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Journal Articles Analytical and Bioanalytical Chemistry Year : 2023

Specific detection of protein carbonylation sites by 473 nm photodissociation mass spectrometry

Abstract

The study of protein oxidation remains a challenge despite the biomedical interest in reliable biomarkers of oxidative stress. This is particularly true for carbonylations although, recently, liquid chromatography-mass spectrometry techniques (LC-MS) have been proposed to detect this non-enzymatic and poorly distributed oxidative modification of proteins using untargeted or carbonyl-reactive probe methods. These methods proved to be feasible but could not preserve the dynamic range of the protein sample, making it impossible to quantify oxidatively modified proteoforms compared with native proteoforms. Here, we propose an innovative method based on the implementation of a reactive carbonyl probe conjugated with a laser-sensitive chromophore, dabcyl-aminooxy, which confers optical specificity to the LC-MS approach. In addition, our protein carbonyl detection method allows us to localize individual carbonylation sites by observing fragments of derivatized oxidized peptides. Two model proteins, alpha-synuclein and beta-lactoglobulin, were oxidized and carbonylation sites were detected, resulting in the identification of respectively 34 and 77 different carbonylated amino acids. Thus, we demonstrated the application of a direct and sensitive method for studying protein carbonylation sites in complex protein extracts.
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Dates and versions

hal-04237901 , version 1 (11-10-2023)

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Romain Ladouce, Luke Macaleese, Karlo Wittine, Mladen Merćep, Marion Girod. Specific detection of protein carbonylation sites by 473 nm photodissociation mass spectrometry. Analytical and Bioanalytical Chemistry, 2023, ABC Highlights: authored by Rising Stars and Top Experts, ⟨10.1007/s00216-023-04956-5⟩. ⟨hal-04237901⟩
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