Limited Proteolysis Mass Spectrometry: A Pipeline for Investigating Loss of Proteostasis with Age (360G-Wellcome-220073_Z_20_Z)

Cell populations are intrinsically changed by age, reaching a point of senescence (arrested division) that has been associated with misregulation of metabolism, circadian rhythm, the extracellular matrix and signalling to the immune system. Crucially, studies have demonstrated age-related abrogation of protein homeostasis – the maintenance of proteins in their functional, folded states. Given that the capacity of cells to maintain a correctly folded proteome, and thus functionality, is attenuated in ageing, understanding the biological mechanisms which underlie this loss of protein homeostasis is paramount. In this project, I will use proteomic and bioinformatical techniques to gain a fundamental understanding of the relationship between age and the loss of protein homeostasis. Limited proteolysis mass spectrometry (LiP-MS) is highly amenable to the study of protein folding, as it facilitates the characterisation of structural changes to the native proteome on both a global and local scale. I will apply LiP-MS to determine the extent of protein unfolding in primary human stem cells. Using replicative senescence as a model of cellular ageing, I will identify a candidate list of proteins whose folding/structure is not maintained at old age and use bioinformatic analyses to predict functional consequences of age-associated protein unfolding.