Dataset Information

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

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			Action	DRS
	230316KA_4pACN_1000ng_n1.raw	Raw
	230316KA_4pACN_1000ng_n2.raw	Raw
	230316KA_4pACN_1000ng_n3.raw	Raw
	230316KA_4pACN_250ng_n1.raw	Raw
	230316KA_4pACN_250ng_n2.raw	Raw

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Two-Step Peptide Solubilization Increases Coverage in High-Sensitivity NanoHILIC/MS/MS-Based Proteomics.

Akamatsu Koshin K Kanao Eisuke E Tomioka Ayana A Ishihama Yasushi Y

Analytical chemistry 20250506 19

Nanoscale hydrophilic-interaction chromatography coupled with tandem mass spectrometry (nanoHILIC/MS/MS) is a promising alternative to reversed-phase liquid chromatography 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 in ...[more]

PMID: 40326241

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