Proteomics is frequently utilized to identify proteins that are differentially expressed under perturbation or disease conditions and has been instrumental in uncovering the mechanisms of the cellular response to such stresses. However, the effects of perturbation on the proteome are often structural and thus will not be captured by measurements of protein abundance alone. Protein-protein interactions, post-translational modifications and small molecule binding are all events that result in protein conformational change and have important functional consequences. Further, perturbation-induced protein structural changes such as unfolding or aggregation can lead to loss of function or even toxic gain of function. Limited proteolysis mass spectrometry (LiP-MS) is an emerging structural proteomics technique capable of evaluating changes to protein conformation on an omics scale in complex biological samples. The LiP-MS pipeline employs a non-specific enzyme and restricted digestion time to nick exposed regions of proteins in their native states. Cleavage sites are then identified by mass spectrometry and subsequent bioinformatic analysis. This thesis first focuses on optimizing and validating LiP-MS for use in primary human cells. The protocol was then utilized to investigate the cellular response to loss of proteostasis, induced through treating cells with heat shock and chaperone inhibitors. This experiment was able to identify hundreds of proteins with altered conformational states. These proteins were significantly enriched for known cellular responses to loss of proteostasis, implying a systematic remodelling of conformations and interactions within the proteostatic machinery in response to stress. Further, specific, peptide level conformational alterations in proteins with well-studied roles in the heat shock response were identified, such as those required for chaperone-client interactions. Moving forward, LiP-MS has the potential to provide fresh insight into the mechanisms of the cellular response to stress through identifying previously undescribed, functionally critical protein structural changes, the presence and consequences of which can then be further investigated using orthogonal biochemical techniques.
|Date of Award||1 Aug 2023|
- The University of Manchester
|Supervisor||Simon Hubbard (Supervisor) & Joe Swift (Supervisor)|