A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called “stoichiometry” (n), a concentration-correction factor.
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M.B. acknowledges the financial support from Fundação para a Ciência e Tecnologia (FCT), Portugal, together with FEDER through “Programa Operacional Competitividade e Internacionalização” (POCI), by COMPETE2020 to Projects POCI-01-0145-FEDER-030579 and UIDB/00081/2020. A.V.C. and O.A. acknowledge financial support from the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (BFU2013-47064-P and BFU2016-78232-P to A.V.C.), the Spanish Ministry of Education and Culture (FPU13/3870 to R.C.G.), Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, “Investing in your future”) (PI15/00663 and PI18/00349 to O.A.), Diputación General de Aragón (Protein Targets and Bioactive Compounds Group E45_20R to A.V.C. and Digestive Pathology Group B25_20R to O.A.) and Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). JBC was supported by NIH Grant GM077422. This work was supported by the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health. MB gratefully acknowledges the support of Frederico Silva of the Biochemical and Biophysical Technologies Scientific Platform of the Instituto de Investigação e Inovação em Saúde, I3S, Universidade do Porto. We all thank COST Action CA15126, Working Group 4, for the support to this study.
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Velazquez-Campoy, A., Claro, B., Abian, O. et al. A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA. Eur Biophys J (2021). https://doi.org/10.1007/s00249-021-01523-7
- Isothermal Titration Calorimetry (ITC)
- Standard reaction
- Benchmark study
- Data treatment
- Sample preparation