Downloaded from orbit.dtu.dk on: Oct 07, 2021 Novel proteomics technologies to decipher protease networks in cells and tissues Savickas, Simonas Publication date: 2020 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Savickas, S. (2020). Novel proteomics technologies to decipher protease networks in cells and tissues. DTU Bioengineering. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. DTU Bioengineering Department of Biotechnology and Biomedicine PhD Thesis 2020 Af Copyright: Simonas Savickas Udgivet af: XXX XXX DTU Rekvireres: www.dtu.dk ISSN: [0000-0000] (elektronisk udgave) ISBN: [000-00-0000-000-0] (elektronisk udgave) ISSN: [0000-0000] (trykt udgave) ISBN: [000-00-0000-000-0] (trykt udgave) Preface This thesis is the result of a PhD project under the supervision of Professor MSO Dr. Ulrich auf dem Keller and Professor Dr. Birte Svensson at Technical University of Denmark Department of Bioengineering. Due to the move of Ulrich auf dem Keller’s laboratory, the experiments were started at ETH Zurich and completed at DTU Bioengineering. This work is subdivided into three sections: a general introduction including three manuscripts that give an overview of mass spectrometry-based degradomics methods and technically introduce a key method. The results section contains four manuscripts describing my experiments. Finally, a general discussion compares findings with the existing published data and provides an outlook how mass spectrometry might be utilized as powerful tool in personalized medicine. I hope you will enjoy reading it. Simonas Savickas Novel proteomics technologies to decipher protease networks in cells and tissues Papers and Manuscripts This PhD thesis contains the following manuscripts: Savickas, S., & auf dem Keller, U. (2017). Targeted degradomics in protein terminomics and protease substrate discovery. Biological chemistry, 399(1), 47-54. Schoof, E. M., Rapin, N., Savickas, S., Gentil, C., Lechman, E., Haile, J. S., ... & Porse, B. T. (2019). A quantitative single-cell proteomics approach to characterize an acute myeloid leukemia hierarchy. bioRxiv, 745679. Savickas, S., Kastl, P., & auf dem Keller, U. (2020). Combinatorial degradomics: Precision tools to unveil proteolytic processes in biological systems. Biochimica et Biophysica Acta (BBA)- Proteins and Proteomics, 140392. Bundgaard, L., Savickas, S., & Auf dem Keller, U. (2020). Mapping the N-Terminome in Tissue Biopsies by PCT-TAILS. In ADAMTS Proteases (pp. 285-296). Humana, New York, NY. Savickas, S., Kastl, P., auf dem Keller, U. (2020). Interconnected activities and functions of matrix metalloproteinases at the wound edge. In preparation. Savickas, S., Kastl, P., Schoof, M.E., auf dem Keller, U. (2020). hybrid Parallel Reaction Monitoring (hPRM) a mass spectrometry based technique for semi-discovery research. In preparation Savickas, S., Schoof, M.E., auf dem Keller, U. (2020). Harnessing a novel targeted single-cell proteomics technique to expand the knowledge of primary leukemia hierarchy. In preparation Additional publications that were outside of the scope of this thesis: Sabino, F., Egli, F. E., Savickas, S., Holstein, J., Kaspar, D., Rollmann, M., ... & Auf dem Keller, U. (2018). Comparative degradomics of porcine and human wound exudates unravels biomarker candidates for assessment of wound healing progression in trauma patients. Journal of Investigative Dermatology, 138(2), 413-422. Yip, M. C., Savickas, S., Gygi, S. P., & Shao, S. (2020). ELAC1 Repairs tRNAs Cleaved during Ribosome-Associated Quality Control. Cell reports, 30(7), 2106-2114. Bundgaard, L. B., Agren, M. S., Holstein, P., Savickas, S., Sabino, F. M. R., & auf dem Keller, U. (2018). A SYSTEM-WIDE STUDY OF THE PROTEOME AND PROTEASE CLEAVAGE PRODUCTS IN CHRONIC SKIN ULCERS. Wound Repair and Regeneration, 26(2) Savickas, S.& Kalogeropoulos, K. Bundgaard, L. B., Larsen, C. A., auf dem Keller, U. (2020). High throughput PRM method for rapid biomarker recording in body fluids. In preparation Novel proteomics technologies to decipher protease networks in cells and tissues Summary Precision processing of protein substrates by limited proteolysis has proven to play a key role in maintaining cellular and tissues homeostasis. Over the past years, an enormous amount of information on protease-substrate interactions has been collected and compiled into a complex network termed the protease web. We are only beginning to understand the connectivity of the protease web but have advanced our perception of the dynamics of proteolysis, complementary pathways and compensatory mechanisms. Identification of cell type- and tissue-specific proteolytic patterns in healthy and perturbed systems has gained much attention to promote generation of fundamental hypothesis for underlying mechanisms of responses to perturbations, thereby drawing a holistic picture of key events. However, we need highly reproducible assays to comprehensively monitor the interconnected protease networks within the protease web and to quantitatively assess protease-protease and protease-substrate relations with high specificity and sensitivity and applicability to samples from cells and tissues. In this project, we have developed novel highly specific and sensitive parallel reaction monitoring (PRM) assays to monitor a network of matrix metalloproteinases (MMPs) in complex samples. To concomitantly analyze the MMP activation status, we have specifically targeted peptides that are generated during propeptide removal, thus resulting in activation of the zymogens. These assays have been applied to study interconnected MMP activation on the molecular, cellular and tissue level. We have tested the hypothesis that MMP10 and MMP3 act as activator MMPs of several other family members and mapped and functionally tested MMP3/10-dependent MMP activation cascades in culture supernatants from fibroblasts and keratinocytes. Mouse models with normal, enhanced and ablated MMP10 activity have been analyzed and its function determined as local MMP activator in the skin. With the arrival of new mass spectrometers, we have extended the PRM analysis of the MMP network by developing a hybrid Parallel Reaction Monitoring (hPRM) method. The workflow utilizes the improved power of the instruments to retain PRM sensitivity to investigate the targeted network and concomitantly perform a global proteome analysis for further proteolytic signatures. This new single-shot method confirmed our previous findings of MMP abundances and simultaneously revealed processing of S100A11 by cathepsin D in response to a perturbation. Investigating proteolytic networks in vitro, in cell cultures and in complex tissues disregards the natural heterogeneity of cellular makeup. Thus, ultimately analysis at the single cell level is needed to understand the protease web in multicellular environments. To address this challenge, we have implemented a workflow and tested if we can identify proteases at the protein level in single cells. By combining fluorescence-activated cell sorting (FACS) and an optimized Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) method, we investigated the proteomic landscape of Leukemia hierarchy. In this single-cell proteomics data set we also identified and Novel proteomics technologies to decipher protease networks in cells and tissues quantified 22 proteases including, but not limited to caspases, aminopeptidase, cathepsins, calpains and proteasome components across three cell types. Due to inherent low abundances, multiple known proteases could not be identified in the previous study. To address this limitation, we further extended our workflow into single-cell targeted proteomics analyses. The upgraded technique combines our optimized SCoPE-MS workflow with internal standard triggered-PRM (IS-PRM) to allow identification and quantification of low-abundance target proteins including proteases. Applying this workflow, we confirmed differential abundances of CD34 and CD38 in Leukemic Blasts, Progenitors and Stem Cells and observed their proportional distribution between the cells. In addition, we monitored HtrA serine proteases (HtrA) 2 and 3 and their flux between the cell types. Thereby, we monitored high abundance of HtrA2 in Leukemic Stem Cells and low levels in Progenitors, which might indicate a role of this protease in modulating differentiation. Having established a workflow for targeted analysis of peptides ultimately opens up the possibility to not only monitor proteases but also proteolytic cleavages by analysis of spanning peptides and neo-termini at the single-cell level. In conclusion, the delineation of protease networks by mass spectrometry-based proteomics methods will elucidate how their components temporally and spatially shape the proteome in complex tissues