by Jessica Nickerson, Research Associate
Every proteomicist has their own list of reasons for avoiding solvent-based precipitation.
Some of the more common objections…
• highly variable recovery
• you are often forced to prioritize either recovery or purity when decanting the supernatant
• And who has time for an overnight incubation? Besides a grad student who wants to call it a day
If only we could eliminate these drawbacks.
Our team favours the philosophy that the simplest answer is the best one, so while there are more sophisticated techniques to deplete mass spec contaminants, we have been working to reimagine simple acetone precipitation. And, we’ve achieved success, in 2 minutes.
In 2013, our team at the Doucette Lab reported that the presence of 1-10 mM salt took the question mark out of protein recovery. Following conventional overnight incubation at -20 °C, we consistently achieved >95% recovery. Knowing that salt played a role in determining protein recovery, we wanted to see if salt could also speed up precipitation.
Yes, it does! We recently published our findings on a rapid precipitation protocol, demonstrating that by controlling the salt (20-100 mM), >99% of the proteome sample is recovered in just 2 minutes of precipitation. And there’s more, it can be done at room temperature. Precipitation has traditionally been conducted at -20°C in an attempt to preserve protein activity. However, we know that acetone routinely causes denaturation, so if we are not preserving activity and actually slowing down the precipitation, what’s the point of working in the cold?
Additional perceived challenges around acetone precipitation involve the nature of the sample. It has been reported that solvent-based precipitation favours the recovery of high molecular weight and hydrophobic proteins and works best when the initial sample is highly concentrated. But isn’t precipitation supposed to be a means of increasing the concentration of a dilute sample? And if we want to characterize the whole proteome, we should certainly be retaining all proteins without biasing structural properties.
Our rapid precipitation protocol is shown to quantitatively recover samples as dilute as 0.01 g L-1 and reveals no bias as to which proteins are present in the pellet. Smaller proteins do precipitate a little bit slower than the big ones, but they are still much more abundant in the pellet than in the supernatant.
To this point, our results disqualify the concerns of low sample throughput as well as variable and biased recovery.
Waiting just a little longer – 30 minutes – ensures a robust recovery over a range of protein concentrations.
The only remaining challenge is achieving quantitative isolation of the pellet from the supernatant, and for that, we have the ProTrap XG. The precipitated pellet is recovered on the Teflon membrane within the Filtration Cartridge as the supernatant flows through during the centrifugation step.
We recognize that each protein mix is different. We currently recommend a starting time of 30 minutes to precipitate your sample, to cover a broad range of samples. The minimum required precipitation time is dependant on the starting protein concentration, and starting buffer composition, of which the salt concentration is key.
We encourage you to read Nicholson and Doucette, J Proteome Res. 2020 May 1;19(5):2035-2042. and apply the concepts to your sample handling and share your experience with us at email@example.com.