For giving highly sensitive and quantitative proteomics data, proteins are required to be completely extracted and digested into peptides. We have developed a sample preparation protocol using a phase transfer surfactant (PTS) cocktail containing sodium deoxycholate (SDC) and sodium lauroyl sarcosinate (SLS) (SDC/SLS). The protease activity and solubilization ability are highly enhanced in the PTS cocktail compared to other solubilizing agents. However, biased in digestion efficiency are still observed. The purpose of the present study was to identify a new PTS cocktail to minimize missed cleavage peptides toward unbiased quantitative proteomics.
Trypsin and lysyl endopeptidase activities in 31 PTS cocktails were measured by the absorbance of p-nitoraniline at 410 nm resulting from digestion of N-benzoyl-lysine-p-nitroanilide. For the evaluation of solubilization ability, proteins solubilized from human liver microsome fraction were quantified by a BCA assay. Proteins were digested at optimized condition and analyzed by using nanoLC-MS/MS.
Results and discussion
In the protease activity comparison, we selected 7 PTS cocktails showing comparable protease activity with SDC/SLS. The solubilization ability of these 7 PTS cocktails were greater than that of SDC/SLS. The protein solubilization ability of a PTS cocktail containing SDC and ursodeoxycholate (UDC) (SDC/UDC) was the greatest and 1.4-fold greater than those of SDC/SLS. Therefore, we used SDC/UDC in subsequent experiments. The number of missed cleavage sites was significantly decreased to 84% in SDC/UDC compared to SDC/SLS. Next, to evaluate whether the intensities of fully digested peptides are increased by the enhancement of digestion efficiency in SDC/UDC, we compared the intensities of these peptides between two PTSs. As expected, the number of peptides showing 2-fold higher in SDC/UDC was increased to 5.5-fold than those in SDC/SLS. These results suggest that the new PTS cocktail with SDC/UDC provides better unbiased quantitative proteomics data than the present PTS cocktail.