And broadly applicable proteolytic assay that probes thermal TA-01 site protein melting ex vivo using common laboratory equipment. We used the thermostable protease Thermolysin (TL) which preferentially cleaves near the hydrophobic residues Phe, Leu, Ile, Val [4,5]. TL showed sufficient specificity for unfolded states to probe protein stability in lysates within seconds. We applied the Fast parallel proteolysis (FASTpp)assay to monitor thermal unfolding of proteins ranging from 10 to 240 kDa and varying in secondary to quarternary structure. FASTpp detected stability alterations due to ligand binding and point mutations. Moreover, FASTpp can probe biophysical protein stability in cell lysates for biomedical screenings without genetic manipulation.Results FASTpp to assay protein stabilityThe unfolding temperature of a protein serves as an intuitive indicator for protein stability. Events that affect stability also affect the unfolding temperature [6,7]. Mutations that compromise protein structure shift, for instance, the point of thermal unfolding to lower temperatures while ligands that recognise the folded but not the unfolded state shift the thermal unfolding temperature to higher values [8?0] (Fig. 1A). A thermostable protease that readily cuts the unfolded but not the folded part of a protein could be used to determine the folded fraction over a wide temperature range. Based on these considerations, we propose a fast parallel proteolysis (FASTpp) assay to determine biophysical protein stability. The principle of the method is the parallel exposure of samples of the protein of choice to a range of different temperatures, in the presence of the thermostable protease. If we choose temperatures just above and below the specific melting temperature of the protein, the temperature-dependent changes of the degradation pattern are readout for the stability of the protein. The precision of the method depends on the precise control of theFast Proteolysis Assay FASTppheating time th, the period for which the protein is exposed to the maximum time (melting time; tm) and the subsequent cooling down period tc (Fig. 1B). Our assay consists of the following steps (Fig. 1C): 1. Sample preparation of the protein of interest at 4uC. 2. Addition of protease. 3. Heating time (th) during which several aliquots of the same sample are heated up in parallel. Each aliquot reaches a specific maximal temperature; for instance the lowest sample 35uC and the highest 42uC. 4. Melting time ™ during which aliquots are kept at defined maximum temperatures of the gradient for defined times. 5. Cooling time (tc) of the protein samples down to 4uC. 6. Stopping proteolysis by EDTA. 7. Analysis of the reaction products by SDS-PAGE. The steps 3? run in a thermal CASIN cycler with gradient control to ensure precision and reproducibility. Variations of th and tc may influence the (absolute) values 1326631 determined by this assay. These variables are instrument dependent, but automation ensures that all samples are reproducibly treated under 
identical conditions. We employed a Bio-Rad C1000 thermal cycler for which th is e. g. 20 s for heating a sample of 10 mL from 4uC to 60uC and tc is e. g. 40 s for cooling a sample of 10 ml from 60uC to 4uC. The C1000 cycler generates a gradient spanning a temperature difference of 22948146 up to 24uC in one block, which allows parallel screening of a sufficiently large temperature range for a broad range of proteins.Thermolysin is suitable for FASTppTo validate thi.And broadly applicable proteolytic assay that probes thermal protein melting ex vivo using common laboratory equipment. We used the thermostable protease Thermolysin (TL) which preferentially cleaves near the hydrophobic residues Phe, Leu, Ile, Val [4,5]. TL showed sufficient specificity for unfolded states to probe protein stability in lysates within seconds. We applied the Fast parallel proteolysis (FASTpp)assay to monitor thermal unfolding of proteins ranging from 10 to 240 kDa and varying in secondary to quarternary structure. FASTpp detected stability alterations due to ligand binding and point mutations. Moreover, FASTpp can probe biophysical protein stability in cell lysates for biomedical screenings without genetic manipulation.Results FASTpp to assay protein stabilityThe unfolding temperature of a protein serves as an intuitive indicator for protein stability. Events that affect stability also affect the unfolding temperature [6,7]. Mutations that compromise protein structure shift, for instance, the point of thermal unfolding to lower temperatures while ligands that recognise the folded but not the unfolded state shift the thermal unfolding temperature to higher values [8?0] (Fig. 1A). A thermostable protease that readily cuts the unfolded but not the folded part of a protein could be used to determine the folded fraction over a wide temperature range. Based on these considerations, we propose a fast parallel proteolysis (FASTpp) assay to determine biophysical protein stability. The principle of the method is the 
parallel exposure of samples of the protein of choice to a range of different temperatures, in the presence of the thermostable protease. If we choose temperatures just above and below the specific melting temperature of the protein, the temperature-dependent changes of the degradation pattern are readout for the stability of the protein. The precision of the method depends on the precise control of theFast Proteolysis Assay FASTppheating time th, the period for which the protein is exposed to the maximum time (melting time; tm) and the subsequent cooling down period tc (Fig. 1B). Our assay consists of the following steps (Fig. 1C): 1. Sample preparation of the protein of interest at 4uC. 2. Addition of protease. 3. Heating time (th) during which several aliquots of the same sample are heated up in parallel. Each aliquot reaches a specific maximal temperature; for instance the lowest sample 35uC and the highest 42uC. 4. Melting time ™ during which aliquots are kept at defined maximum temperatures of the gradient for defined times. 5. Cooling time (tc) of the protein samples down to 4uC. 6. Stopping proteolysis by EDTA. 7. Analysis of the reaction products by SDS-PAGE. The steps 3? run in a thermal cycler with gradient control to ensure precision and reproducibility. Variations of th and tc may influence the (absolute) values 1326631 determined by this assay. These variables are instrument dependent, but automation ensures that all samples are reproducibly treated under identical conditions. We employed a Bio-Rad C1000 thermal cycler for which th is e. g. 20 s for heating a sample of 10 mL from 4uC to 60uC and tc is e. g. 40 s for cooling a sample of 10 ml from 60uC to 4uC. The C1000 cycler generates a gradient spanning a temperature difference of 22948146 up to 24uC in one block, which allows parallel screening of a sufficiently large temperature range for a broad range of proteins.Thermolysin is suitable for FASTppTo validate thi.