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Identification of the Whole Blood Protein ADP-Ribosylome Under Sepsis Conditions in Pigs

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Identification of the Whole Blood Protein ADP-Ribosylome Under Sepsis Conditions in Pigs Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2020 Identification of the whole blood protein ADP-ribosylome under sepsis conditions in pigs Lüthi, Stephanie Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-188727 Dissertation Published Version Originally published at: Lüthi, Stephanie. Identification of the whole blood protein ADP-ribosylome under sepsis conditions in pigs. 2020, University of Zurich, Vetsuisse Faculty. Institut für molekulare Mechanismen bei Krankheiten der Vetsuisse-Fakultät Universität Zürich Direktor: Prof. Dr. med. vet. Dr. phil. II Michael O. Hottiger Arbeit unter wissenschaftlicher Betreuung von Dr. sc. nat. Deena Leslie Pedrioli Identification of the whole blood protein ADP-ribosylome under sepsis conditions in pigs Inaugural-Dissertation zur Erlangung der Doktorwürde der Vetsuisse-Fakultät Universität Zürich vorgelegt von med. vet. Stephanie Lüthi Tierärztin von Lauperswil, Bern und aus den Vereinigten Staaten von Amerika genehmigt auf Antrag von Prof. Dr. med. vet. Dr. phil. II Michael O. Hottiger, Referent Prof. Dr. sc. nat. Urs Meyer, Korreferent 2020 Institut für molekulare Mechanismen bei Krankheiten der Vetsuisse-Fakultät Universität Zürich Direktor: Prof. Dr. med. vet. Dr. phil. II Michael O. Hottiger Arbeit unter wissenschaftlicher Betreuung von Dr. sc. nat. Deena Leslie Pedrioli Identification of the whole blood protein ADP-ribosylome under sepsis conditions in pigs Inaugural-Dissertation zur Erlangung der Doktorwürde der Vetsuisse-Fakultät Universität Zürich vorgelegt von med. vet. Stephanie Lüthi Tierärztin von Lauperswil, Bern und aus den Vereinigten Staaten von Amerika genehmigt auf Antrag von Prof. Dr. med. vet. Dr. phil. II Michael O. Hottiger, Referent Prof. Dr. sc. nat. Urs Meyer, Korreferent 2020 Table of Contents Summary 4 Zusammenfassung 5 Title page 6 1. Abstract 7 2. Introduction 8 3. Materials and Methods 11 4. Results 17 5. Discussion 26 6. References 32 7. Figures 34 8. Tables 44 Acknowledgements Curriculum vitae 3 Summary While blood proteins are heavily investigated as biomarkers for different diseases, the modification state of such proteins is rarely analyzed. Here, we established a novel mass spectrometry-based workflow to identify in vivo ADP-ribosylated blood proteins of LPS- treated pigs as a model for human systemic inflammatory response syndrome. We found that ADP-ribosylated proteins are rapidly demodified after sample collection by phosphodiesterases and ADP-ribosylhydrolases in the plasma, and thus to prevent demodification, we immediately denatured blood samples after collection with guanidine hydrochloride. Moreover, we improved the enrichment protocol of modified peptides via ADP-ribosylome specific LC/MSMS by depleting free ADP-ribose from samples prior to enrichment. This led to the identification of 44 ADP-ribosylated proteins in LPS-treated and 38 in control pigs. These proteins were cross-referenced with the plasma proteome, to determine which of the modified proteins belong to the plasma fraction of the blood, leading to the identification 12 ADP- ribosylated plasma proteins in LPS-treated pigs and 13 in control group pigs. Overall, we developed a novel method for identifying ADP-ribosylated blood proteins and characterized the whole blood and plasma protein ADP-ribosylomes of LPS-treated and control pigs. Further characterizing the blood ADP-ribosylome can help unravel the molecular mechanisms that drive sepsis. Keywords: ADP-ribosylation, sepsis, whole blood lysate, NAD 4 Zusammenfassung Blutproteine werden oft als Biomarker für verschiedene Krankheiten analysiert, aber deren post-translationelle Modifikationen werden selten untersucht. Wir entwickelten ein neues, Massenspektrometrie-basiertes Protokoll um in vivo ADP-ribosylierte Blutproteine in LPS- behandelten Schweinen zu identifizieren, welche als Modell für humanes systemisches inflammatorisches response Syndrom dienen. Da ADP-ribosylierte Proteine nach der Blutprobenentnahme rasch durch Phosphodiesterasen und ADP-ribosylhydrolasen demodifiziert werden, denaturierten wir die Blutproben direkt nach der Entnahme mit Guanidinhydrochlorid. Ein optimiertes Anreicherungsprotokoll bei welchem freie ADP-ribose depletiert wird, wurde entwickelt um die Peptidanreicherung für die Detektion via ADP- ribosylom-spezifischer LC-MS/MS zu verbessern. Mit diesem Protokoll wurden 38 modifizierte Blutproteine in gesunden und 44 in LPS-behandelten Schweinen identifiziert. Durch Vergleichen dieser Blutproteine mit dem Plasmaproteom, definierten wir das plasmaspezifische ADP-ribosylom: 13 modifizierte Proteine wurden in gesunden und 12 in LPS-behandelten Schweinen identifiziert. Zusammengefasst entwickelten wir eine neue Methode um ADP-ribosylierte Blutproteine zu identifizieren und charakterisierten erstmals das Vollblut- und Plasma-ADP-ribosylom von gesunden und LPS-behandelten Schweinen. Weiterführende Untersuchungen dieser Proteine könnten helfen, die molekularen Mechanismen von Sepsis besser zu verstehen. Stichwörter: ADP-ribosylierung, Sepsis, Vollblut, NAD 5 Identification of the whole blood protein ADP-ribosylome under sepsis conditions in pigs Stephanie C. Lüthi1, Anna Howald1, Kathrin Nowak1, Robert Graage2, Giody Bartolomei3, Christine Neupert3, Xaver Sidler2, Deena M. Leslie Pedrioli1,4 and Michael O. Hottiger1,4 1 Department of Molecular Mechanisms of Disease, Vetsuisse Faculty and Faculty of Science, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland 2 Department of Farm Animals, Division of Swine Medicine, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland 3 Malcisbo AG, Wagistrasse 27A, 8952 Schlieren, Switzerland 4 equal last authors Corresponding author: Michael O. Hottiger ([email protected]) 6 1. ABSTRACT While the analysis of blood proteins as biomarkers for different diseases is common nowadays, the modification state of such proteins is only rarely addressed. We observed that ADP- ribosylated proteins are rapidly demodified after blood sample collection due to active phosphodiesterases and ADP-ribosylhydrolases in the plasma, thus, requiring an optimized collection procedure for this PTM to prevent demodification. Here, we established a novel mass spectrometry based workflow to identify blood proteins that are ADP-ribosylated in healthy and lipopolysaccharide treated pigs in vivo, that serves as a model for human systemic inflammatory response syndrome. To this extent, blood samples including all cells and soluble proteins were immediately denatured with guanidine hydrochloride after collection to prevent post-sampling demodification. Moreover, an improved enrichment protocol that depletes free ADP-ribose from samples was established to enhance ADP-ribosylated peptide enrichment and identification rates in the whole blood lysates via ADP-ribosylome specific LC/MS-MS. Application of this modified protocol led to the identification of a total of 38 ADP-ribosylated proteins in healthy pigs and 44 in LPS-treated pigs. By cross-referencing these proteins with the corresponding plasma proteome, we determined which of the ADP-ribosylated proteins belong to the plasma fraction of the blood. This led to the identification of 13 ADP-ribosylated plasma proteins in healthy pigs and 12 in LPS-treated pigs. In summary, we developed a novel method for identifying ADP-ribosylated blood proteins and characterized the whole blood and plasma protein ADP-ribosylomes of healthy and LPS-treated pigs. This study thus contributes to the rapidly developing field of ADP-ribosylation and further characterization of the newly identified blood ADP-ribosylome will potentially help unravel the molecular mechanisms that drive SIRS and sepsis, two of the major health threats today. Keywords: ADP-ribosylation, systemic inflammatory response syndrome, sepsis, whole blood lysate, plasma, histidine 7 2. INTRODUCTION The clinical condition of systemic inflammatory response syndrome (SIRS) or sepsis is caused by pathogen-associated molecular pattern (PAMP) molecules, including lipopolysaccharide (LPS) [1]. These molecules interact with toll-like receptors (TLRs) on host immune cells, which triggers increased production of pro-inflammatory cytokines by immune cells circulating in the blood [2, 3]. Studies suggest that during sepsis, control of the primarily physiological host response to the infection is lost, which causes a multitude of systemic dysfunctions that ultimately lead to multi-organ failure and the resulting high mortality rates [4]. Although sepsis has been extensively studied, the specific molecular mechanisms that drive multi-organ failure and eventual death are not fully understood [4, 5]. As sepsis is one of the leading causes of death in humans, developing a reproducible study system and accurate diagnostic tools that allow sepsis detection at early stages is critical to facilitate appropriate therapeutic interventions and further therapeutic developments [5]. Porcine pleuropneumonia, a highly infectious disease in pigs that is caused by Actinobacillus pleuropneumoniae, could serve as a model for human SIRS and sepsis [6]. LPS derived from A. pleuropneumoniae has been shown to induce inflammatory responses by triggering alveolar macrophages to overproduce proinflammatory cytokines like IL-1B, IL-6 and TNFα [7]. This proinflammatory cytokine influx, combined with the occurrence of typical clinical symptoms
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