biostudies-literatureHellman LMNCATS NIH HHSNIAID NIH HHSNCI NIH HHSNational Institutes of HealthNIGMS NIH HHS95-101https://www.ebi.ac.uk/biostudies/studies/S-EPMC4837003biostudies-literatureUnknown432Measurements of thermal stability by circular dichroism (CD) spectroscopy have been widely used to assess the binding of peptides to MHC proteins, particularly within the structural immunology community. Although thermal stability assays offer advantages over other approaches such as IC50 measurements, CD-based stability measurements are hindered by large sample requirements and low throughput. Here we demonstrate that an alternative approach based on differential scanning fluorimetry (DSF) yields results comparable to those based on CD for both class I and class II complexes. As they require much less sample, DSF-based measurements reduce demands on protein production strategies and are amenable for high throughput studies. DSF can thus not only replace CD as a means to assess peptide/MHC thermal stability, but can complement other peptide-MHC binding assays used in screening, epitope discovery, and vaccine design. Due to the physical process probed, DSF can also uncover complexities not observed with other techniques. Lastly, we show that DSF can also be used to assess peptide/MHC kinetic stability, allowing for a single experimental setup to probe both binding equilibria and kinetics.Journal of immunological methodsDifferential scanning fluorimetry based assessments of the thermal and kinetic stability of peptide-MHC complexes.PMC4837003R01 GM103773CA154778R37 AI038996CA153789TR001108R21 CA153789F30 CA180731CA15378R56 AI038996CA180731R01 GM067079GM103773GM067079AI038996UL1 TR001108P01 CA154778R01 AI038996Belden OSRiley TPHellman LMBaker BMBlevins SJSpear TTStern LJNishimura MIWang YYin LfalseDifferential scanning fluorimetry based assessments of the thermal and kinetic stability of peptide-MHC complexes.Measurements of thermal stability by circular dichroism (CD) spectroscopy have been widely used to assess the binding of peptides to MHC proteins, particularly within the structural immunology community. Although thermal stability assays offer advantages over other approaches such as IC50 measurements, CD-based stability measurements are hindered by large sample requirements and low throughput. Here we demonstrate that an alternative approach based on differential scanning fluorimetry (DSF) yields results comparable to those based on CD for both class I and class II complexes. As they require much less sample, DSF-based measurements reduce demands on protein production strategies and are amenable for high throughput studies. DSF can thus not only replace CD as a means to assess peptide/MHC thermal stability, but can complement other peptide-MHC binding assays used in screening, epitope discovery, and vaccine design. Due to the physical process probed, DSF can also uncover complexities not observed with other techniques. Lastly, we show that DSF can also be used to assess peptide/MHC kinetic stability, allowing for a single experimental setup to probe both binding equilibria and kinetics.2016-01-01T00:00:00Z2016 May2024-11-09T01:51:55.883Z2019-03-27T02:11:57ZS-EPMC48370032690608910.1016/j.jim.2016.02.016