Presenting Tycho data

The most common way to present Tycho data is to use the raw profiles of the ratio, since these contain the most information – in addition to inflection temperatures (T_is) they also show initial ratio and ∆ ratio. See below for an example figure and figure legend (Figure 1).

Other representations of the data such as smoothed, first derivative and single wavelength can also be useful and informative ways to display and interpret the results. The first derivative can sometimes be easier to analyze visually, since identifying peaks in a graph is typically easier than identifying transitions. In the first derivative, T_is can be clearly seen, but initial ratio and ∆ ratio information is lost. Raw (i.e. unsmoothed) data is usually preferred over smoothed data since it shows the unvarnished truth, but in some cases smoothed can be useful for reasons of clarity. Single wavelength data is most useful in rare cases where unfolding events are not visible in the ratio signal due to the position and fluorescence properties of the involved amino acid residues.

For data that differs greatly with regards to the absolute ratio values or the ∆ ratio values, normalization can be useful. Normalization can be handled in different ways. One way is to normalize all unfolding profiles to start at the same value. For an example of normalized data and first derivative data, see Tullman, J., Callahan, N., Ellington, B. et al. Appl Microbiol Biotechnol (2019). https://doi.org/10.1007/s00253-019-09624-2  

Tycho allows to export measurements into convenient file formats. The exported zip file contains raw data compiled in a Microsoft Excel spreadsheet file (.xlsx) as well as ready-to-use profile images (.svg and .png formats). For presentation and publication purposes, the profile images can be used the way they are. The .svg file is a vector-based file and can be modified in many common graphics editor software applications, for example to change colors or adjust font sizes. Alternatively, you can use the exported spreadsheet to modify graphs any way you like: combine data across runs or  adapt colors and fonts.

Example texts for describing Tycho results for different applications

Protein functionality: Protein functionality was tested using Tycho NT.6. All proteins showed a thermal shift of the inflection temperature (T_i) in the presence of their natural ligand, indicating that the proteins were correctly folded and functional.

Ligand binding: To test for a potential interaction of the target proteins with molecule X, thermal unfolding profiles were recorded on Tycho NT.6. All proteins showed a thermal shift of the inflection temperature (T_i) in the presence of molecule X, indicating molecular interactions. To further characterize these interactions, dissociation constants (K_ds) were determined using a Monolith system.

Figure 1: Thermal unfolding profiles of samples 1-6. Tycho records intrinsic protein fluorescence at 330 nm and 350 nm over a 30 °C/min temperature ramp from 35 °C to 95 °C. The figure shows the ratio of the signal at the two wavelengths. The vertical lines indicate inflection temperatures (T_is), which represent unfolding of the proteins. The data indicates that the tested samples are of the same quality, as can be seen in the highly similar T_is and initial ratio values (at 35 °C). The latter indicate that all samples contain the same portion of folded vs unfolded protein.

Concentration: Protein concentration was routinely checked on Tycho NT.6. For this, the sample brightness parameter was used and compared to a previously purified sample of known concentration in the same buffer.

Quality checks – presence, purity, similarity: Tycho NT.6 was used to monitor protein expression and purification. All through the purification process, aliquots were compared and their unfolding profiles analyzed for similarity to a previously purified batch. Sufficient similarity (in this case >80%) confirmed that the correct protein was purified and was present at all steps at sufficient concentration and purity.

Example text for a Materials and Methods section

Tycho experiments: 10 µL of each protein sample were run on a Tycho NT.6 system in Tycho capillaries (both by NanoTemper Technologies; capillaries cat# TY-C001) to analyze protein quality and functionality. The platform runs experiments which utilize Tycho technology, a modified version of nanoDSF (nano Differential Scanning Fluorimetry). It runs a thermal ramp from 35 °C to 95 °C at a defined rate of 30 °C/min and records intrinsic protein fluorescence from tryptophan and tyrosine residues at 330 nm and 350 nm throughout the run. The data is automatically analyzed by the system's software to calculate inflection temperatures (T_i), a measure for the temperature(s) at which the protein unfolds. In addition, Tycho outputs the sample's brightness, a measure of its concentration. Tycho data can be used to draw conclusions about a protein's quality by looking at the protein's presence, purity, concentration, functionality, and similarity to other samples.

Tycho concentration determination: For this, the sample brightness parameter was used and compared to a previously purified sample of known concentration (1 mg/mL) in the same buffer. Assuming a linear correlation of sample brightness and concentration, the concentration of the unknown sample can easily be calculated using the equation c_u = b_u * c_r / b_r  where c is the concentration of a sample, b is its sample brightness, u stands for unknown (the sample to be quantified) and r stands for reference (the sample with known concentration). The buffer used was previously tested not to have any fluorescence signal of its own, so no blank measurements were needed.