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Determining Biophysical Protein Stability in Lysates by a Fast Proteolysis Assay, FASTpp 1 2 ¨ 1 David P. Minde , Madelon M. Maurice *, Stefan G. D. Rudiger * 1 Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands, 2 Department of Cell Biology, University Medical Center Utrecht (UMCU), Utrecht, The Netherlands Abstract The biophysical stability is an important parameter for protein activity both in vivo and in vitro. Here we propose a method to analyse thermal melting of protein domains in lysates: Fast parallel proteolysis (FASTpp). Combining unfolding by a temperature gradient in a thermal cycler with simultaneous proteolytic cleavage of the unfolded state, we probed stability of single domains in lysates. We validated FASTpp on proteins from 10 kDa to 240 kDa and monitored stabilisation and coupled folding and binding upon interaction with small-molecule ligands. Within a total reaction time of approximately 1 min, we probed subtle stability differences of point mutations with high sensitivity and in agreement with data obtained by intrinsic protein fluorescence. We anticipate a wide range of applications of FASTpp in biomedicine and protein engineering as it requires only standard laboratory equipment. ¨ Citation: Minde DP, Maurice MM, Rudiger SGD (2012) Determining Biophysical Protein Stability in Lysates by a Fast Proteolysis Assay, FASTpp. PLoS ONE 7(10): e46147. doi:10.1371/journal.pone.0046147 Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America Received July 6, 2012; Accepted August 27, 2012; Published October 3, 2012 Copyright: 2012 Minde et al. This is an open-access article distri