mass spectrometry-based assay, FLEXITau, is presented to measure phosphorylation stoichiometry and provide an unbiased quantitative view of the tau post-translational modification (PTM) landscape. The power of this assay is demonstrated by measuring the state of hyperphosphorylation from tau in a cellular model for AD pathology, mapping, and calculating site occupancies for over 20 phosphorylations. We further employ FLEXITau to define the tau PTM landscape present in AD post-mortem brain. As shown in this study, the application of this assay provides mechanistic understanding of tau pathology that could lead to novel therapeutics, and we envision its further use in prognostic and diagnostic approaches for tauopathies

Global, in situ analysis of the structural proteome in individuals with Parkinson's disease to identify a new class of biomarker

Parkinson’s disease (PD) is a prevalent neurodegenerative disease for which robust biomarkers are needed. Because protein structure reflects function, we tested whether global, in situ analysis of protein structural changes provides insight into PD pathophysiology and could inform a new concept of structural disease biomarkers. Using limited proteolysis–mass spectrometry (LiP–MS), we identified 76 structurally altered proteins in cerebrospinal fluid (CSF) of individuals with PD relative to healthy donors. These proteins were enriched in processes misregulated in PD, and some proteins also showed structural changes in PD brain samples. CSF protein structural information outperformed abundance information in discriminating between healthy participants and those with PD and improved the discriminatory performance of CSF measures of the hallmark PD protein α-synuclein. We also present the first analysis of inter-individual variability of a structural proteome in healthy individuals, identifying biophysical features of variable protein regions. Although independent validation is needed, our data suggest that global analyses of the human structural proteome will guide the development of novel structural biomarkers of disease and enable hypothesis generation about underlying disease processes