TY - JOUR
T1 - A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA
AU - Velazquez-Campoy, Adrian
AU - Claro, Bárbara
AU - Abian, Olga
AU - Höring, Jonas
AU - Bourlon, Louis
AU - Claveria-Gimeno, Rafael
AU - Ennifar, Eric
AU - England, Patrick
AU - Chaires, Jonathan Brad
AU - Wu, Di
AU - Piszczek, Grzegorz
AU - Brautigam, Chad
AU - Tso, Shih Chia
AU - Zhao, Huaying
AU - Schuck, Peter
AU - Keller, Sandro
AU - Bastos, Margarida
N1 - Publisher Copyright:
© 2021, European Biophysical Societies' Association.
PY - 2021/5
Y1 - 2021/5
N2 - 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.
AB - 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.
KW - Benchmark study
KW - Data treatment
KW - Isothermal Titration Calorimetry (ITC)
KW - Ligand-binding
KW - Sample preparation
KW - Standard reaction
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U2 - 10.1007/s00249-021-01523-7
DO - 10.1007/s00249-021-01523-7
M3 - Article
C2 - 33864101
AN - SCOPUS:85104703666
SN - 0175-7571
VL - 50
SP - 429
EP - 451
JO - European Biophysics Journal
JF - European Biophysics Journal
IS - 3-4
ER -