Development of mass spectrometry-based proteome analysis workflows for the systematic study of cardiac disease research models
by Hugo Alejandro Amedei
Date of Examination:2024-08-12
Date of issue:2024-09-26
Advisor:Dr. Christof Lenz
Referee:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Stephan E. Lehnart
Persistent Address:
http://dx.doi.org/10.53846/goediss-10763
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Description:In his thesis work, Hugo Amedei describes the development of both analytical workflows and data processing and evaluation approaches for an approach termed Complexome Profiling (CP). In CP, cells or tissue are lysed under non-denaturing (or ‘native’) conditions that do not disrupt non-covalent protein-protein interactions. Consequently, protein-protein complexes (PPCs) can be fractionated by e.g. Blue Native-PAGE or Size Exclusion Chromatography (SEC) according to their apparent molecular weight, and their presence and abundance profiled across fractions using mass spectrometry-based bottom-up proteomics approaches. Shared or at least similar intensity profiles across fractions (co-elution, co-migration) indicate biophysical interaction and, by extension, joint biological function in PPCs. application/pdf
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Abstract
English
A specific variant of genetic mutation in the phospholamban gene expresses a cardiac phospholamban protein lacking the arginine amino acid at position 14 (R14Δ-PLN). The mutation is believed to have a founder in the Netherlands, with over 1500 cases detected. It is also present in Greece, the United States, Canada, China, and Japan. The variant potentially leads to dilated cardiomyopathy and/or arrhythmogenic cardiomyopathy. A clinical study in the Netherlands suggests that there is a 12–15% correlation between the R14Δ-PLN mutation and dilative cardiomyopathy or arrhythmogenic cardiomyopathy, respectively. The clinically documented cases for the R14Δ-PLN mutation exhibit heterozygosity. Patient carriers are at risk of developing biventricular abnormalities, and fatty and fibrofatty infiltration of the outer compact layer of the ventricular myocardium, with a poor prognosis. Under unknown treatment, efforts are focused on detecting early disease events in cardiomyopathy prior to the manifestation of the initial symptoms. Complexome profiling and crosslinking mass spectrometry are two systems wide mass spectrometry-based proteome methods with proven capabilities for the detection of protein protein interactions. Complexome profiling was applied to study atrial fibrillation by Alsina et al., 2019. Crosslinking mass spectrometry was studied in mouse cardiac tissue in a pioneering study by Chavez et al., 2018; there are, however, only a few reports of the application of complexome profiling and crosslinking mass spectrometry to whole-cell or tissue studies in cardiovascular research. While less explored in cardiology, the methods hold promise for revealing system-wide changes with further development and optimization. We hypothesized that the R14Δ-PLN mutation causes a systemic affection that leads to cardiomyopathy through changes or alterations in the composition and function of different protein-protein complexes and critical cardiac pathways. Therefore, we developed novel systems for wide mass spectrometry-based proteome analysis workflows and investigated the R14Δ-PLN cardiomyopathy in a well-established heterozygous mouse disease-causing variant research model of the R14Δ-PLN mutation. Here, I extend the state-of-the-art of complexome profiling and crosslinking mass spectrometry workflows with enhanced application to the cardiology field. I introduce two platforms for complexome profiling and one for crosslinking mass spectrometry, along with their respective bioinformatics analysis approaches: 1- mini-Complexome Profiling (mCP), a precast blue native polyacrylamide gel electrophoresis data independent acquisition mass spectrometry workflow. Along with software for FDR-controlled global targeted detection of protein complexes. 2- Extended-range SEC-DIA/MS workflow, utilizing size exclusion chromatography (SEC). Along with a bioinformatics workflow for unbiased differential identification of altered protein profiles between two conditions. 3- A crosslinking mass spectrometry workflow. Along with an integrative workflow for mutual validation of crosslinking and complexome profiling of the detected protein protein interactions. I conducted a systematic analysis of high-molecular weight protein complexes and supercomplexes in young, presymptomatic R14Δ/+ mouse hearts by CP workflows. We identified alterations in inner mitochondrial membrane complexes and preliminary intercalated disk supercomplexes, which are involved in cardiomyocyte-to-cardiomyocyte electrical contacts, as early indicators of disease. Additionally, our findings suggest MICOS complexes alterations, rather than changes in calcium handling components, are associated with disruptions at ER-Mitochondria contact sites. The impaired cardiomyocyte connections and resulting energetic imbalance could explain the arrhythmic events in the disease. These discoveries highlight the impact of mitochondrial dysfunction and ICD cell-cell contact disruption in the early stages of R14Δ-PLN cardiomyopathy, offering potential early-disease therapeutic targets, particularly crucial given the lack of effective treatments for advanced stages of the disease.
Keywords: Complexome profiling; proteome; protein complex; protein interaction; targeted proteomics; Blue Native; PLNR14del; cardiomyophaty; ; Size Exclusion
