ITC data analysis contest – New submission Date

A few days ago AFFINImeter launched an Isothermal Titration Calorimetry (ITC) data analysis contest. At this point no one has been able to finish the challenge, eventhough there is a considerable amount of participants. Since the main goal of the contest is to give all our AFFINImeter users an educational tool to get experience in the process of ITC data analysis.

Therefore, we would not be satisfied if some of you ran  out of time. For this, we have decided to extend the contest deadline to January 31st

Join the Challenge

We encourage all people interested in molecular binding analysis, not only based on ITC data but also on any other experimentaltechnique, to participate in this challenge. If you are an expert in the analysis of thermodynamic data this is a nice opportunity for you!

Find the best combination of model & parameters for the target data and win a Kindle Fire HD 7 just sending us your results.

tablet kindle fire HD7
Isothermal Titration Calorimetry Data Analysis Contest

If you are a beginner in the analysis of ITC or, in general, of binding experiments, this challenge is also perfect for you since it will give you the opportunity to learn how to analyse actual ITC measurements. Our scientific team has selected some really interesting experiments and prepared some instructions that will guide you in the analysis process.

Learn the difference between stoichiometric and site equilibrium constants

By trying to solve this contest you will learn the difference between stoichiometric and site equilibrium constants, you will see how to easily perform a global analysis of several isotherms, and even how to find the, eventually unknown, concentration of active protein in a given experiment. Sign up HERE  and you will get free access to AFFINImeter. Please, do not hesitate to encourage your labmates to join us. People from the whole world are already participating!

Download Poster here

The importance of the treatment of ITC raw data in calorimetry experiments

Isothermal titration calorimetry (ITC) is an extremely sensitive technique to assess for the formation/disruption of complex chemical/biological species in solution. During the last years, the increase in instrument sensitivity as well as the reduction of the sample concentration required to perform experiments, have made possible to expand the application range of ITC, which is expected to continue growing.

Quality of the ITC Raw Data?

The amount and the quality of useful information that can be obtained from an ITC experiment depend on several factors including the purity of the samples, the concentration of the solutions prepared, the choice of injection volume and its length in time. The researcher handling the instrument is responsible for the appropiate selection of these variables as part of the experimental setup. They can be optimized on the basis of previous experience and also taking advantage of computational simulations. A key factor for this is that ITC is an incremental technique and so the results depend strongly on the injection volume employed to perform the experiment.

Kinetic information from ITC Experiments

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Direct reading of Microcal Origin OPJ files with AFFINImeter

Direct reading of Microcal Origin OPJ files with AFFINImeter

The direct reading  Microcal Origin OPJ files has been recently implemented in AFFINImeter. Initially, only OPJ files containing a single datasheet were allowed. Our code has just been modified to make possible the reading of OPJ files with multiple datasheets. Additionally the uploaded OPJ files are now being filtered to permit just the selection of datasheets suitable for analysis with AFFINImeter (i.e. datasheets of ITC isotherms). Some minor improvements that optimize this process have also been implemented.
Soon you will also be able to directly upload Microcal Origin itc files. In collaboration with our scientific advisor Prof. Philippe Dumas, from the University of Strasbourg, we are currently implementing a procedure to automate the integration of the raw ITC data, including removal of noise and baseline correction.

How to perform a Global Fitting Analysis?

The Global fitting of multiple isotherms is one of the advanced tools that AFFINImeter offers to facilitate the analysis and interpretation of isothermal titration experiments and to expand the range of applications of this technique.

The following video tutorial describes the global fitting of three isotherms of a displacement assay describing, a receptor interacting with a tight ligand, with a weak ligand, or with both ligands simultaneously, in a competitive experiment where the ligands are mixed in the syringe of the ITC equipment.

 

If you want to know more about global fittings with AFFINImeter you can also download the case of use “Global Analysis in ITC Displacement Titrations with AFFINImeter” that describes a Displacement Titration Assay to determine the thermodynamics of HIV-protease with indinavir, a high-affinity binder, and with acetyl-pepstatin, a weaker ligand.

ITC displacement titrations offer an attractive alternative to standard assays when working with ultra-high or ultra-low- affinity interacting systems. The method requires the fitting of at least two isotherms that share various adjustable parameters. The case study exemplifies the potential advantages of using AFFINImeter in ITC displacement assays. The software offers unique advanced tools that enhance the robustness of the method and makes it more versatile, facilitating the acquisition of reliable thermodynamic data from ultra-high of ultra-low affinity systems. Thus, it opens a door for new applications of the displacement assay.

 

 

 

Competitive Binding Assay

Competition assays

Competitive binding assays where two (or more) ligands bind to the same receptor have become common experiments in many research areas, from basic investigations to innovation in the pharmaceutical industry. These assays can be done in different formats, i.e. through a displacement assay where ligand “L1” is displaced by ligand “L2” from a preformed complex “L1-receptor” or via titration of a receptor solution with a mixture of L1 +L2. Either way, the competitive binding assay provides rich thermodynamic and structural information of the various binding events taking place during the course of the experiment. Thus, Isothermal Titration Calorimetry (ITC) competition assays performed in a displacement format have been revealed as an efficient tool for the quantitative analysis of very high- / low- interactions, with application in the field of fragment based drug screening (ref).

Analysis of competition experiments with AFFINImeter

The versatility of the experimental setup in AFFINImeter-ITC permits the analysis of ITC competition experiments in its various formats. As an illustration, the following lines describe the analysis of an ITC isotherm resulting from a competitive experiment where a solution of a receptor in titrated with mixture of two competing ligands.

AFFINImeter contains a series of examples with which users can practice and learn the overall process of data fitting: from equipment and data uploading to fitting model design and data fitting.

In this post we will review an example of a competitive model fitting model used to analyze the experiment data of two ligands in the syringe competing for binding to the same receptor.

Practical case: competitive binding model

The AFFINImeter example “competitive binding model” illustrates an ITC experiment where two ligands, “A” and “B” compete with each other for binding to the receptor “M”.

Drawing of a competitive binding model
Competitive Binding model scheme

This situation corresponds to a binding model consisting of three free species (A, B and M) two binding equilibria representing the interaction of M with A and M with B

The model was designed with the “reaction builder” and stored in “models”.The equipment used is decribed and stored in “equipments”. The dataseries is uploaded and stored in “dataseries”. When the dataseries is uploaded, the user has to complete the information relative to the equipment used and the species concentration. In this particular case (where there is a competitor “B”).

How to fit a project with AFFINImeter

 

1- Go to PROJECT MANAGEMENT and create a new PROJECT an a new FIT SUBPROJECT.

  • Add the dataseries created previously to the subproject.
  • Select the model created previously.
  • Keep all the default values in FitSetting.

2- Press Run button.

This Steps are described in the following Video Tutorial:

You can follow this tutorial in AFFINImeter, the Experimental Data and Binding model are stored in your own AFFINImeter account. If you hasn’t registered yet go to the AFFINImeter Software WebPage to get your account.

 

 

Ref: W. B. Turnbull, Divided we fall? Studying low-affinity fragments of ligands by ITC. GE Healthcare Life Sciences protocol, 2011, pp 1-11.

 

THANK YOU TO ALL PARTICIPANTS OF THE WIDEN-ITC OPEN DAY

THANK YOU TO ALL PARTICIPANTS OF THE WIDEN-ITC OPEN DAY: Over 100 researchers joined us to know more of the potential and latest advances in Isothermal Titration Calorimetry.

Speakers and invited researchers.
Speakers and invited researchers to WIDEN-ITC Open Day.

The WIDEN-ITC Open Day, has confirmed the awareness of the Scientific Community in exploring the full potential of the Isothermal Titration Calorimetry technique. Researchers from 18 countries attended on-site in Santiago de Compostela or via streaming this Open Day, consisting in 8 scientific talks and a tutorial of the ITC analysis software AFFINImeter (developed by the organizers of the workshop). Outstanding Pharmaceutical Companies (Lilly, Astra Zeneca,…..), microcalorimeters manufacturers (Malvern Microcal) and numerous Universities and Research Institutes worldwide participated in this interactive day,  dedicated  to share knowledge and exchange ideas around the present and future of ITC. The WIDEN-ITC Open Day discussed topics of actual interest like the current state of both ITC instruments and data analysis, common errors and misuses, and new analytical and technical developments to exploit the full potential of the technique and expand the application range of ITC. The morning session consisted in 6 invited talks of distinguished experts in the field: Adrián Velázquez-Campoy (University of Zaragoza, Spain), Phillipe Dumas (CNRS, Strassbourg, France), Eric Ennifar (CNRS, Strassbourg, France), Natalia Markova (Malvern Microcal), Niek Burma (Cardiff University, UK) and Daniel Ondo (Institute of Chemical Technology, Prague). The evening session of the Open Day was conducted by members of the scientific team of AFFINImeter, who presented 2 talks and an interactive tutorial to show the full potential of this new analysis software. Moreover, the workshop counted with the key participation of the invited researchers Margarita Bastos (University of Porto, Portugal), Maria Joao Moreno (University of Coimbra, Portugal), Luis García (University of Santiago de Compostela, Spain), Enrique García Hernández (National Autonomous University of Mexico) and Luis Pablo Calle (Analytical Technologies, Lilly, Spain)

Invited Speakers to WIDEN-ITC Open Day
Invited Speakers to WIDEN-ITC Open Day

We would like to specially thank all the presenters and invited researchers, who did an outstanding job sharing their expertise with us and again, to all participants for attending the WIDEN-ITC Open Day.

We would also like to extend our thanks to all the AFFINImeter members, to the University of Santiago de Compostela and the Galician Supercomputing Center (CESGA) for all the support during the organization of this event.

 

WIDEN-ITC: WIDENING KNOWLEDGE

WIDEN-ITC 2014

Worldwide reference research scientists on molecular interactions analyse technical advances in Isothermal Titration Calorimetry (ITC) at WIDEN-ITC.

Organised by AFFINImeter, a spin-off of the Universidad de Santiago de Compostela (USC), WIDEN-ITC will take place  September 10th at the Faculty of Physics of USC, with participation of about ten worldwide experts on specialised scientific software.

Advances in research for the development and analysis of chemical products, with applications in areas such as the pharmaceutical industry or material sciences, have been showing the shortcomings of available software for the efficient analysis of data from experimental techniques such as Isothermal Titration Calorimetry (ITC). The AFFINImeter software, a result of progress recently made to overcome these limitations, was launched in the international marketplace with a Software as a Service model, and is now being tested by 40 researchers from 25 countries.

The creators of AFFINImeter have invited at WIDEN-ITC some of the best worldwide reference scientists in ITC, with expertise in both current use and in the development of technological innovations that will shape the future of research with this particular technique. Prestigious researchers such as Philippe Dumas from University of Strasbourg, Adrián Velázquez from University of Zaragoza, and Ángel Piñeiro from USC and creator of AFFINImeter, among others, will work for three days with experts from industry, such as Natalia Markova from Malvern Microcal, to exchange knowledge and experience on the limitations of current software and new possibilities offered by technological advances.

Logo of the Workshop WIDEN-ITC 2014
Invitation to WIDEN-ITC, International Workshop on Isothermal Titration Calorimetry (ITC)

WIDEN-ITC will have an “Open Day” on the 10th of September at the Faculty of Physics of USC, which can be attended in person or online upon registration free of charge. The workshop will include a tutorial on the use of AFFINImeter, given by Eva Muñoz and Juan Sabín, researchers at AFFINImeter.

All the information about WIDEN-ITC is available on the website www.affinimeter.com.

WIDEN-ITC: Ensanchando el Conocimiento

WIDEN-ITC 2014

En WIDEN-ITC científicos de referencia mundial en la investigación de interacciones moleculares se reunen para analizar los avances de la técnica de Calorimetría de Titulación Isotérmica (Isothermal Titration Calorimetry, ITC).

Organizado por AFFINImeter, spin-off de la Universidad de Santiago de Compostela (USC), una decena de expertos mundiales en software científico participarán en el WIDEN-ITC, un workshop que se celebrará el 10 de septiembre en la Facultad de Físicas de la USC.
Los avances en la investigación para el descubrimiento y análisis de productos químicos, con aplicaciones en áreas como la industria farmacéutica o ciencia de materiales, han puesto en evidencia en los últimos años las carencias del software disponible para el análisis con técnicas experimentales como la Calorimetría de Titulación Isotérmica (ITC). Fruto de los avances para superar esas limitaciones es el software AFFINImeter, que acaba de presentarse en el mercado internacional con un modelo de Software as a Service, y que está siendo probado en su versión inicial por 40 investigadores de 25 países.
Los creadores de AFFINImeter convocan en el WIDEN-ITC a algunos de los expertos de referencia mundial en esta técnica, tanto en su uso actual como en el desarrollo de innovaciones tecnológicas que marcarán el futuro de la investigación con ITC. Prestigiosos investigadores como Philippe Dumas, de la Universidad de Estrasburgo, Adrián Velázquez, de la Universidad de Zaragoza o Ángel Piñeiro, creador de AFFINImeter e investigador de la USC, entre muchos otros, trabajarán durante tres días con expertos de la industria, como Natalia Markova, de Malvern Microcal, para intercambiar conocimientos y experiencias sobre las limitaciones del software actual y las nuevas posibilidades que permiten los avances tecnológicos.

Logo of the Workshop WIDEN-ITC 2014
Invitación a WIDEN-ITC, Workshop de Calorimetría de Titulación Isotérmica (ITC)

El WIDEN-ITC contará con un “Open Day” el 10 de septiembre, que podrá seguirse presencialmente en la Facultad de Físicas de la USC, o bien por internet desde cualquier lugar, con sólo realizar una inscripción gratuita. La jornada incluirá un tutorial para el uso de AFFINImeter impartido por Eva Muñoz y Juan Sabín, investigadores de AFFINImeter.
Toda la información del WIDEN-ITC está disponible en la web www.affinimeter.com.

Multiple Independent Sites: Advanced tools for a successful analysis.

Isothermal titration Calorimetry experiments of a ligand binding to a macromolecule with multiple independent sites

A successful Isothermal Titration Calorimetry (ITC) experiment requires the acquisition of high quality experimental data together with a careful analysis. Choosing the right binding model to fit the ITC isotherm is critical in order to get the true thermodynamic profile of the interaction. Often, the main limitation to achieve good results arises when the evaluation software lacks of the mathematical model that best describes our binding experiment. A good example is the case of a ligand binding to a macromolecule with multiple independent sites, i.e ligand – DNA interactions (1). Until now the readily available mathematical models to fit such experiments was limited to one or two sets of “n” independent identical sites; frequently, these models offer a poor description of the interaction due to the inherent higher complexity of the system, where many distinct binding equilibria coexist.

 

Multiple Sets of Independent Sites

AFFINImeter ITC offers an unlimited number of user-defined binding models. Particularly, it counts with a feature to easily design models based on multiple independent binding sites. Here, a model with a number of sets of independent sites can be created with no limitation in the number of sets or sites. Noteworthy, the number of sites in each set can be considered as a fitting parameter throughout the data analysis. As an illustration, the following figure shows the reaction parameters of a model generated with AFFINImeter that describes a ligand binding to a receptor having 3 sets of sites, each set having an unknown number of sites. Fitting the experimental data to such model yields the microscopic association constant (K) and the change in enthalpy (ΔH) of the ligand binding to each site type, and the number of sites in each set (n).

Scheme of the interaction of a ligand with a multisite receptor
Reaction parameters table of a model that describes the interaction of a ligand (in syringe) with a multi-site receptor (in cell) having 3 sets of independent sites. Note that the option “Fit” was checked to consider the number of sites (n) as fitting parameters.

These binding models, described by numerous variable parameters, may end up in an over-parameterized fitting function. Thus, the best strategy to achieve a robust and consistent analysis involves the global fitting of several ITC curves acquired under different experimental conditions. In this sense, AFFINImeter also supports global fitting of multiple isotherms wherein parameter linkage between curves is used to decrease the relative number of estimated parameters per experiment.

References:
(1) Methods 2007, 42, 162–172.

Use of precise standard reactions for Isothermal Titration Calorimeter validation

Use of  precise Standard Reactions for Isothermal Titration  Calorimeter Validation

Many published papers report inconsistent thermodynamic values of the same interactions between chemical reactants or macromolecular binding. One of the reasons for this discrepancies is the difficulty of repeating the same conditions in the ITC experiments (buffer, pH, concentrations, ionic strength, source of the materials…). But users start to be more aware that some systematic errors of the calorimeters may also have an important effect in the reported values.

For instance, the interaction between 4-carboxybenzenesulfonamide and bovine carbonic anhydrase II is considered a standard reaction to be measured by ITC and its enthalpy has been measured by 14 operator using different calorimeters (1). The resulting value considering all these independent measurements is -10.4±2.5 kcal·mol-1. The error of the enthalpy is surprisingly high and significantly higher than those typically reported for ITC measurements.

Baranauskiené and co-workers (1) suggest the use of precise standard reactions for  Isothermal Titration calorimeter validation after the calibration. The table below shows the series of chemical reaction they propose as standards where the enthalpy of binding has been determined to high precision and the reagents are readily available from commercial sources.

 

table-chemical-reactions-calorimeters-reference

They also used these standards reactions to compare the results obtained with different micro calorimeter. Their study concluded that Microcal calorimeters are more reliable than TA Calorimeters; and the most recent Microcal ITC200 is less accurate than Microcal VP-ITC. Nano ITC-III calorimeter results were very reproducible, but enthalpy values were systematically underestimated. To learn more about Isothermal Titration Calorimeter validation,, visit the references from where this article was taken.

VP-ITC-NanoITC-ITC200-calorimeter-comparison-entalphie
Enthalpy of the interaction between Tris base and HNO3 as a function of temperature and measured in different ITC calorimeter. Literature values were taken from (2).

References:

(1) Int. J. Mol. Sci 2009, 10, 2752-2762.

(2) Handbook of proton Ionization Heats Wiley-Interscience: Hoboken, NJ, USA, 1979