Alternative Ion-Pairing Modifiers Should Be Investigated in Low-Input and Single-Cell Proteomics.

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Shotgun proteomics traditionally uses formic acid (FA) as an ion pairing modifier.
• Limited material in single-cell proteomics necessitates optimized analytical methods.
• Acetic acid (AA) has shown potential for increased peptide signal.

Purpose of the Study:

• To investigate acetic acid (AA) as an alternative ion pairing modifier for low-input and single-cell proteomics.
• To evaluate the impact of AA on proteome coverage and signal enhancement at subnanogram levels.
• To assess the reproducibility and quantification improvements offered by AA.

Main Methods:

• Analysis of subnanogram proteome dilutions and single cancer cells using liquid chromatography-tandem mass spectrometry.
• Comparison of 0.5% acetic acid (AA) with 0.1% formic acid (FA) as ion pairing modifiers.
• Evaluation of peptide signal, proteome coverage, quantification accuracy (%CVs), and scan times.

Main Results:

• Acetic acid (AA) consistently increased proteome coverage across all peptide loads assessed, with a 1.8-fold increase at 20 pg peptide digest.
• AA enabled a 1.7x increase in scans per peak by reducing ramp times, improving quantification accuracy.
• Analysis of single cancer cells identified ~13% more peptide groups using AA compared to FA.

Conclusions:

• Acetic acid (AA) is a superior ion pairing modifier for low-input and single-cell proteomics, enhancing signal and coverage.
• The use of AA allows for reduced analysis times and improved quantification in complex proteomic samples.
• Re-evaluation of ion pairing modifiers and additives is recommended for optimizing low-input proteomic workflows.