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Abstract
We here present a new method to measure the degree of protein-bound methionine sulfoxide formation at a proteome-wide scale. In human Jurkat cells that were stressed with hydrogen peroxide, over 2000 oxidation-sensitive methionines in more than 1600 different proteins were mapped and their extent of oxidation was quantified. Meta-analysis of the sequences surrounding the oxidized methionine residues revealed a high preference for neighboring polar residues. Using synthetic methionine sulfoxide containing peptides designed according to the observed sequence preferences in the oxidized Jurkat proteome, we discovered that the substrate specificity of the cellular methionine sulfoxide reductases is a major determinant for the steady-state of methionine oxidation. This was supported by a structural modeling of the MsrA catalytic center. Finally, we applied our method onto a serum proteome from a mouse sepsis model and identified 35 in vivo methionine oxidation events in 27 different proteins.
Highlights
Reactive oxygen species (ROS)1 are involved in a broad range of processes including signal transduction and gene expression [1], receptor activation [2], antimicrobial and cytotoxic actions of immune cells [3], and aging and age-related degenerative diseases [4]
Thephysiological importance of this modification is reflected by the methionine sulfoxide reductases (Msr) that are present in most organisms [8, 9]: decreased activity of these enzymes was associated with aging and Alzheimer disease [10], and abnormal dopamine signaling was recently found in the methionine sulfoxide reductase A knockout mouse [11]
COFRADIC consists of three consecutive steps: [1] a first fractionation of peptides based on their hydrophobicity (RPHPLC), [2] a chemical or enzymatic reaction that modifies a subset of peptides in each primary fraction, and [3] a series of identical RP-HPLC fractionations of individual, or pooled, modified primary fractions aimed at isolating specific peptides
Summary
Reactive oxygen species (ROS)1 are involved in a broad range of processes including signal transduction and gene expression [1], receptor activation [2], antimicrobial and cytotoxic actions of immune cells [3], and aging and age-related degenerative diseases [4]. 2626 methionine sulfoxide containing peptides in 1655 proteins were identified and their degree of oxidation was quantified.
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