Single-cell proteomics is a key technology for the understanding of tissue and cell heterogeneities. However, it is hardly possible to perform single-cell proteomics because of low peptide recovery during sample preparation. To reduce the contact area of a sample solution with a plastic tube, we have developed a water droplet in oil (WinO) protocol. WinO is an inexpensive and easily manual protocol for small-scale preparations. In this presentation, we evaluated and optimized the WinO to improve the tryptic peptides recovery toward single-cell proteomics.
HEK293 cells were solubilized by 100 mM Tris-HCl (pH9.0) containing 12 mM sodium deoxycholate and 12 mM sodium lauroyl sarcosinate. The droplet was formed by adding 1 μL of the whole cell lysate into ethyl acetate. Proteins were reduced and alkylated, and digested with Lys-C followed with trypsin for overnight. Tryptic peptides were analyzed by a nanoLC-MS/MS.
Results and discussion
To evaluate the improvement of peptides recovery in WinO, 10 ng of proteins were digested by WinO or in-solution digestion (InSol). The identified number and total signal intensities of proteins in WinO were 1.7-fold greater than those in InSol, indicating that the peptides recovery was improved in WinO. However, the total signal intensities of 10 ng of peptides digested by WinO were only 38% compared with those of 10 ng of peptides divided from 10 μg of peptides prepared by InSol. We have assessed the sample recovery in each preparation step, and the lowest recovery step was trypsin digestion. Trypsin activity in WinO was decreased by 34% compared with that in InSol, suggested that the low recovery was due to low trypsin digestion efficiency in the droplet. In conclusion, WinO provides higher peptides recovery than InSol. WinO can be the novel technology for small-scale preparations, including single-cell analysis, by improving trypsin digestion efficiency.