Unrestricted Mass Spectrometric Data Analysis for Identification, Localization, and Quantification of Oxidative...

Area of Science:

• Proteomics
• Biochemistry
• Mass Spectrometry

Background:

• Oxidation causes diverse protein post-translational modifications, impacting function and disease.
• Carbonylations are common markers of oxidative damage, but other modifications may be more prevalent physiologically.
• Understanding the full spectrum of oxidative modifications is crucial for disease research.

Purpose of the Study:

• To develop a comprehensive mass spectrometry approach for identifying, localizing, and quantifying various oxidative protein modifications.
• To investigate the relative abundance and distribution of different oxidative modifications under controlled oxidation conditions.

Main Methods:

• A holistic mass spectrometry-based strategy using unrestricted database searches and rigorous filtering for oxidative modifications.
• Application of the method to bovine serum albumin and human serum proteins subjected to metal ion-catalyzed oxidation.
• Analysis of "dependent peptides" for simultaneous identification, localization, and quantification.

Main Results:

• Identification of a wide array of oxidative modifications, including hydroxylation, carbonylation, decarboxylation, and dihydroxylation.
• Hydroxylation was the most common modification, while carbonylation showed the greatest relative increase.
• Specific "oxidation hotspots" were identified on histidine, tryptophan, methionine, glutamate, and aspartate residues, though most modifications occurred at low occupancy.

Conclusions:

• The developed mass spectrometry approach provides a broad view of oxidative protein modifications.
• Hydroxylation and carbonylation are significant oxidative modifications, with carbonylation showing a notable increase upon oxidation.
• The study identifies key residues prone to oxidation, offering insights into disease mechanisms related to protein oxidative damage.