Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Sample preparation is critical for high-quality mass spectrometry data in bottom-up proteomics.
• Common reagents can inhibit digestion, cause ion suppression, or interfere with liquid chromatography-tandem mass spectrometry (LC-MS/MS).
• These issues complicate reproducible quantitative proteomics, especially with label-free methods like HDMS(E) and SWATH.

Purpose of the Study:

• To validate a reproducible sample preparation method for proteomics.
• To ensure sufficient protein yield without LC-MS/MS interfering substances.
• To address challenges in quantitative proteomics, particularly label-free quantification.

Main Methods:

• Detailed validation of a novel sample preparation protocol.
• Utilized 1% sodium deoxycholate (SDC) for both cell lysis and in-solution digestion.
• Focused on minimizing interfering substances and maximizing protein yield.

Main Results:

• The validated method provides reproducible results with sufficient protein yield.
• The use of SDC avoids common interfering substances problematic for LC-MS/MS.
• The method is suitable for quantitative proteomics, including HDMS(E) and SWATH.

Conclusions:

• A reproducible and efficient sample preparation method using SDC has been validated.
• This method overcomes limitations of traditional reagents, improving quantitative proteomics accuracy.
• The SDC-based approach is recommended for label-free quantitative proteomics workflows.