Rapid Quantification of Peptide Oxidation Isomers From Complex Mixtures

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Hydroxyl radical protein footprinting (HRPF) probes protein surface accessibility.
• Quantifying residue-level oxidation in HRPF is challenging due to MS/MS fragmentation and isomeric products.
• Existing methods struggle with separating and analyzing these complex oxidation isomers.

Purpose of the Study:

• To develop an improved method for residue-level quantification of HRPF oxidation products.
• To overcome the limitations of chromatographic separation for isomeric peptide oxidation products.
• To simplify data acquisition and analysis in HRPF studies.

Main Methods:

• Development of a zwitterionic hydrophilic interaction capillary chromatography (ZIC-HILIC) method.
• Coelution of isomeric peptide oxidation products.
• Utilizing electron transfer dissociation tandem mass spectrometry (ETD MS/MS) for analysis.

Main Results:

• The ZIC-HILIC method successfully coelutes all isomeric peptide oxidation products.
• Different peptides are effectively separated by the ZIC-HILIC method.
• Relative quantification of peptide oxidation isomers is achievable from a single ETD MS/MS spectrum.

Conclusions:

• The developed ZIC-HILIC method significantly simplifies HRPF data acquisition and analysis.
• This advancement facilitates more accurate residue-level protein topography analysis.
• The method enables efficient quantification of complex peptide oxidation isomers.