Caroline Mas, Grzegorz Piszczek, Marjetka Podobnik, Kathryn Perez, Timothy Sharpe, Stephen H. McLaughlin, Roland Montserret, Stefan H. Knauer, Bruno Baron, Christopher M. Johnson, Quyen Nguyen, Mark A. Williams, Katja Pirc, Arthur Sedivy, Celeste Abreu, Tina Daviter, David Ortega-Alarcon, Daumantas Matulis, Rouba Nasreddine, Carlo Carolis, Thomas A. Jowitt, Stephan Uebel, David Staunton, Andrew P. Leech, Maria Garcia-Alai, June Southall, Alexander Fish, Jitka Holková, Alexander K. Buell, Michael Weyand, Jasmina Rokov-Plavec, Blanca López-Méndez, Sylvie Berger, Josef Houser, Natalia Rodrigo, Wojciech Bal, Chad A. Brautigam, Josef Hamacek, Reine Nehmé, Olga Abian, Christian Guenther, Ondrej Vanek, Susanne Schaefer, Sharon M. Kelly, Malgorzata Adamczyk, Di Wu, Pedro Tavares, University of Copenhagen = Københavns Universitet (KU), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), University of Texas Southwestern Medical Center [Dallas], University of Manchester [Manchester], Universität Bayreuth, Max Planck Institute of Biochemistry (MPIB), Max-Planck-Gesellschaft, University of London [London], Vienna Biocenter Core Facilities, Partenaires INRAE, University of Zaragoza - Universidad de Zaragoza [Zaragoza], Charles University [Prague] (CU), Warsaw University of Technology [Warsaw], Institute of Biochemistry and Biophysics, Polish Academy of Sciences (PAN), Institut de Recherches SERVIER (IRS), Technical University of Denmark [Lyngby] (DTU), Centre for Genomic Regulation [Barcelona] (CRG), Universitat Pompeu Fabra [Barcelona] (UPF)-Centro Nacional de Analisis Genomico [Barcelona] (CNAG), The institute of cancer research [London], Netherlands Cancer Institute (NKI), Antoni van Leeuwenhoek Hospital, EMBL Hamburg, Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Central European Institute of Technology [Brno] (CEITEC MU), Brno University of Technology [Brno] (BUT), MRC Laboratory of Molecular Biology [Cambridge, UK] (LMB), University of Cambridge [UK] (CAM)-Medical Research Council, University of Glasgow, University of York [York, UK], Integrated Structural Biology Grenoble (ISBG - UMS 3518 ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Vilnius University [Vilnius], Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie Organique et Analytique (ICOA), Université d'Orléans (UO)-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Institut National de la Santé et de la Recherche Médicale (INSERM), EMBL Heidelberg, National Institute of Chemistry, National Heart, Lung, and Blood Institute [Bethesda] (NHLBI), University of Zagreb, University of Bayreuth, University of Basel (Unibas), University of Oxford [Oxford], Faculdade de Ciências e Tecnologia [Faro] (FCT), Universidade do Algarve (UAlg), This research was funded by Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, 'Investing in your future' PI18/00349 and Diputación General de Aragón Digestive Pathology Group B25_17R to O.A. Spanish Ministry of Science, Innovation and Universities, FPI Predoctoral Research Contract BES-2017-080739 to D.O.A. This research was funded by project no 2015/17/B/NZ2/01160 (granted to MA), we also acknowledge financial support, from the Faculty of Chemistry at Warsaw University of Technology. As part of the Federal FTI strategy, this work was generously supported by the City of Vienna and the Austrian Ministry for Education, Science and Research., Frapart, Isabelle, University of Copenhagen = Københavns Universitet (UCPH), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Max-Planck-Institut für Biochemie = Max Planck Institute of Biochemistry (MPIB), Danmarks Tekniske Universitet = Technical University of Denmark (DTU), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Integrated Structural Biology Grenoble (ISBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and University of Oxford
Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method. All authors acknowledge the COST Action project ARBRE-MOBIEU CA15126 under the auspices of whose Working Group 4 this study was carried out. ARBRE-MOBIEU COST Action CA15126 funded meetings in the Center for Protein Research, University of Copenhagen (ECOST-MEETING-CA15126-280618-098884) to organize this study and in the Vienna Biocenter Core Facilities GmbH (ECOST-MEETING-CA15126-121119-111544) to discuss the results of the benchmark. Ondřej Vaněk acknowledges support from the Ministry of Education, Youth and Sports of the Czech Republic (LTC17065 in frame of the COST Action CA15126). Jitka Holková and Josef Houser acknowledge CF Biomolecular Interactions and Crystallization of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2018127). Jasmina Rokov-Plavec acknowledges support from the Croatian Science Foundation (IP-2016-06-6272). Alexander K. Buell thanks the Novo Nordisk Foundation (grant number NNFSA170028392) for funding. Li Peng Lundgren acknowledges for preliminary MST/TRIC experiments (STMS 37745 in the frame of the COST Action CA15126) and the COST Action (CA15126) for a STMS (STMS 44783) for the data analysis. The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (Grant agreement NNF14CC0001). Katja Pirc and Marjetka Podobnik acknowledge the grant by the Slovenian Research Agency P1-0391 (Title: Molecular Interactions). This research was funded by Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, “Investing in your future” PI18/00349 and Diputación General de Aragón Digestive Pathology Group B25_17R to O.A. Spanish Ministry of Science, Innovation and Universities, FPI Predoctoral Research Contract BES-2017-080739 to D.O.A. This research was funded by project no 2015/17/B/NZ2/01160 (granted to MA)