Quantitative Analysis of in Vivo Methionine Oxidation of the Human Proteome

Abstract

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 18 O-labeled hydrogen peroxide and quantifying the relative ratios of 18 O-and 16 O-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.

Methionine oxidation within the prion protein

“…It should be noted that newer methodologies have since been developed that circumvent artifactual oxidation by 'blocking' unoxidized methionines using isotopically labelled oxidizing agents prior to mass spectrometric analysis ( Figure 2) [36,87,88]. This approach allows for accurate quantitation of in vivo methionine oxidation from tissue or cell extracts.…”

“…Catalytic activities of oxidized Msrs are subsequently restored by the reduction of disulfide cysteine intermediates by thioredoxin and thioredoxin reductases, with NADPH acting as the terminal electron donor to produce one molecule of NAD + per catalytic Figure 2. A schematic overview of a novel method for the accurate quantification of methionine oxidation [36]. As shown in the left panel, methionine oxidation can typically occur in vitro during sample preparation, and the measured levels of oxidation may not be reflective of levels of methionine oxidation in vivo.…”

“…ROS are redox reactive metabolites that can oxidize amino acids in proteins and result in protein misfolding. The sulfur containing amino acid methionine has been demonstrated to be uniquely prone to oxidation by ROS, resulting in the formation of protein bound methionine sulfoxide residues [ 36 ]. Unmodified methionine is a non-polar hydrophobic amino acid whereas methionine sulfoxide is a polar hydrophilic amino acid.…”

“…Indeed, the issue of potentially artifactual in vitro oxidation of methionine residues is at the core of the controversy surrounding measurements of methionine oxidation in PrP Sc . As described below, some studies have found strong enrichment for methionine oxidation in prion infected samples while others report no significant differences in the extent of PrP oxidation between normal and prion infected samples [ 36 , 74 , 84 – 86 ].…”

Susceptibility of Protein Methionine Oxidation in Response to Hydrogen Peroxide Treatment–Ex Vivo Versus In Vitro: A Computational Insight

“…On the other hand, there is abundant evidence that the dysregulation of this methionine oxidation-reduction cycle could be the basis of multiple diseases [ 17 ]. Not surprisingly, the interest in characterizing methionine oxidation has been revived and a number of proteome-wide studies of methionine oxidation has been reported in the past few years [ 18 , 19 , 20 , 21 , 22 , 23 , 24 ]. In an additional effort to identify, classify and document sulfoxidized proteins/sites in different organisms and under different experimental conditions, we have recently built a database, MetOSite, that provides easy access to information on experimentally confirmed sulfoxidized methionine sites [ 25 ].…”