Cells have an extensive quality control network responsible for maintaining proteostasis. This network regulates protein synthesis, folding and transport. Proteostasis is often severely imbalanced in neurodegenerative diseases such as Alzheimer’s and Motor Neuron diseases, permitting the characteristic aggregation and deposition of vulnerable proteins. After decades of dedicated examination, the folding and stability characteristics of many individual proteins are well understood in vitro. Most often, this is achieved using a single purified protein in a test tube. However, understanding the process of protein folding and unfolding in the cellular milieu remains a grand challenge.
A key limitation to probing protein stability within a biological context is the capacity to quantitatively assess the folding status of individual proteins at the proteome-wide scale. We have recently developed a fluorogenic thiol-binding dye (TPE-MI) that can capture a snapshot of the balance of unfolded protein relative to folded states [1]. This approach offers single-protein folding information for endogenous proteins at a proteome-wide scale. Here, we demonstrate the use of this probe to yield denaturation curves of the cys proteome in whole-cell lysate. We then investigate the altered stability of a subset of proteins in response to pharmacological proteostasis challenge. This probe represents an invaluable tool which will empower researchers to quantitatively probe proteome foldedness across diverse disease states in a variety of model organisms.