Neuroproteomics: method development and applications

With this Thesis I have exploited several aspects of proteomics and phosphoproteomics related to neurologic diseases, such as glioblastoma, Alzheimer disease and globoid cell leukodystrophy (GLD or Krabbe disease). The work was focused both on methodological and applicative aspects. I worked mostly with murine cerebrospinal fluid and cell pellets and experimented several digestion methods for different starting protein amount, such as SP3, Urea and SDC digestion protocols. The longitudinal analysis of murine cerebrospinal fluid allowed to identify some molecular changes between the baseline, the asymptomatic stage and the symptomatic stage of the glioblastoma, such as macrophage capping protein, measured using a linear mixed effect model. I also focused on the relevance of phosphorylation as post-translational modification in different contexts, such as Alzheimer or Krabbe disease. As potential biomarker for Alzheimer disease I identified the phosphorylation of centrin-2, indicating a role of phosphocentrin-2 in the early phases of Alzheimer disease, never fully investigated before. From a methodological point of view, I also implemented an automated on-column TMT labeling procedure for both peptides and phosphopeptides, using AssayMAP BRAVO liquid handling robot, and I applied it for the analysis of differentiated neurons belonging to treated and untreated GLD cell lines, to explain better the multimodal action of psychosine in the pathogenesis of Krabbe disease.