Efficient sample preparation is key to any proteomics workflow, with three priorities at the forefront: protein recovery, purity, and sample throughput. Acetone precipitation is a common method of purifying proteins from a complex sample prior to LC-MS/MS analysis, but is sometimes set aside due to seemingly variable yields.1 Our group previously established that the addition of 1-30 mM salt with 80% v/v acetone facilitates recoveries >95%.2 We later developed a filtration cartridge (ProTrap XG) which automates precipitation and SDS depletion.3 In order to maximize recovery, purity and sample throughput, we investigated the kinetics of protein precipitation with respect to structural properties of proteins, including molecular weight, hydrophobicity, and charge.
Extracted yeast lysates were combined with various concentrations of salt and 80% v/v acetone at a defined temperature (-20 to +37 oC), within the ProTrap XG. The samples were incubated for varying times, followed by centrifugation and pellet re-solubilization. Protein recovery was quantified by the bicinchoninic acid assay, followed by a bottom-up proteomics workflow, which was used to identify precipitated proteins after varying incubation times. Structural properties of the recovered proteins were compared across the various incubation conditions. Purity was assessed using the methylene blue active substances assay to quantify residual SDS.
By incorporating ≥10 mM NaCl, acetone precipitation recovered >95% of protein following 2 minutes incubation at room temperature. Protein identifications made by bottom-up LC-MS/MS analysis indicate no difference in the structural properties of proteins recovered after 2 minutes, 60 minutes and 24 hours incubation.
Under optimal conditions, acetone precipitation quantitatively recovers all types of protein within a sample in 2 minutes while depleting >99% of the initial SDS.