Applications of mass spectrometry-based proteomics to characterize preclinical animal models

Abstract

Proteomics, regarded as the comprehensive study of the gene expression at protein level at a particular time in different organs, tissues and cell types is a key enabling technology for the systems biology approach. The aim of this Ph.D. work is to establish quantification and discovery approaches to measure and study proteins involved in the pulmonary system and associated models of disease to be implemented in drug discovery processes. Progression or establishment of a pulmonary disease, normal lung development and different pharmacodynamic preclinical models were taken into account to explore the applicability of mass spectrometry-based proteomics to identify new key molecular players -or to confirm the existing ones-, potential biomarkers and to help drug target prioritization. To achieve this aim, a comprehensive proteomic workflow, involving protein extraction protocols optimization, chromatographic and mass spectrometry methods and bioinformatic analysis setup, has been optimized. The whole work has been performed in collaboration with Chiesi Farmaceutici S.p.A. Both bottom up targeted and discovery proteomics approaches, were considered precious tools to quantify target proteins through targeted experiments (MRM), and to lead to high-throughput discovery datasets, respectively. Both platforms were applied to answer to different key questions on pulmonary disease preclinical models. Targeted proteomics allows to focus on specific proteins, based on proteotypic peptide detection. With the accurate selection of proteotypic peptides, it is possible to isolate and quantify a specific protein isoform, or to detect post-translational modifications on peptides without enrichment protocols. In this work, as proof of concept, three examples of targeted proteomics are described, applied to established biomarkers of fibrosis (fibrillar collagen and α-sma) and applied to pharmacodynamic study of a known HDACs inhibitor. These methods required and optimization of protein extraction protocol from lung, which has peculiar characteristics specific of the fibrotic tissue, and of protein enrichment. Fibrillar collagen detection in fibrotic lung: a targeted method was developed to quantify collagen type I and collagen type III in rats treated with bleomycin with the focus on a time course analysis. A specific sample preparation was improved to enrich and digest extracellular matrix proteins. An MRM method was set up to investigate proteotypic peptides of the alpha chains of fibrillar collagens. The results confirm the knowledge about bleomycin-induced pulmonary fibrosis. Detection of α-sma in fibrotic lung: a targeted method was developed to investigate this isoform of actin, selecting proteotypic peptides unique for α-sma different from other actin isoforms. MRM method was set up and the results show that actin is not overexpressed at 28 days after bleomycin treatments, results supported by literature knowledge about the early expression in the bleomycin model. Quantitation of acetylation on histone proteins after treatment with HDACs inhibitor: a new targeted method was developed for this aim. Acetylated peptides and unmodified peptides of Histone H3 and H4 were detected in the MRM method, after an enrichment protocol for histone proteins. The results show a reduction of Histone acetylation from 1 hour to 72 hours after drug administration, confirming the effect of the pan-HDACs inhibitors over time. Discovery proteomics allows to identify and quantify thousands of proteins within a sample. Bioinformatic analysis leads to a deep understanding of the biological meaning and the processes involved. Two methods of quantitative high-throughput proteomics were studied and tested in this work. Proteome profiling through TMT quantitative proteomics: the quantitative labeling technique was used to characterize two different experiments, (1) a bleomycin-induced pulmonary fibrosis experiment in rat at 28 days, testing Nintendanib, the approved drug for pulmonary fibrosis; and (2) the characterization of lung developmental stages studied in a rabbit model, from canalicular stage to alveolar stage. Quantitative proteomics were coupled with bioinformatic analysis, in order to understand the biological processes involved in the system. Investigation of nintedanib mode of action through phosphoproteomics: As known, protein kinases and their substrates have been gaining increasing attention as therapeutic targets for the treatment of cancer and chronic inflammatory diseases. A specific sample preparation, based on enrichment of phosphopeptides, was employed to study the phosphoproteome of a multikinase inhibitor (Nintedanib), and three different bioinformatic approaches were applied to investigate the quantified phosphopeptides/proteins and to evaluate the mode of action of Nintedanib.