Investigating protein expression, modifications and interactions in the brain: Protocol for preparing rodent brain tissue for mass spectrometry-based quantitative- and phospho-proteomics analysis.

Abstract Neurodegenerative diseases remain a major global health challenge, affecting millions of individuals worldwide. Despite significant research efforts, the etiology and pathological mechanisms of most neurodegenerative disorders remain uncertain. This indicates that new disease models and research strategies need to be developed to enhance biomarker discovery and drug development processes. Studies have shown that dysregulation of protein-protein interactions in the brain may be key to the pathophysiological processes in a number of neurodegenerative diseases. Consequently, a deeper understanding of protein-protein interactions in the brain in both healthy and neurodegenerative states may provide greater insights into disease mechanisms and progress, and aid the development of preventive, management and treatment strategies. Efforts to investigate protein-protein interactions in the brain have largely depended on purifying the protein, together with its interacting partner proteins (interactome) and then analyzing these interactions by mass spectrometry. However, factors including abundance and phosphorylation status of the protein of interest and its interactome can lead to identification of false protein-protein interactions. Moreover, studies have shown that combining quantitative proteomics data with data from affinity-purification-mass spectrometry data can reduce these false positives and also provide more insights into protein-protein interactions. Thus, we have developed a protocol for preparing rodent brain tissue for quantitative- and phospho-proteomics analysis, combining in-house and commercially available kits. Data from this protocol will enhance the analysis and interpretation of protein-protein interactions in both physiological and pathological states.