| Timetable |
| Download Conference Program |
| Download All Abstracts |
| Zoom Access |
| Corporate Program |
Poster Presentations
Day 3, June 24（Tue.）
Room P (Maesato East, Foyer, Ocean Wing)
- 3P-PM-48（4B-O1-1210）
Two-step peptide solubilization increases coverage in high-sensitivity nanoHILIC/MS/MS-based proteomics
(1Kyoto Univ., 2NIBIOHN)
oKoshin Akamatsu1, Eisuke Kanao1,2, Ayana Tomioka1, Yasushi Ishihama1,2
Nanoscale hydrophilic-interaction chromatography coupled with tandem mass spectrometry (nanoHILIC/MS/MS) is a promising alternative to reversed-phase liquid chromatography (RPLC) for proteomics, but its application is limited by the poor solubility of peptides in organic solvent-rich sample solutions. To address this solubility problem, we have developed a two-step solubilization method, in which peptides are first solubilized in an aqueous solvent with an optimal acetonitrile (ACN) concentration of 25 %, and then diluted into a high ACN concentration solution of 95 %. This procedure increases the peptide solubility without compromising compatibility with nanoHILIC/MS/MS. Compared to one-step solubilization in 95 % ACN, this approach increased the intensity of 82.8 % of commonly quantified peptides in nanoHILIC/MS/MS, with an average intensity gain of 20.9 %. Furthermore, nanoHILIC/MS/MS with this two-step solubilization outperformed nanoRPLC/MS/MS, identifying 8.47 times more peptides and 3.54 times more protein groups from 2.5 ng (equivalent to 10 cells) of tryptic peptides extracted from HeLa cells. In this presentation, we will also report further improvement of two-step solubilization method and optimization of separation conditions of nanoHILIC/MS/MS for highly-sensitive proteomics.