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Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry
Published on: April 4, 2018
11.7K
Acetic acid is a superior ion pairing modifier for sub-nanogram and single cell proteomics
Colten D Eberhard1,2, Benjamin C Orsburn1,2
1The Department of Pharmacology and Molecular Sciences.
Biorxiv : the Preprint Server for Biology
|August 14, 2023
Summary
Using acetic acid (AA) instead of formic acid in proteomics significantly boosts peptide signal and protein identification, especially for single-cell proteomics (SCP) and low-sample amounts.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Traditional shotgun proteomics relies on formic acid (FA) as an ion pairing modifier.
- Acetic acid (AA) has shown potential to enhance peptide signal in prior studies.
- The efficacy of AA in low-input and single-cell proteomics (SCP) requires further investigation.
Purpose of the Study:
- To evaluate the impact of acetic acid (AA) as an ion pairing modifier in sub-nanogram and single-cell proteomics (SCP).
- To compare the performance of AA against formic acid (FA) in enhancing peptide signal and proteome coverage.
- To assess the utility of AA for reducing analysis time and improving quantification.
Main Methods:
- Tryptic digest standards were analyzed using a TIMSTOF SCP system at varying low concentrations (down to 20 picograms).
- Acetic acid (0.5%) was employed as the ion pairing modifier, replacing the traditional 0.1% formic acid.
- Single cancer cells were analyzed to assess peptide group identification rates.
Main Results:
- Acetic acid (AA) consistently increased peptide signal and proteome coverage across all tested peptide loads.
- A 20 picogram peptide digest showed a 1.8-fold increase in identified protein groups (over 2,000) in 30 minutes with AA.
- AA enabled reduced gradient times, increasing scan counts by 1.7x and improving quantification precision (%CVs).
- Single cancer cell analysis yielded an average of 13% more identified peptide groups using AA.
Conclusions:
- Acetic acid (AA) is a superior ion pairing modifier for low-input and single-cell proteomics (SCP) compared to formic acid (FA).
- The use of AA enhances proteome coverage and identification sensitivity, particularly at low sample amounts.
- AA facilitates faster analyses and more robust quantification in complex proteomic workflows.
