We all know that we should aliquot reagents and samples, and that leaving samples sitting on a bench in the lab isn’t ideal, but just how quickly do these precious proteins degrade and how many freeze-thaw cycles can you put your sample through before you need to call it quits?
Sample degradation can contribute to analytical variation in biological samples, meaning unreliable results and more time and money spent repeating assays. Pre-analytical variation in samples and reagents is also important to consider and can arise from many sources including the nature of the sample and freezing conditions, such as the temperature, the rate of freezing, thaw events and time in storage.
During freezing the formation of ice crystals from the water component of your sample can leave high salt or protein concentrations in the aqueous phase. This is known as freeze concentration and causes severe stresses to protein stability. It has even been shown to cause protein unfolding at the ice: aqueous interface and the aggregation of unfolded proteins, making your samples or reagents useless.
To overcome this it is recommended that we flash freeze proteins in buffers with a cryoprotectant, such as glycerol to support protein stability. Avoiding phosphate buffers is important as these undergo a phase transition upon freezing which can leave proteins open to damage. Another important step in preventing this kind of damage is to avoid numerous freeze-thaw cycles to your sample by storing it in working aliquots.
The results of a proteomics study by the National Cancer Institute, which addressed the stability of proteins in stored samples, were presented at the 2015 ISBER conference. The authors were able to measure protein changes such as deamidation, changes in disulphide bonds, how much protein was remaining in solution and how much precipitated and relative protein concentrations, in order to assess protein degradation across different sample storage conditions.
Power outage of freezers can leave samples open to freeze-thawing. This was simulated in the study by removing human blood samples from the -80°C freezer and leaving them at room temperature for an hour before being returned to the freezer for 24 hours. Through an increasing number of freeze-thaw cycles, ranging from 1-5 cycles, an increasing level of sample degradation was noted. A significant increase in protein degradation was found in the samples that underwent more rounds of freeze-thawing. However, though certain protein markers were identified that allowed protein degradation to be easily spotted in samples, not all proteins appeared susceptible to degradation by freeze-thawing. Surprisingly, only 5-10% of the proteins analysed showed degradation from freeze-thawing in this study.
The study also looked at the effects of long term storage on protein degradation. Human blood samples were incubated at -20°C and -80°C for different time periods ranging from 0-18 months. As expected protein degradation was significantly greater in the samples stored at -20°C compared to -80°C, as at lower temperatures less energy is available for reactions or precipitation to occur. A panel of marker proteins was developed that indicate long term storage damage to proteins. In the case of IgGFc-binding protein it was clear that degradation was evident after 6 months in storage at both -20°C and -80°C, and the level of protein degradation increased with increasing time in storage.
While protein stability is a function of its biophysical characteristics, including its sequence, how it folds and how much of it is exposed, it has been shown that freeze-thawing and long term storage of samples results in protein degradation. Solutions to avoiding protein sample degradation may include:
- making sure samples are stored correctly- in the correct buffer and flash frozen
- aliquoting samples on first use so they don’t undergo repeated freeze-thaw cycles
- making sure samples aren’t left at room temperature for long periods of time
- storing at -80°C rather than -20°C where possible to increase sample longevity
- not storing samples long term
- using proteomics to check samples remain fit-for-purpose following freezer storage
- using alternative reagents that are stable at room temperature where possible to avoid the issue altogether.
Whatever method you use it is always good to be mindful of the changes you may be causing in your samples with your freezing practices.