High-throughput method for on-target performic acid oxidation of MALDI-deposited samples

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Disulfide bonds are crucial for protein structure and function.
• Accurate characterization of disulfide bonds is essential in proteomics.
• Existing methods for disulfide bond analysis can be laborious and time-consuming.

Purpose of the Study:

• To develop an efficient on-target performic acid oxidation method for peptide and protein analysis.
• To enable differentiation of free cysteine from disulfide-containing peptides.
• To improve throughput and reduce sample handling in mass spectrometry workflows.

Main Methods:

• On-target oxidation using performic acid vapor on matrix-assisted laser desorption/ionization (MALDI) sample deposits.
• Simultaneous oxidation of multiple MALDI sample spots.
• Compatibility with subsequent alkylation for peptide analysis.

Main Results:

• Successful oxidation of disulfide-containing peptides and small proteins directly on MALDI targets.
• Demonstrated utility with model disulfide-linked peptides, intact bovine insulin, and a bovine ribonuclease A digest.
• Significant increase in oxidation throughput compared to solution-phase methods.
• Reduced ion suppression effects due to direct application to MALDI spots.

Conclusions:

• The on-target performic acid oxidation method is a rapid and efficient technique for analyzing disulfide bonds in peptides and proteins.
• This method simplifies sample preparation and enhances throughput for mass spectrometry-based proteomics.
• The approach is versatile and applicable to various biological samples, including complex digests.