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Two-Step Peptide Solubilization Increases Coverage in High-Sensitivity NanoHILIC/MS/MS-Based Proteomics
Koshin Akamatsu1, Eisuke Kanao1,2, Ayana Tomioka1
1Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Analytical Chemistry
|May 6, 2025
Summary
We developed a two-step peptide solubilization method to improve nanoscale hydrophilic-interaction chromatography coupled with tandem mass spectrometry (nanoHILIC/MS/MS). This technique enhances peptide solubility and boosts protein identification in proteomics research.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Nanoscale hydrophilic-interaction chromatography coupled with tandem mass spectrometry (nanoHILIC/MS/MS) offers potential for proteomics.
- Peptide solubility in organic solvent-rich solutions is a major limitation for nanoHILIC/MS/MS.
- Reversed-phase liquid chromatography (RPLC) is a common but sometimes less sensitive method.
Purpose of the Study:
- To develop an improved peptide solubilization method for nanoHILIC/MS/MS.
- To enhance peptide solubility and compatibility with high organic solvent concentrations.
- To increase the sensitivity and scope of nanoHILIC/MS/MS for proteomics applications.
Main Methods:
- A two-step solubilization procedure was developed: initial dissolution in 25% acetonitrile (ACN), followed by dilution to 95% ACN.
- Peptide samples were analyzed using nanoHILIC/MS/MS and compared to direct solubilization in 95% ACN.
- Performance was benchmarked against nanoscale reversed-phase liquid chromatography coupled with tandem mass spectrometry (nanoRPLC/MS/MS) using HeLa cell tryptic peptides.
Main Results:
- The two-step method significantly increased peptide solubility and intensity in nanoHILIC/MS/MS, with 82.8% of peptides showing increased intensity (average gain of 20.9%).
- nanoHILIC/MS/MS with the new method identified 8.47 times more peptides and 3.54 times more protein groups compared to nanoRPLC/MS/MS using minimal sample (2.5 ng).
- Enhanced peptide loading and superior electrospray ionization (ESI) efficiency in ACN-rich mobile phases contributed to the high sensitivity.
Conclusions:
- The developed two-step solubilization method effectively overcomes peptide solubility limitations in nanoHILIC/MS/MS.
- This approach significantly enhances sensitivity, enabling deeper proteome coverage.
- The optimized nanoHILIC/MS/MS system presents a powerful platform for sensitive proteomics, including clinical and single-cell analyses.
