Perfluorooctanoic acid for shotgun proteomics.

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Traditional shotgun proteomics methods often face challenges with membrane protein solubilization and sample cleanup.
• Surfactants like sodium dodecyl sulfate (SDS) are effective but can interfere with downstream mass spectrometry analysis.
• Perfluorooctanoic acid (PFOA) is a volatile surfactant with potential for improved proteomics workflows.

Purpose of the Study:

• To introduce and validate a novel, single-tube shotgun proteomics method utilizing the volatile surfactant perfluorooctanoic acid (PFOA).
• To demonstrate PFOA's efficacy in solubilizing membrane proteins and its compatibility with enzymatic digestion.
• To develop a streamlined proteomics workflow that minimizes sample loss and simplifies analysis by removing all volatile chemicals via evaporation.

Main Methods:

• Proteins were extracted using 2% PFOA, followed by reduction and S-alkylation.
• Trypsin digestion was performed in 0.5% PFOA, and the PFOA was subsequently removed by evaporation.
• Peptide analysis was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS), with an option for proteolytic (18)O labeling for quantitative experiments.

Main Results:

• PFOA effectively solubilized membrane proteins, comparable to SDS.
• PFOA concentrations up to 0.5% did not significantly inhibit trypsin activity.
• A single-tube method identified 75 proteins from 1 µg of tryptic peptides in a 1-hour LC-MS/MS run, with 67% being membrane proteins.
• The method eliminated the need for sample clean-up devices, preventing protein/peptide loss.

Conclusions:

• The developed single-tube shotgun proteomics method using volatile PFOA overcomes major drawbacks of traditional surfactant-based approaches.
• This streamlined workflow enhances efficiency and sensitivity, particularly for membrane protein analysis.
• The method is broadly applicable and can be integrated with quantitative proteomic techniques like (18)O labeling.