Oxidation of methionine residues in recombinant human interleukin-1 receptor antagonist: implications of conformational stability on protein oxidation kinetics

Oxidation of methionine residues is involved in several biochemical processes and in degradation of therapeutic proteins. The relationship between conformational stability and methionine oxidation in recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was investigated to document how thermodynamics of unfolding affect methionine oxidation in proteins. Conformational stability of rhIL-1ra was monitored by equilibrium urea denaturation, and thermodynamic parameters of unfolding (DeltaGH2O, m, and Cm) were estimated at different temperatures. Methionine oxidation induced by hydrogen peroxide at varying temperatures was monitored during "coincubation" of rhIL-1ra with peptides mimicking specific regions of the reactive methionine residues in the protein. The coincubation study allowed estimation of oxidation rates in protein and peptide at each temperature from which normalized oxidation rate constants and activation energies were calculated. The rate constants for buried Met-11 in the protein were lower than for methionine in the peptide with an associated increase in activation energy. The rate constants and activation energy of solvent exposed methionines in protein and peptide were similar. The results showed that conformational stability, monitored using the Cm value, has an effect on oxidation rates of buried methionines. The rate constant of buried Met-11 correlated well with the Cm value but not DeltaGH2O. No correlation was observed for the oxidation rates of solvent-exposed methionines with any thermodynamic parameters of unfolding. The findings presented have implications in protein engineering, in design of accelerated stability studies for protein formulation development, and in understanding disease conditions involving protein oxidation.     

Molecular dynamics simulations and oxidation rates of methionine residues of granulocyte colony-stimulating factor at different pH values

To understand the connection between the conformation of a protein molecule and the oxidation of its methionine residues, we measured the rates of oxidation of methionine residues by H(2)O(2) in granulocyte colony-stimulating factor (G-CSF) as a function of pH and also studied the structural properties of this protein as a function of pH via molecular dynamics simulations. We found that each of the four methionine groups in G-CSF have significant and different rates of oxidation as a function of pH. Moreover, Met(1), in the unstructured N-terminal region, has a rate of oxidation as low as half that of free methionine. The structural properties of G-CSF as a function of pH are evaluated in terms of properties such as hydrogen bonding, deviations from X-ray structure, helical/helical packing, and the atomic covariance fluctuation matrix of alpha-carbons. We found that dynamics (structural fluctuations) are essential in explaining oxidation and that a static picture, such as that resulting from X-ray data, fails in this regard. Moreover, the simulation results also indicate that the solvent-accessible area, traditionally used to measure solvent accessibility of a protein site, of the sulfur atom of methionine residues does not correlate well with the rate of oxidation. Instead, we identified a structural property, average two-shell water coordination number, that correlates well with measured oxidation rates.     

Tertiary structural rearrangements upon oxidation of Methionine145 in calmodulin promotes targeted proteasomal degradation

The selectivity underlying the recognition of oxidized calmodulin (CaM) by the 20S proteasome in complex with Hsp90 was identified using mass spectrometry. We find that degradation of oxidized CaM (CaMox) occurs in a multistep process, which involves an initial cleavage that releases a large N-terminal fragment (A1-F92) as well as multiple smaller carboxyl-terminus peptides ranging from 17 to 26 amino acids in length. These latter small peptides are enriched in methionine sulfoxides (MetO), suggesting a preferential degradation around MetO within the carboxyl-terminal domain. To confirm the specificity of CaMox degradation and to identify the structural signals underlying the preferential recognition and degradation by the proteasome/Hsp90, we have investigated how the oxidation of individual methionines affect the degradation of CaM using mutants in which all but selected methionines in CaM were substituted with leucines. Substitution of all methionines with leucines except Met144 and Met145 has no detectable effect on the structure of CaM, permitting a determination of how site-specific substitutions and the oxidation of Met144 and Met145 affects the recognition and degradation of CaM by the proteasome/Hsp90. Comparable rates of degradation are observed upon the selective oxidation of Met144 and Met145 in CaM-L7 relative to that observed upon oxidation of all nine methionines in wild-type CaM. Substitution of leucines for either Met144 or Met145 promotes a limited recognition and degradation by the proteasome that correlates with decreases in the helical content of CaM. The specific oxidation of Met144 has little effect on rates of proteolytic degradation by the proteasome/Hsp90 or the structure of CaM. In contrast, the specific oxidation of Met145 results in both large increases in the rate of degradation by the proteasome/Hsp90 and significant circular dichroic spectral shape changes that are indicative of changes in tertiary rather than secondary structure. Thus, tertiary structural changes resulting from the site-specific oxidation of a single methionine (i.e., Met145) promote the degradation of CaM by the proteasome/Hsp90, suggesting a mechanism to regulate cellular metabolism through the targeted modulation of CaM abundance in response to oxidative stress.     

In vitro methionine oxidation of Escherichia coli-derived human stem cell factor: effects on the molecular structure, biological activity, and dimerization.

The effect of oxidation of the methionine residues of Escherichia coli-derived recombinant human stem cell factor (huSCF) to methionine sulfoxide on the structure and activity of SCF was examined. Oxidation was performed using hydrogen peroxide under acidic conditions (pH 5.0). The kinetics of oxidation of the individual methionine residues was determined by quantitation of oxidized and unoxidized methionine-containing peptides, using RP-HPLC of Asp-N endoproteinase digests. The initial oxidation rates for Met159, Met-1, Met27, Met36, and Met48 were 0.11 min-1, 0.098 min-1, 0.033 min-1, 0.0063 min-1, and 0.00035 min-1, respectively, when SCF was incubated in 0.5% H2O2 at room temperature. Although oxidation of these methionines does not affect the secondary structure of SCF, the oxidation of Met36 and Met48 affects the local structure as indicated by CD and fluorescence spectroscopy. The 295-nm Trp peak in the near-UV CD is decreased upon oxidation of Met36, and lost completely following the oxidation of Met48, indicating that the Trp44 environment is becoming significantly less rigid than it is in native SCF. Consistent with this result, the fluorescence spectra revealed that Trp44 becomes more solvent exposed as the methionines are oxidized, with the hydrophobicity of the Trp44 environment decreasing significantly. The oxidations of Met36 and Met48 decrease biological activity by 40% and 60%, respectively, while increasing the dissociation rate constant of SCF dimer by two- and threefold. These results imply that the oxidation of Met36 and Met48 affects SCF dimerization and tertiary structure, and decreases biological activity.     

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.     

Local flexibility facilitates oxidization of buried methionine residues

In proteins, all amino acid residues are susceptible to oxidation by various reactive oxygen species (ROS), with methionine and cysteine residues being particularly sensitive to oxidation. Methionine oxidation is known to lead to destabilization and inactivation of proteins, and oxidatively modified proteins can accumulate during aging, oxidative stress, and in various age-related diseases. Although the efficiency of a given methionine oxidation can depend on its solvent accessibility (evaluated from a protein structure as the accessible surface area of the corresponding methionine residue), many experimental results on oxidation rate and oxidation sites cannot be unequivocally explained by the methionine solvent accessible surface area alone. In order to explore other possible mechanisms, we analyzed a set of seventy-one oxidized methionines contained in thirty-one proteins by various bioinformatics tools. In which, 41% of the methionines are exposed, 15% are buried but with various degree of flexibility, and the rest 44% are buried and structured. Buried but highly flexible methionines can be oxidized. Buried and less flexible methionines can acquire additional local structural flexibility from flanking regions to facilitate the oxidation. Oxidation of buried and structured methionine can also be promoted by the oxidation of neighboring methionine that is more exposed and/or flexible. Our data are consistent with the hypothesis that protein structural flexibility represents another important factor favoring the oxidation process.     

Selective degradation of oxidized calmodulin by the 20 S proteasome

We have investigated the mechanisms that target oxidized calmodulin for degradation by the proteasome. After methionine oxidation within calmodulin, rates of degradation by the 20 S proteasome are substantially enhanced. Mass spectrometry was used to identify the time course of the proteolytic fragments released from the proteasome. Oxidized calmodulin is initially degraded into large proteolytic fragments that are released from the proteasome and subsequently degraded into small peptides that vary in size from 6 to 12 amino acids. To investigate the molecular determinants that result in the selective degradation of oxidized calmodulin, we used circular dichroism and fluorescence spectroscopy to assess oxidant-induced structural changes. There is a linear correlation between decreases in secondary structure and the rate of degradation. Calcium binding or the repair of oxidized calmodulin by methionine sulfoxide reductase induces comparable changes in alpha-helical content and rates of degradation. In contrast, alterations in the surface hydrophobicity of oxidized calmodulin do not alter the rate of degradation by the proteasome, indicating that changes in surface hydrophobicity do not necessarily lead to enhanced proteolytic susceptibility. These results suggest that decreases in secondary structure expose proteolytically sensitive sites in oxidized calmodulin that are cleaved by the proteasome in a nonprocessive manner.     

FTIR markers of methionine oxidation for early detection of oxidized protein therapeutics

The biological activity of therapeutic proteins is strongly dependent on the stability of their folded state, which can easily be compromised by degradation. Oxidation is one of the most common causes of degradation and is typically associated with impairment of the native protein structure. Methionine residues stand out as particularly susceptible to oxidation by reactive oxygen intermediates even under mild conditions. Consequently, methionine oxidation has profound effects on protein activity up to the point of adverse biological responses. Of immediate importance therefore is finding affordable approaches for rapid detection of methionine oxidation before any substantial structural changes can ensue. Herein we report that vibrational bands at 1,044 and 1,113 cm⁻¹ in the mid-infrared region can serve as characteristic markers of methionine oxidation in oxidatively stressed protein therapeutics, monoclonal antibodies (IgG1 and its antigen-binding fragment). Such Fourier-transform infrared (FTIR) markers underpin rapid detection assays and hold particular promise for correlation of methionine oxidation with protein structure and function.     

Structural analysis of gelsolin using synchrotron protein footprinting

Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron "white" beam results in the formation of stable side chain oxidation products, which can be digested with proteases for mass spectrometry (MS) analysis. Liquid chromatography-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its "inactive" and Ca2+-activated state. Oxidation rate data for 81 peptides derived from the trypsin digestion of gelsolin are presented; 60 of these peptides were observed not to be oxidized, and 21 had detectable oxidation rates. We also report the Ca2+-dependent changes in oxidation for all 81 peptides. Fifty-nine remained unoxidized, five increased their oxidation rate, and two experienced protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca2+-insensitive and Ca2+-dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand-induced structural changes, and the data provide a detailed model for gelsolin activation.     

Site-specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity of apolipoproteins A-I, C-II, and C-III.

ApoA-I and apoC-II are eluted in two isoforms and apoC-III2 is eluted in three isoforms by reversed phase high performance liquid chromatography (HPLC). The structural basis of these nongenetic heterogeneities was unravelled using HPLC of proteolytic peptides and time-of-flight secondary ion mass spectrometry (TOF-SIMS). In apoA-I, the chromatographic microheterogeneity was caused by the formation of methionine sulfoxides (MetSO). However, only residues Met112 and Met148 were found oxidized, whereas Met86 was unaffected and also resistant towards artificial oxidation. To assess whether and to what extent amino acid substitutions in apoA-I might affect methionine sulfoxidation, the tryptic peptides of 13 different mutant apoA-I proteins from 24 heterozygous apoA-I variant carriers were analyzed by HPLC. In normal apoA-I, the ratios MetSO112/Met112 and MetSO148/Met148 were highly variable. By contrast, the relative ratio of oxidation of methionine residues 112 and 148 was constant. The amino acid changes Lys107----Met, Lys107----O, Glu139----Gly, Glu147----Val, and Pro165----Arg resulted in the preferential oxidation of Met112, and Asp103----Asn resulted in a preferential oxidation of Met148; whereas Pro3----Arg, Pro3----His, Pro4----Arg, Asp89----Glu, Ala158----Asp, Glu198----Lys, and Asp213----Gly had no impact. ApoC-II and apoC-III isoforms differed by the oxidation of the two methionine residues in these proteins. Whereas in apoC-II both methionine residues were oxidized in parallel, in apoC-III the two methionine residues differed in their susceptibility towards oxidation. We conclude that the formation of MetSO depends on the molecular microenvironment within a protein.     

Identification of tryptophan oxidation products in bovine alpha-crystallin.

Oxidation is known to affect the structure, activity, and rate of degradation of proteins, and is believed to contribute to a variety of pathological conditions. Metal-catalyzed oxidation (MCO) is a primary oxidizing system in many cell types. In this study, the oxidative effects of a MCO system (the Fenton reaction) on the structure of the tryptophan residues of alpha-crystallin were determined. Tandem mass spectrometry (MS/MS) was utilized to identify specific tryptophan and methionine oxidation products in the bovine alpha-crystallin sequence. After oxidative exposure, alpha-crystallin was digested with trypsin, and the resulting peptides were fractionated by reverse-phase HPLC. Structural analysis by mass spectrometry revealed that tryptophan 9 of alphaA- and tryptophan 60 of alphaB-crystallin were each converted into hydroxytryptophans (HTRP), N-formylkynurenine (NFK), and kynurenine (KYN). However, only HTRP and KYN formation were detected at residue 9 of alphaB-crystallin. Oxidation of methionine 1 of alphaA- and methionine 1 and 68 of alphaB-crystallin was also detected. The products NFK and KYN are of particular importance in the lens, as they themselves are photosensitizers that can generate reactive oxygen species (ROS) upon UV light absorption. The unambiguous identification of HTRP, NFK, and KYN in intact alpha-crystallin represents the first structural proof of the formation of these products in an intact protein, and provides a basis for detailed structural analysis of oxidized proteins generated in numerous pathological conditions.     

Structure and stability changes of human IgG1 Fc as a consequence of methionine oxidation

The Fc region has two highly conserved methionine residues, Met 33 (C(H)3 domain) and Met 209 (C(H)3 domain), which are important for the Fc's structure and biological function. To understand the effect of methionine oxidation on the structure and stability of the human IgG1 Fc expressed in Escherichia coli, we have characterized the fully oxidized Fc using biophysical (DSC, CD, and NMR) and bioanalytical (SEC and RP-HPLC-MS) methods. Methionine oxidation resulted in a detectable secondary and tertiary structural alteration measured by circular dichroism. This is further supported by the NMR data. The HSQC spectral changes indicate the structures of both C(H)2 and C(H)3 domains are affected by methionine oxidation. The melting temperature (Tm) of the C(H)2 domain of the human IgG1 Fc was significantly reduced upon methionine oxidation, while the melting temperature of the C(H)3 domain was only affected slightly. The change in the C(H)2 domain T m depended on the extent of oxidation of both Met 33 and Met 209. This was confirmed by DSC analysis of methionine-oxidized samples of two site specific methionine mutants. When incubated at 45 degrees C, the oxidized Fc exhibited an increased aggregation rate. In addition, the oxidized Fc displayed an increased deamidation (at pH 7.4) rate at the Asn 67 and Asn 96 sites, both located on the C(H)2 domain, while the deamidation rates of the other residues were not affected. The methionine oxidation resulted in changes in the structure and stability of the Fc, which are primarily localized to the C(H)2 domain. These changes can impact the Fc's physical and covalent stability and potentially its biological functions; therefore, it is critical to monitor and control methionine oxidation during manufacturing and storage of protein therapeutics.     

Identification of potential sites for tryptophan oxidation in recombinant antibodies using tert-butylhydroperoxide and quantitative LC-MS

Amino acid oxidation is known to affect the structure, activity, and rate of degradation of proteins. Methionine oxidation is one of the several chemical degradation pathways for recombinant antibodies. In this study, we have identified for the first time a solvent accessible tryptophan residue (Trp-32) in the complementary-determining region (CDR) of a recombinant IgG1 antibody susceptible to oxidation under real-time storage and elevated temperature conditions. The degree of light chain Trp-32 oxidation was found to be higher than the oxidation level of the conserved heavy chain Met-429 and the heavy chain Met-107 of the recombinant IgG1 antibody HER2, which have already been identified as being solvent accessible and sensitive to chemical oxidation. In order to reduce the time for simultaneous identification and functional evaluation of potential methionine and tryptophan oxidation sites, a test system employing tert-butylhydroperoxide (TBHP) and quantitative LC-MS was developed. The optimized oxidizing conditions allowed us to specifically oxidize the solvent accessible methionine and tryptophan residues that displayed significant oxidation in the real-time stability and elevated temperature study. The achieved degree of tryptophan oxidation was adequate to identify the functional consequence of the tryptophan oxidation by binding studies. In summary, the here presented approach of employing TBHP as oxidizing reagent combined with quantitative LC-MS and binding studies greatly facilitates the efficient identification and functional evaluation of methionine and tryptophan oxidation sites in the CDR of recombinant antibodies.     

Hydrogen peroxide regulates the cholinergic signal in a concentration dependent manner

The human epidermis holds the full capacity for autocrine synthesis, transport and degradation of acetylcholine as well as the muscarinic (m1-m5) and nicotinic signal transduction in keratinocytes and melanocytes. This cholinergic cascade is severely affected in patients with the depigmentation disorder vitiligo due to accumulation of hydrogen peroxide (H(2)O(2)) in the mM range as shown by in vivo FT-Raman spectroscopy. These high levels can oxidise susceptible amino acid residues such as methionine, tryptophan, cysteine and selenocysteine in the structure of proteins and peptides which in turn can severely affect the function. Here the effect of this reactive oxygen species was followed on the production and degradation of acetylcholine using immunofluorescence, enzyme kinetics, in vivo and in vitro FT-Raman and fluorescence spectroscopy as well as computer modelling. The results showed that both epidermal acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) are target to H(2)O(2)-mediated oxidation of methionine and tryptophan residues close to the catalytic triad, while cholineacetyltransferase (chAT) is not affected. Enzyme kinetics revealed concentration dependent activation/deactivation of both degrading enzymes by H(2)O(2). Oxidation of methionine to methionine sulfoxide was confirmed by FT-Raman spectroscopy while oxidation of tryptophan to 5OH-tryptophan was identified by fluorescence spectroscopy. H(2)O(2)-mediated oxidation of both enzymes takes place in acute vitiligo yielding accumulation of acetylcholine in the epidermis of these patients. This process is reversible with a narrowband UVB activated pseudocatalase PC-KUS leading to recovery of epidermal and systemic enzyme activities as well as restoration of the lost skin colour.     

Identification and characterization of oxidation and deamidation sites in monoclonal rat/mouse hybrid antibodies

Oxidation of methionine residues and deamidation of asparagine residues are the major causes of chemical degradation of biological pharmaceuticals. The mechanism of these non-enzymatic chemical reactions has been studied in great detail. However, the identification and quantification of oxidation and deamidation sites in a given protein still remains a challenge. In this study, we identified and characterized several oxidation and deamidation sites in a rat/mouse hybrid antibody. We evaluated the effects of the sample preparation on oxidation and deamidation levels and optimized the peptide mapping method to minimize oxidation and deamidation artifacts. Out of a total number of 18 methionine residues, we identified six methionine residues most susceptible to oxidation. We determined the oxidation rate of the six methionine residues using 0.05% H₂O₂ at different temperatures. Methionine residue 256 of the mouse heavy chain showed the fastest rate of oxidation under those conditions with a half life of approximately 200 min at 4 °C and 27 min at 37 °C. We identified five asparagine residues prone to deamidation under accelerated conditions of pH 8.6 at 37 °C. Kinetic characterization of the deamidation sites showed that asparagine residue 218 of the rat heavy chain exhibited the fastest rate of deamidation with a half live of 1.5 days at pH 8.6 and 37 °C. Analysis of antibody isoforms using free flow electrophoresis showed that deamidation is the major cause of the charged variants of this rat/mouse hybrid antibody.     

Spontaneous degradation of polypeptides at aspartyl and asparaginyl residues: effects of the solvent dielectric.

We have investigated the spontaneous degradation of aspartate and asparagine residues via succinimide intermediates in model peptides in organic co-solvents. We find that the rate of deamidation at asparagine residues is markedly reduced in solvents of low dielectric strength. Theoretical considerations suggest that this decrease in rate is due to the destabilization of the deprotonated peptide bond nitrogen anion that is the postulated attacking species in succinimide formation. This result suggests that asparagine residues in regions with low dielectric constants, such as the interior of a protein or in a membrane bilayer, are protected from this type of degradation reaction. On the other hand, we found little or no effect on the rate of succinimide-mediated isomerization of aspartate residues when subjected to the same changes in dielectric constant. In this case, the destabilization of the attacking peptide bond nitrogen anion may be balanced by increased protonation of the aspartyl side chain carboxyl group, a reaction that results in a superior leaving group. Consequently, any protein structure or conformation that would increase the protonation of an aspartate side chain carboxyl group can be expected to render that residue more labile. These results may help explain why particular aspartate residues have been found to degrade in proteins at rates comparable to those of asparagine residues, even though aspartyl-containing peptides degrade more slowly than corresponding asparaginyl-containing peptides in aqueous solutions.     

Chemical de‐O‐glycosylation of glycoproteins for application in LC‐based proteomics

We describe a cyclic on‐column procedure for the sequential degradation of complex O‐glycans on proteins or peptides by periodate oxidation of sugars and cleavage of oxidation products by elimination. Desialylated glycoproteins were immobilized to alkali‐stable, reversed‐phase Poros 20 beads followed by two degradation cycles and the eluted apoproteins were either separated by SDS gel electrophoresis or digested with trypsin prior to LC/ESI‐MS. We demonstrate on the peptide and protein level that even complex glycan moieties are removed under mild conditions with only minimal effects on structural integrity of the peptide core by fragmentation, dehydration or by racemization of the Lys/Arg residues. The protocol is applicable on gel‐immobilized glycoproteins after SDS gel electrophoresis. Conversion of O‐glycoproteins into their corresponding apoproteins should result in facilitated accessibility of tryptic cleavage sites, increase the numbers of peptide fragments, and accordingly enhance protein coverage and identification rates within the subproteome of mucin‐type O‐glycoproteins.     

Formation of methionine sulfoxide during glycoxidation and lipoxidation of ribonuclease A

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 Met29>Met30>Met13, with Met79 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.     

Protein oxidation and aging

Organisms are constantly exposed to various forms of reactive oxygen species (ROS) that lead to oxidation of proteins, nucleic acids, and lipids. Protein oxidation can involve cleavage of the polypeptide chain, modification of amino acid side chains, and conversion of the protein to derivatives that are highly sensitive to proteolytic degradation. Unlike other types of modification (except cysteine oxidation), oxidation of methionine residues to methionine sulfoxide is reversible; thus, cyclic oxidation and reduction of methionine residues leads to consumption of ROS and thereby increases the resistance of proteins to oxidation. The importance of protein oxidation in aging is supported by the observation that levels of oxidized proteins increase with animal age. The age-related accumulation of oxidized proteins may reflect age-related increases in rates of ROS generation, decreases in antioxidant activities, or losses in the capacity to degrade oxidized proteins.     

Protein oxidation and aging

Organisms are constantly exposed to various forms of reactive oxygen species (ROS) that lead to oxidation of proteins, nucleic acids, and lipids. Protein oxidation can involve cleavage of the polypeptide chain, modification of amino acid side chains, and conversion of the protein to derivatives that are highly sensitive to proteolytic degradation. Unlike other types of modification (except cysteine oxidation), oxidation of methionine residues to methionine sulfoxide is reversible; thus, cyclic oxidation and reduction of methionine residues leads to consumption of ROS and thereby increases the resistance of proteins to oxidation. The importance of protein oxidation in aging is supported by the observation that levels of oxidized proteins increase with animal age. The age-related accumulation of oxidized proteins may reflect age-related increases in rates of ROS generation, decreases in antioxidant activities, or losses in the capacity to degrade oxidized proteins.