How DSC can complement your protein thermal stability studies
In the final installment of our three-part blog series on how differential scanning calorimetry (DSC) can help speed up the selection of vaccine candidates, we explain how DSC can be used as a screening tool for other protein thermal stability techniques.
Using fluorescence to measure protein unfolding
In our last blog [link] we took a deep dive into exactly how DSC works to fingerprint proteins. MicroCal PEAQ-DSC microcalorimeters are flexible and measure in temperatures as high as 130°C. As well as making it suitable for measuring almost any protein, these features also make it effective as a screening tool for other protein stability techniques, including differential scanning fluorimetry (DSF) and circular dichroism (CD).
DSF is a useful method to measure protein unfolding. Like DSC, DSF investigates unfolding as a function of temperature, but here it measures changes in fluorescence rather than in the protein’s melting point.
In DSF, the thermal unfolding of protein samples is followed with an intrinsic fluorescence signal. The protein will start to unfold, and fluorescent groups in the protein structure get exposed to the aqueous solution. This causes their fluorescence spectra to experience redshift to higher wavelength positions for the spectral maxima.
You can also use an extrinsic fluorescence detection method for DSC, by adding a fluorescent dye to protein samples in a real-time PCR machine. The dye will remain quenched by the water solution when the protein is still in its folded state. But once the protein starts to unfold as temperature increases, the dye begins to bind to the protein’s hydrophobic core. This binding of the dye intensifies the fluorescence, giving a nice profile of the protein’s unfolding with increasing temperature.
How can DSC support DSF?
DSF is a robust tool for getting data on protein unfolding. It only needs small samples to work, and can work on a pretty large scale, running up to 96 tests at the same time. However, the applicability of the DSF assay needs validating, as DSF has a limited temperature range. Additionally, the dye used may alter the molecules, or the intrinsic fluorescence signal might be complex.
This is where MicroCal PEAQ-DSC comes in. A DSC profile can be run initially on the protein sample: once this profile is established, you can compare your DSF tests against this DSC standard. The wide temperature range of MicroCal PEQQ-DSC also means that it can be a useful alternative in situations where protein folding temperatures are outside the range of DSF.
DSC as an orthogonal tool for CD
CD is another great technique that has the advantages of being highly sensitive and high-throughput. It works by using a wide range of UV light to analyze the secondary structure of proteins.
CD’s UV power can complicate matters: a lot of molecules are UV-active, so the technique is limited by buffer conditions. This means many standard buffers are unsuitable for application here, and additives are required to maintain stability. However, using DSC can help to identify any anomalies generated by the CD technique, helping you to get better overall results. Perfect.
Characterization and profiling using DSC
When it comes to characterizing and profiling the assay development stage, DSC can be your best friend alongside high-throughput, small sample volume techniques. We know that MicroCal PEAQ-DSC offers wide operating temperatures, but did you know it can also give ultra-clear pinpointing of the transition stage and high resolution of domains or protein components?
One of the advantages of DSC is that it can offer these benefits without using any dyes or buffer conditions. This avoids compromise of the protein stability and gives a stable reference point for any further analysis techniques you need to use on your proteins. So, MicroCal PEAQ-DSC scans can provide precise and detailed characterization of protein structures and their thermal stability parameters.
Other methods such as DSF and CD can then be used for wider testing on multiple batches. They can also confirm the critical material attributes (CMAs) for the quality target product profile (QTPP), as part of the QbD process we mentioned in our first blog here [link].
So, as well as being a powerful tool on its own, MicroCal PEAQ-DSC is a fantastic addition to your protein screening studies alongside DSF and CD. In the final blog of this series, we’ll provide an overview of how DSC provides a stable base for accelerated vaccine development.
Download the full guide here to find out more about how DSC can change your vaccine development program.
Read other blogs in this series
Part 1: How “fingerprinting” vaccine candidates saves time
Part 2: Testing thermal stability of proteins with DSC