Output parameters
Prometheus Panta software automatically analyzes data and outputs several stability parameters. For more sophisticated data analysis in PR.Panta Analysis, however, regions of interest can be defined in which a Fit is applied to the data. These fits then yield additional stability parameters.
List of all output parameters
| Parameter | Source data | Description |
|---|---|---|
| σ | DLS | Standard deviation (calculated from all DLS acquisitions of a capillary, which were not marked as outliers). |
| ø | DLS | Average value (calculated from all DLS acquisitions of a capillary, which were not marked as outliers). |
| Amplitude | DLS | The amplitude of the autocorrelation function. To calculate it, the y-axis intercept of the autocorrelation function is subtracted from the baseline. |
| Baseline | DLS | The baseline of the autocorrelation function, calculated from the median of the last third of the autocorrelation values. |
| Cumulant PDI | DLS | Polydispersity index determined with cumulant fit applied to a single acquisition. |
| Cumulant radius | DLS | Mean hydrodynamic radius rH determined with cumulant fit applied to a single acquisition. |
| D | DLS | Mean diffusion coefficient determined with cumulant fit applied to a single acquisition. |
| D0 | DLS | Diffusion coefficient at infinite dilution; y-axis intercept from linear fit to obtain diffusion interaction parameter kD. |
| InitialRatio | fluorescence | The ratio 350 nm/ 330 nm at the start temperature. |
| IP | fluorescence | Inflection point of the unfolding profile of the protein (or domain), often also called Tm (melting point). |
| kD | DLS | First-order diffusion interaction parameter calculated from the concentration dependence of the diffusion coefficient at a single temperature. The diffusion coefficient is calculated using the size distribution fit. |
| ON | all | The onset of unfolding, size increase, or aggregation (from scattering or turbidity). The temperature at which the protein first starts to unfold or aggregate. |
| Peak | DLS | Mean hydrodynamic radius of peak from intensity distribution calculated from size distribution fit. Values for up to three peaks are output. |
| Peak PDI | DLS | Mean polydispersity index of peak from intensity distribution calculated from size distribution fit. Values for up to three peaks are output. |
| R0 | DLS | Hydrodynamic radius calculated from diffusion coefficient at infinite dilution, D0. |
| R2 | DLS | Coefficient of determination of linear fit to obtain diffusion interaction parameter kD. |
| Scattering intensity | DLS | Average scattering intensity calculated from the intensity fluctuations of a single DLS acquisition. |
| Signal/Noise | DLS | Parameter indicating quality of the autocorrelation function, determined from the amplitude and the scattering noise originating from the instrumental setup. |
| The following parameters are only available in PR.Panta Analysis. | ||
| AlphaD | fluorescence | y-axis intercept of the fit's final baseline (D stands for denatured) (at T=0 °C). |
| AlphaI | fluorescence | The y-value of an inflection point between two Tms (only applicable for three-state fits) (I stands for intermediate). |
| AlphaN | fluorescence | y-axis intercept of the fit's first baseline (N stands for native) (at T=0 °C). |
| Amplitude | fluorescence | The amplitude of a transition. To calculate the amplitude, the distance between the fit's baselines is measured on a straight line passing through the Tm. For a three-state fit, the intermediate baseline is always assumed to be horizontal. Not to be confused with the amplitude of the autocorrelation function in DLS data. |
| BetaD | fluorescence | The slope of the fit's final baseline (D stands for denatured). |
| BetaN | fluorescence | The slope of the fit's first baseline (N stands for native). |
| dH | fluorescence | Theoretical unfolding enthalpy ΔHm in J/mol calculated from the fit. Please note that dH is calculated using the approximation that ΔCp (the change in heat capacity) equals 0. This approximation is necessary because ΔCp cannot be directly calculated from Prometheus data. |
| EndValue | fluorescence | The y-value of the data at the end temperature or the y-value of a fit function at the end of regions of interest. |
| Inflection Increase | turbidity | Measure of the total amount of aggregation in a sample. |
| InitialValue | fluorescence, turbidity | The y-value of the data at the start temperature or the y-value of a fit function at the start of regions of interest. |
| Number of IPs | fluorescence | The total number of IPs detected in this sample for this detector. |
| Turbidity Average | turbidity | Measure of the total amount of aggregation in a sample. |
| Similarity | fluorescence, DLS | The similarity of an unfolding profile or a cumulant radius profile compared to a reference. |
| Slope | fluorescence, turbidity | The slope of the unfolding profile at a specific point (at an IP or a Tm). |
| Tm | fluorescence | The melting point of a protein (or domain). The temperature at which 50% of the protein (or domain) is unfolded. |
Be aware that scattering data is only available if data was generated with measurement types supporting DLS and with Dynamic Light Scattering set to on.
For all thermodynamic parameters derived from these fits, note that care must be taken in their use. Strictly speaking, thermodynamic parameters can only be derived from equilibrium measurements, which thermal unfolding measurements often aren't. However, the same is commonly done for data collected with other non-equilibrium measurements (not just Prometheus), and the extracted parameters can still provide useful information.
Please also be aware that the PR.Panta Analysis data table by default shows only a subset of columns. If a desired parameter columns is not shown, use the functionality to unhide the relevant column.