Top Down Proteomics on TDP-43 and its interaction partners

Proteins are key functional entities in the cell, regulating cellular processes by protein variation, stemming from proteoforms, such as splice variants, truncations, and proteins with post-translational modifications (PTMs). Although the number of proteoforms that are reported to be functional in disease related processes is rapidly increasing, they remain poorly characterized due to the lack of appropriate analytical methods. Hence, this project has the long-term goal to decipher proteoforms, their PTM codes and cross-talk dynamics, on individual proteins and between interacting proteins, by means of a combination of novel proteomics technology with mouse model systems. Short-term research objectives are to further develop and optimize intact protein top-down proteomic methods (Aim A), addressing the main challenges of the field today. In particular, the work aims at (i) evaluating different strategies for the reduction of sample complexity, (ii) improving protein sequence coverage and thus PTM localization by implementation of a dual fragmentation technique developed in the Heck lab, combining electron transfer dissociation (ETD) and higher-energy collision dissociation (HCD), termed EThcD, and (iii) comparing different protein deconvolution, identification, and quantification software for top down proteomics data. The developed methods will then be applied for characterizing the PTM codes on transactive response DNA-binding protein (TDP-43) and its interacting proteins to elucidate their role in the neurodegenerative disease amyotrophic lateral sclerosis (ALS, Aim B). More specifically, I seek to generate an inventory of ALS-related proteoforms, a better understanding of the cross-talk between interacting proteoforms, and a quantitative view of the PTMs regulating the development of inclusions in ALS. The project will be undertaken in the Biomolecular Mass Spectrometry and Proteomics group lead by Professor Albert Heck at Utrecht University. Due to the strong proteomics expertise of this group, in particular in the fields of PTM analysis and ETD fragmentation, the Heck lab is the ideal location for my research.