Novel (pospho-)proteomics approaches to study aging and senescence

This thesis describes the application of proteomics technologies to further increase the knowledge about aging and cancer suppression mechanisms. The core tool in all performed experiments is mass spectrometry based proteomics. In addition to the application of these techniques, newly developed proteomics techniques are being introduced that help to increase the analytical power of proteomics experiments. In chapter one, a general introduction is given into proteomics and mass spectrometry-based proteomics workflows. The type of research question strongly determines the type of experimental workflow and equipment needed. Therefore, there is no standard one method fits all approach resulting in highly dynamic experimental workflow with a myriad of different techniques/approaches. In chapter two, the influence of DNA-damage on tissue aging was studied for a specific part of the mouse brain over time. Quantitative shotgun proteomics discovery experiments were performed in order to find biomarkers and mechanisms of aging in the cerebellum. After the identification of several interesting differentially regulated proteins associated with age and neuron-deterioration by proteomics, a more targeted validation was performed using immunohistochemistry. By using this complementary method, changes over time as well as changes in protein localization could be monitored for a set of differentially regulated proteins. Major observations of this study include a DNA-damage induced down regulation of a tightly interconnected synaptic signaling network associated with a morphological transformation of Purkinje cells and a new molecular link between DNA-damage and motoric diseases such as spinocerebellar ataxia. Chapter three describes a technical method development to optimize the transition from a discovery orientated proteomics approach to a targeted proteomics experiment. The work demonstrates that CID spectra acquired on SRM instruments are more similar to spectra acquired in the HCD cell than those acquired in the ion trap of hybrid LTQ-Orbitrap instruments. Concomitantly, SRM assays generated using HCD spectra showed a higher sensitivity when compared to ion trap spectra-generated SRM assays. Therefore, when planning a targeted MS experiment, choosing for HCD fragmentation in the discovery phase can help facilitate SRM assay development later on. In chapter four, the phosphopeptide enrichment robustness of a new Ti4+-IMAC method was assessed. First it was established that Ti4+-IMAC enrichment resulted in a highly reproducible quantification of phosphorylation sites in HeLa cells. Subsequently, this strategy was applied to monitor the phosphoproteome of Jurkat T-cells upon Prostaglandin E2 stimulation over the timespan of 60 minutes. It was demonstrated that using this enrichment strategy and label-free quantification a comprehensive temporal phosphoproteome of Jurkat T-cells could be constructed, indicating differential regulation of different kinases over time. The proved straight-forward yet comprehensive phosphoproteomics workflow and its’ applicability to every sample type could form a good alternative method for the phosphoproteomics community. Chapter five describes a study performed to increase knowledge about the mechanisms involved in BRAFV600E oncogene induced senescence (OIS). Using both whole proteome and phosphoproteome analysis, differential protein expression and phosphorylation were compared between cycling, OIS and OIS bypassed (OISb) cells. Proteome analysis revealed a strong resemblance between proliferating BRAF-activated (OISb) cells and senescent BRAF-activated (OIS) cells. In both cell lines an up regulation of the retinoblastoma tumor suppressor mechanism was observed that was more pronounced in OIS. Proteins specifically regulated in OIS included senescence markers, inflammatory proteins and extracellular processing factors amongst other previously senescence-unassociated proteins. Phosphorylation screening identified strong reduction in cyclin-dependent kinase activity and numerous sites to be differentially regulated in OIS. This work forms an extensive resource of protein and phosphorylation regulations associated with OIS, benefitting researchers in the field of tumor suppressor research. In chapter six an outlook is described regarding the validation of proteomics-derived target lists with the emphasis on cell signaling analysis. Currently, antibody-based methods are used predominantly to validate and investigate the involvement of proteins and post translation modifications (PTMs) in different biological processes. Limitation of antibody-based methods are described and targeted mass spectrometry-based methods are proposed as an alternative approach to overcome these problems. Especially for PTM analysis, targeted mass spectrometry has a high potential that will likely become a standard methodology for dynamic cell signaling analysis.