Accurate Proteomewide Measurement of Methionine Oxidation in Aging Mouse Brains.

The oxidation of methionine has emerged as an important post-translational modification of proteins. A number of studies have suggested that the oxidation of methionines in select proteins can have diverse impacts on cell physiology, ranging from detrimental effects on protein stability to functional roles in cell signaling. Despite its importance, the large-scale investigation of methionine oxidation in a complex matrix, such as the cellular proteome, has been hampered by technical limitations. We report a methodology, methionine oxidation by blocking (MobB), that allows for accurate and precise quantification of low levels of methionine oxidation typically observed in vivo. To demonstrate the utility of this methodology, we analyzed the brain tissues of young (6 m.o.) and old (20 m.o.) mice and identified over 280 novel sites for in vivo methionine oxidation. We further demonstrated that oxidation stoichiometries for specific methionine residues are highly consistent between individual animals and methionine sulfoxides are enriched in clusters of functionally related gene products including membrane and extracellular proteins. However, we did not detect significant changes in methionine oxidation in brains of old mice. Our results suggest that under normal conditions, methionine oxidation may be a biologically regulated process rather than a result of stochastic chemical damage.

Protein folding stabilities are a major determinant of oxidation rates for buried methionine residues.

The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications, making it important to delineate factors that influence methionine oxidation rates within a given protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility have been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of proteins. We surveyed the Escherichia coli proteome using several proteomic methodologies and globally measured oxidation rates of methionine residues in the presence and absence of tertiary structure, as well as the folding stabilities of methionine-containing domains. These data indicated that buried methionines have a wide range of protection factors against oxidation that correlate strongly with folding stabilities. Consistent with this, we show that in comparison to E. coli, the proteome of the thermophile Thermus thermophilus is significantly more stable and thus more resistant to methionine oxidation. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and propose a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures. Overall, these results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability.

Quantitative Analysis of in Vivo Methionine Oxidation of the Human Proteome.

The oxidation of methionine is an important post-translational modification of proteins with numerous roles in physiology and pathology. However, the quantitative analysis of methionine oxidation on a proteome-wide scale has been hampered by technical limitations. Methionine is readily oxidized in vitro during sample preparation and analysis. In addition, there is a lack of enrichment protocols for peptides that contain an oxidized methionine residue, making the accurate quantification of methionine oxidation difficult to achieve on a global scale. Herein, we report a methodology to circumvent these issues by isotopically labeling unoxidized methionines with 18O-labeled hydrogen peroxide and quantifying the relative ratios of 18O- and 16O-oxidized methionines. We validate our methodology using artificially oxidized proteomes made to mimic varying degrees of methionine oxidation. Using this method, we identify and quantify a number of novel sites of in vivo methionine oxidation in an unstressed human cell line.

Global analysis of methionine oxidation provides a census of folding stabilities for the human proteome.

The stability of proteins influences their tendency to aggregate, undergo degradation, or become modified in cells. Despite their significance to understanding protein folding and function, quantitative analyses of thermodynamic stabilities have been mostly limited to soluble proteins in purified systems. We have used a highly multiplexed proteomics approach, based on analyses of methionine oxidation rates, to quantify stabilities of ∼10,000 unique regions within ∼3,000 proteins in human cell extracts. The data identify lysosomal and extracellular proteins as the most stable ontological subsets of the proteome. We show that the stability of proteins impacts their tendency to become oxidized and is globally altered by the osmolyte trimethylamine N-oxide (TMAO). We also show that most proteins designated as intrinsically disordered retain their unfolded structure in the complex environment of the cell. Together, the data provide a census of the stability of the human proteome and validate a methodology for global quantitation of folding thermodynamics.