Formation of methionine sulfoxide during glycoxidation and lipoxidation of ribonuclease A

Abstract

Chemical modification of proteins by reactive oxygen species affects protein structure, function and turnover during aging and chronic disease. Some of this damage is direct, for example by oxidation of amino acids in protein by peroxide or other reactive oxygen species, but autoxidation of ambient carbohydrates and lipids amplifies both the oxidative and chemical damage to protein and leads to formation of advanced glycoxidation and lipoxidation end-products (AGE/ALEs). In previous work we have observed the oxidation of methionine during glycoxidation and lipoxidation reactions, and in the present work we set out to determine if methionine sulfoxide (MetSO) in protein was a more sensitive indicator of glycoxidative and lipoxidative damage than AGE/ALEs. We also investigated the sites of methionine oxidation in a model protein, ribonuclease A (RNase), in order to determine whether analysis of the site specificity of methionine oxidation in proteins could be used to indicate the source of the oxidative damage, i.e. carbohydrate or lipid. We describe here the development of an LC/MS/MS for quantification of methionine oxidation at specific sites in RNase during glycoxidation or lipoxidation by glucose or arachidonate, respectively. Glycoxidized and lipoxidized RNase were analyzed by tryptic digestion, followed by reversed phase HPLC and mass spectrometric analysis to quantify methionine and methionine sulfoxide containing peptides. We observed that: 1) compared to AGE/ALEs, methionine sulfoxide was a more sensitive biomarker of glycoxidative or lipoxidative damage to proteins; 2) regardless of oxidizable substrate, the relative rate of oxidation of methionine residues in RNase was Met 29 > Met 30 > Met 13 , with Met 79 being resistant to oxidation; and 3) arachidonate produced a significantly greater yield of MetSO, compared to glucose. The methods developed here should be useful for assessing a protein's overall exposure to oxidative stress from a variety of sources in vivo.

The Reductive Desulfurization of Met and Cys Residues in Bovine RNase A Is Associated with trans Lipids Formation in a Mimetic Model of Biological Membranes

“…It is worth mentioning that Met oxidation has been reported to result from protein manipulation. 36 In contrast, clear differences were observed as result of irradiation progress. In addition to components reported above, four additional protein species were visible in the RNase A sample irradiated in 0.2 M t-BuOH with 84 Gy (Figure 3, panel B).…”

Detection and identification of arginine modifications on methylglyoxal‐modified ribonuclease by mass spectrometric analysis

“…Modification of amino acid residues is not uniform, but is site‐directed. Studies on RNase indicate, for example, that Lys‐41 is the primary site of early glycation and carboxymethylation,3 while Arg‐39 is the main site of reaction with glyoxal (GO),4 and Met‐29 is the principal site of methionine sulfoxide formation 5. It is important to know the distribution of modifications on the protein because, although the overall degree of modification may be low, location of a high proportion of the adducts at a few sites may amplify effects on protein structure and function, e.g.…”

“…loss of enzyme activity and/or modified protein–ligand interactions 6, 7. Peptide mapping using mass spectrometry is the most efficient means of determining the nature of AGE/ALEs and their distribution on the protein 3–5, 7, 8…”

Mass spectrometry to detect the site specificity of advanced glycation/lipoxidation end-product formation on protein: some challenges and solutions

“…Our recent studies using RNase A as a model protein indicate, for example, that Lys-41 is the primary site of early glycation and carboxymethylation [9], whereas Arg-39 and Arg-85 are the main sites of reaction with GO and MGO [10,11]. We have also shown Met-29 to be the principal site of methionine sulfoxide formation [12]. Peptide mapping using MS is the most efficient means of determining the nature of AGE/ALEs and their distribution on protein.…”