Two-step peptide solubilization increases coverage in high-sensitivity nanoHILIC/MS/MS-based proteomics

ABSTRACT: 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 overcome this issue, we have developed a two-step solubilization method, in which peptides are first solubilized in a 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 direct 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 of tryptic peptides extracted from HeLa cells. The high sensitivity of nanoHILIC/MS/MS can be attributed to enhanced loading of peptides as a result of the two-step solubilization, together with superior ESI efficiency arising from the use of the ACN-rich mobile phase. This high-sensitivity proteomics system is a promising platform for clinical and single-cell applications.

ORGANISM(S): Homo Sapiens (human) Bos Taurus

SUBMITTER: Yasushi Ishihama 

PROVIDER: PXD059304 | JPOST Repository | Thu May 08 00:00:00 BST 2025

REPOSITORIES: jPOST