Characterization of PFAS Binding Effects on Protein Structure Using Collision-Induced Unfolding

Per- and poly fluoroalkyl substances (PFAS) have become a global concern due to their persistence in the environment, contaminating drinking water, air, and soil. Human exposure to PFAS can potentially cause adverse effects due to its bioaccumulation and nonbiodegradability. To fully understand the role of PFAS in human health conditions, it is important to elucidate their roles in cellular toxicity and biotransformation pathways. Noncovalent complexation of PFAS to proteins is one potential mode of toxicity that can be investigated by comparing structural differences between native and bound proteins. In this work, we perform collision-induced unfolding (CIU) using a cyclic ion mobility-mass spectrometer (cIM-MS) to measure the effects of PFAS binding on protein structure. CIU characterizes the unfolding pathway of analytes by measuring changes in analyte size and shape as a function of increasing activation energy. The CIU results of different species can then be compared to determine potential structural changes. This method is demonstrated using ubiquitin as a model protein and three related PFAS: perfluorobutanesulfonic acid (PFBS), perfluorohexanesulfonic acid (PFHxS), and perfluorooctanesulfonic acid (PFOS). All three PFAS have the same sulfonate headgroup but different fluorinated chain lengths. We observed both qualitative and quantitative differences in ubiquitin unfolding based on the number of bound PFAS molecules as well as the PFAS chain length, suggesting that these molecules are not necessarily passive when associated with protein. Primarily, our results demonstrate a rapid, targeted analysis that can characterize the noncovalent complexation of toxins to biological molecules.