Abstract

Young Researchers' Sessions

Day 3, May 17（Wed.）　09:45-10:00　Room C (Room 1009)

3C-O-0945（2P-16）　　PDF

Advancement of Water Droplet-in Oil Digestion Method for High-Throughput Single-Cell Proteomics

(Kumamoto Univ.)
^oMaki Yamahiro, Takeshi Masuda, Shingo Ito, Sumio Ohtsuki

Protein expression varies between cells, and observing this heterogeneity helps to understand biological phenomena. We have developed a water droplet-in-oil digestion (WinO) method for single-cell proteomics. The WinO method reduces adsorption losses of proteins and peptides during sample preparation and allows parallel sample preparation in 96-well plates. However, the original WinO method requires a liquid-liquid extraction during the peptide desalting step and a manual sample combining after tandem mass tag labeling, which are major obstacles to automating sample preparation. The purpose of this present study was to improve the throughput of the WinO method. For eliminating the liquid-liquid extraction step, we used a polystyrene-divinylbenzene reversed-phase sulfonated disk to peptide desalting instead of a polystyrene-divinylbenzene reversed-phase disk (Fig.1). With this approach, peptides were successfully purified, and the number of quantified peptides was increased 1.45-fold compared to the original method. In addition, we used a liquid handling robot for sample combining. Compared to the manual combining, this approach reduced the sample preparation time to one-seventh and the peptide recovery rate was 1.04-fold. This modified WinO method enables automation of the sample preparation process and is expected to be applied to a large-scale single-cell proteomics.