High-performance liquid chromatographic determination of imidazole dipeptides,histidine, 1-methylhistidine and 3-methylhistidine in equine and camel muscle and individual muscle fibres

The combined solid-phase extraction (Isolute PRS columns) and reversed-phase gradient HPLC method presented provides a sensitive, reproducible and selective quantification of carnosine, anserine, balenine, homocarnosine, histidine, 1-methylhistidine and 3-methylhistidine in equine and camel muscle and individual muscle fibres. Recoveries were 91–115%. Lower limits of detection were 0.005–0.010 mmol kg⁻ dry muscle. The compounds were isolated from other physiological amino acids and small peptides and resolved within a single chromatographic run of 55 min. Concentrations of these compounds in equine myocardium, diaphragm, skeletal muscle, camel muscle and individual muscle fibres of both species are presented for the first time.     

Carnosine is a quencher of 4-hydroxy-nonenal: through what mechanism of reaction?

The aim of this study was to understand the mechanism of action through which carnosine (beta-alanyl-L-histidine) acts as a quencher of cytotoxic alpha,beta-unsaturated aldehydes, using 4-hydroxy-trans-2,3-nonenal (HNE) as a model aldehyde. In phosphate buffer solution (pH 7.4), carnosine was 10 times more active as an HNE quencher than L-histidine and N-acetyl-carnosine while beta-alanine was totally inactive; this indicates that the two constitutive amino acids act synergistically when incorporated as a dipeptide and that the beta-alanyl residue catalyzes the addition reaction of the histidine moiety to HNE. Two reaction products of carnosine were identified, in a pH-dependent equilibrium: (a) the Michael adduct, stabilized as a 5-member cyclic hemi-acetal and (b) an imine macrocyclic derivative. The adduction chemistry of carnosine to HNE thus appears to start with the formation of a reversible alpha,beta-unsaturated imine, followed by ring closure through an intra-molecular Michael addition. The biological role of carnosine as a quencher of alpha,beta-unsaturated aldehydes was verified by detecting carnosine-HNE reaction adducts in oxidized rat skeletal muscle homogenate.     

A re-evaluation of the antioxidant activity of purified carnosine

The antioxidant activity of carnosine has been re-evaluated due to the presence of contaminating hydrazine in commercial carnosine preparations. Purified carnosine is capable of scavenging peroxyl radicals. Inhibition of the oxidation of phosphatidylcholine liposomes by purified carnosine is greater in the presence of copper than iron, a phenomenon likely to be due to the copper chelating properties of carnosine. Purified carnosine is capable of forming adducts with aldehydic lipid oxidation products. Adduct formation is greatest for alpha,beta-monounsaturated followed by polyunsaturated and saturated aldehydes. While the ability of carnosine to form adducts with aldehydic lipid oxidation products is lower than other compounds such as glutathione, the higher concentrations of carnosine in skeletal muscle are likely to make it the most important molecule that forms aldehyde adducts. Monitoring changes in carnosine concentrations in oxidizing skeletal muscle shows that carnosine oxidation does not occur until the later stages of oxidation suggesting that carnosine may not be as effective free radical scavenger in vivo as other antioxidants like alpha-tocopherol.     

Copper and cobalt complexes of carnosine and anserine: production of active oxygen species and its enhancement by 2-mercaptoimidazoles.

Phosphate buffer solutions of two dipeptides prevalent in striated muscle, L-carnosine (beta-alanyl-L-histidine) and L-anserine (beta-alanyl-L-1-methylhistidine), produce active oxygen species as measured by bleaching of N,N-dimethyl-4-nitrosoaniline (RNO). Activity is enhanced 5-14-fold in the presence of 2-mercaptoimidazoles such as ergothioneine, carbimazole (3-methyl-2-mercaptoimidazole-1-carboxylate), methimazole (2-mercapto-1-methylimidazole) and 2-mercaptoimidazole but only slightly by thiourea and dimethylthiourea. Activity is proportional to carnosine concentration and to mercaptoimidazole concentration at a fixed concentration of the second component. A variety of imidazoles closely related to carnosine and anserine are inactive, even after addition of transition metal ions. Activity is moderately increased above the pKa of the carnosine imidazole ring (pH 7.2, 7.5 and 8.0) versus below the pKa (pH 6.5 and 6.8). Activity is slightly increased by addition of copper or cobalt ions but not by addition of ferrous or ferric ions. Activity is decreased by Chelex 100 pretreatment of phosphate buffer and stimulated when copper or cobalt ions are added to the chelated buffer but there is no significant stimulation by ferric ions. Catalase eliminates most activity but superoxide dismutase has little effect. We propose that metal-carnosine and metal-anserine complexes produce superoxide and also serve as superoxide dismutases with resultant accumulation of hydrogen peroxide. An unidentified radical produced from hydrogen peroxide subsequently bleaches RNO. From the biological distributions of carnosine, anserine and ergothioneine, we infer that deleterious effects are probably minimal under normal physiological circumstances due to tissue and cellular compartmentalization and to sequestration of these compounds and transition metal ions.     

Detection, characterisation, and quantification of carnosine and other histidyl derivatives in cardiac and skeletal muscle

Isocratic reverse phase analytical high performance liquid chromatography (HPLC) has been used to examine naturally occurring imidazoles of cardiac and skeletal muscles. Elution of muscle extracts with a phosphate buffer mobile phase from columns packed with hypersil ODS (5 micron) resulted in good separation of the skeletal muscle imidazole-containing dipeptides carnosine and anserine. Measured concentrations corresponded to published values. N-Acetyl forms that were not commercially available were prepared from their parent compounds and their identities verified by NMR-spectroscopy. Examination of frog cardiac muscle confirmed the presence of N-acetylhistidine and also indicated the presence of its 1-methyl derivative. Extracts of mammalian cardiac muscle were examined by HPLC which indicated the presence of low concentrations of carnosine but substantial amounts of N-acetyl forms of histidine, 1-methylhistidine, carnosine and anserine. Fractions corresponding to the numerous peaks were examined using staining systems specific for certain chemical features and compared to results obtained for commercial or synthetic standards. Results of these tests supported the chromatographic data. The total concentrations in cardiac muscle of these imidazole-containing substances (approx. 10 mM) is sufficient to alter significantly the sensitivity of their contractile apparatus to calcium ions.     

肌肽、丙氨酸和组氨酸抗氧化性的比较研究

肌肽是一种发现于脊椎动物骨骼肌和大脑中的二肽(β-丙氨酰-L-组氨酸).为了探讨肌肤的抗氧化性与其结构之间的关系,试验研究了肌肽、丙氨酸和组氨酸对DPPH自由基的清除作用和对牛血清白蛋白(BSA)氧化修饰的抑制作用.结果表明肌肽对DPPH自由基有显著的清除效果(P＜0.01),组氨酸清除率低于肌肤,而丙氨酸基本无清除自...     

Reduced muscle carnosine content in type 2, but not in type 1 diabetic patients

Carnosine is present in high concentrations in skeletal muscle where it contributes to acid buffering and functions also as a natural protector against oxidative and carbonyl stress. Animal studies have shown an anti-diabetic effect of carnosine supplementation. High carnosinase activity, the carnosine degrading enzyme in serum, is a risk factor for diabetic complications in humans. The aim of the present study was to compare the muscle carnosine concentration in diabetic subjects to the level in non-diabetics. Type 1 and 2 diabetic patients and matched healthy controls (total n=58) were included in the study. Muscle carnosine content was evaluated by proton magnetic resonance spectroscopy (3 Tesla) in soleus and gastrocnemius. Significantly lower carnosine content (-45%) in gastrocnemius muscle, but not in soleus, was shown in type 2 diabetic patients compared with controls. No differences were observed in type 1 diabetic patients. Type II diabetic patients display a reduced muscular carnosine content. A reduction in muscle carnosine concentration may be partially associated with defective mechanisms against oxidative, glycative and carbonyl stress in muscle.     

Proteomics of lipid oxidation-induced oxidation of porcine and bovine oxymyoglobins

Myoglobin (Mb) redox state affects meat color and is destabilized by lipid oxidation products such as 4-hydroxy-2-nonenal (HNE). Our objective was to investigate lipid oxidation-induced oxymyoglobin (OxyMb) oxidation in Mb from two major meat-producing livestock species utilizing MS and proteomics tools. Porcine OxyMb was incubated with HNE and analyzed for metmyoglobin (MetMb) formation. MetMb formation was greater in the presence of HNE than controls at pH 7.4 and 37 degrees C (p <0.05). MALDI-TOF MS was used to identify adduct formation; only mono-adducts of HNE (via Michael addition) with porcine Mb were detected. LC-ESI-MS/MS identified three histidine (HIS) residues in porcine Mb that were readily adducted by HNE (HIS 24, 36 and 119), whereas in bovine Mb seven histidine residues (HIS 24, 36, 81, 88, 93, 119 and 152) were adducted. Quantitation of HNE-adducted peptides using isotope-labeled phenyl isocyanate indicated that, initially, HIS 36 was preferentially adducted in porcine Mb whereas HIS 81, 88 and 93 were the predominant sites of early HNE adduction in bovine Mb. Preferential HNE adduction at the proximal histidine (HIS 93) was observed exclusively in bovine OxyMb and may explain why lipid oxidation-induced OxyMb oxidation appears more extensive in beef, than in pork.     

Role of Aldose Reductase in the Metabolism and Detoxification of Carnosine-Acrolein Conjugates

Oxidation of unsaturated lipids generates reactive aldehydes that accumulate in tissues during inflammation, ischemia, or aging. These aldehydes form covalent adducts with histidine-containing dipeptides such as carnosine and anserine, which are present in high concentration in skeletal muscle, heart, and brain. The metabolic pathways involved in the detoxification and elimination of these conjugates are, however, poorly defined, and their significance in regulating oxidative stress is unclear. Here we report that conjugates of carnosine with aldehydes such as acrolein are produced during normal metabolism and excreted in the urine of mice and adult human non-smokers as carnosine-propanols. Our studies show that the reduction of carnosine-propanals is catalyzed by the enzyme aldose reductase (AR). Carnosine-propanals were converted to carnosine-propanols in the lysates of heart, skeletal muscle, and brain tissue from wild-type (WT) but not AR-null mice. In comparison with WT mice, the urinary excretion of carnosine-propanols was decreased in AR-null mice. Carnosine-propanals formed covalent adducts with nucleophilic amino acids leading to the generation of carnosinylated proteins. Deletion of AR increased the abundance of proteins bound to carnosine in skeletal muscle, brain, and heart of aged mice and promoted the accumulation of carnosinylated proteins in hearts subjected to global ischemia ex vivo. Perfusion with carnosine promoted post-ischemic functional recovery in WT but not in AR-null mouse hearts. Collectively, these findings reveal a previously unknown metabolic pathway for the removal of carnosine-propanal conjugates and suggest a new role of AR as a critical regulator of protein carnosinylation and carnosine-mediated tissue protection.     

Protein targets for carbonylation by 4-hydroxy-2-nonenal in rat liver mitochondria

Protein carbonylation has been associated with various pathophysiological processes. A representative reactive carbonyl species (RCS), 4-hydroxy-2-nonenal (HNE), has been implicated specifically as a causative factor for the initiation and/or progression of various diseases. To date, however, little is known about the proteins and their modification sites susceptible to "carbonyl stress" by this RCS, especially in the liver. Using chemoprecipitation based on a solid-phase hydrazine chemistry coupled with LC-MS/MS bottom-up approach and database searching, we identified several protein-HNE adducts in isolated rat liver mitochondria upon HNE exposure. The identification of selected major protein targets, such as the ATP synthase β-subunit, was further confirmed by immunoblotting and a gel-based approach in combination with LC-MS/MS. A network was also created based on the identified protein targets, which showed that the main protein interactions were associated with cell death, tumor morphology and drug metabolism, implicating the toxic nature of HNE in the liver mitoproteome. The functional consequence of carbonylation was illustrated by its detrimental impact on the activity of ATP synthase, a representative major mitochondrial protein target for HNE modifications.     

Basic aspects of the biochemical reactivity of 4-hydroxynonenal

4-hydroxynonenal (HNE), a major lipid peroxidation product of n-6 polyunsaturated fatty acids, which was discovered by the late Hermann Esterbauer, is a remarkable trifunctional molecule. Both the hydroxy group and the conjugated system consisting of a C=C double bond and a carbonyl group contribute to the high reactivity of HNE. Most of the biochemical effects of HNE can be explained by its rapid reactions with thiol and amino groups. Among the primary reactants for HNE are the amino acids cysteine, histidine and lysine, which--either free or protein-bound--undergo readily Michael additions to the C=C bond. After this primary reaction, which confers rotational freedom to the C2-C3 bond, secondary reactions may occur involving the carbonyl and the hydroxy group. Primary amines may alternatively react with the carbonyl group to form Schiff bases. Reactions which do not fit into this scheme are the oxidation and the reduction respective of the carbonyl group and the epoxidation of the C=C double bond. Examples will be presented for the interaction of HNE with various classes of biomolecules such as proteins and peptides, lipids and nucleic acids and the biochemical consequences will be discussed.     

Profiling histidine dipeptides in plasma and urine after ingesting beef, chicken or chicken broth in humans

The in vitro metabolic stability of histidine-dipeptides (HD), carnosine (CAR) and anserine (ANS), in human serum, and their absorption kinetics after ingesting pure carnosine or HD rich foods in humans have been investigated. Healthy women (n = 4) went through four phases of taking one dose of either 450 mg of pure carnosine, 150 g beef (B), 150 g chicken (C), or chicken broth (CB) from 150 g chicken with a >2-week washout period between each phase. Blood samples were collected at 0, 30, 60, 100, 180, 240, and 300 min, and urine samples before and after (up to 7 h) ingesting pure carnosine or food. Both plasma and urine samples were analyzed for HD concentrations using a sensitive and selective LC–ESI-MS/MS method. CAR was undetectable in plasma after ingesting pure carnosine, B, C or CB. By contrast, plasma ANS concentration was significantly increased (P < 0.05) after ingesting C or CB, respectively. Urinary concentrations of both CAR and ANS were 13- to 14-fold increased after ingesting B, and 14.8- and 243-fold after CB ingestion, respectively. Thus, dietary HD, which are rapidly hydrolyzed by carnosinase in plasma, and excreted in urine, may act as reactive carbonyl species sequestering agents.     

Effect of carnosine and related compounds on the inactivation of human Cu,Zn-superoxide dismutase by modification of fructose and glycolaldehyde

Glycolaldehyde, an intermediate of the Maillard reaction, and fructose, which is mainly derived from the polyol pathway, rapidly inactivate human Cu,Zn-superoxide dismutase (SOD) at the physiological concentration. We employed this inactivation with these carbonyl compounds as a model glycation reaction to investigate whether carnosine and its related compounds could protect the enzyme from inactivation. Of eight derivatives examined, histidine, Gly-His, carnosine and Ala-His inhibited the inactivation of the enzyme by fructose (p<0.001), and Gly-His, Ala-His, anserine, carnosine, and homocarnosine exhibited a marked protective effect against the inactivation by glycolaldehyde (p<0.001). The carnosine-related compounds that showed this highly protective effect against the inactivation by glycolaldehyde had high reactivity with glycolaldehyde and high scavenging activity toward the hydroxyl radical as common properties. On the other hand, the carnosine-related compounds that had a protective effect against the inactivation by fructose showed significant hydroxyl radical-scavenging ability. These results indicate that carnosine and such related compounds as Gly-His and Ala-His are effective anti-glycating agents for human Cu,Zn-SOD and that the effectiveness is based not only on high reactivity with carbonyl compounds but also on hydroxyl radical scavenging activity.     

In vitro and in vivo inhibition of muscle lipid and protein oxidation by carnosine.

Carnosine, a beta-alanyl-L-histidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe2+, H2O2)-generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and beta-alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fe-nitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.     

Susceptibility of amyloid beta peptide degrading enzymes to oxidative damage: a potential Alzheimer's disease spiral

Insulysin (IDE) and neprilysin (NEP) were found to be inactivated by oxidation with hydrogen peroxide, an iron-ascorbate oxidation system, and by treatment with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). In each case reaction led to the introduction of protein carbonyl groups as judged by reaction with 2,4-dintrophenylhydrazine. IDE was inactivated by reaction with 4-hydroxy-2-nonenal (HNE) with the concomitant formation of protein adducts. NEP was not inactivated to a significant extent by HNE, but some HNE-adduct formation did occur. Prior reaction with hydrogen peroxide or AAPH led to enhanced formation of HNE adducts. Treatment of IDE with AAHP or hydrogen peroxide increased its susceptibility to proteolysis, while treatment of NEP with iron/ascorbate or hydrogen peroxide increased its susceptibility to proteolysis. Since IDE and NEP play a prominent role in the clearance of amyloid beta peptides, their oxidative inactivation and enhanced proteolysis can contribute to the onset and/or progression of Alzheimer's disease.     

The Sod Like Activity of Copper: Arnosine,Copper: Anserine and Copper: Homocarnosine Complexes

Carnosine, anserine and homocarnosine are natural compounds which are present in high concentrations (2–20 mM) in skeletal muscles and brain of many vertebrates. We have demonstrated in a previous work that these compounds can act as antioxidants, a result of their ability to scavenge peroxyl radicals, singlet oxygen and hydroxyl radicals. Carnosine and its analogues have been shown to be efficient chelating agents for copper and other transition metals. Since human skeletal muscle contains one-third of the total copper in the body (20–47 mmol/kg) and the concentration of carnosine in this tissue is relatively high, the complex of carnosine:copper may be of biological importance. We have studied the ability of the coppenarnosine (and other carnosine derivatives) complexes to act as superoxide dismutasc. The results indicate that the complex of copper:carnosine can dismute superoxide radicals released by neutrophils treated with PMA in an analogous mechanism to other amino acids and copper complexes. Copper:anserine failed to dismute superoxide radicals and coppwhomocarnosine complex was efficient when the cells were treated with PMA or with histone-opsonized streptococci and cytochalasine B. The possible role of these compounds to act as physiological antioxidants that possess superoxide dismutase activity is discussed.     

LC-ESI-MS/MS determination of 4-hydroxy-trans-2-nonenal Michael adducts with cysteine and histidine-containing peptides as early markers of oxidative stress in excitable tissues

A sensitive, selective, specific and rapid liquid chromatographic-electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination in skeletal muscle of the Michael adducts between 4-hydroxy-trans-2-nonenal (HNE), one of the most reactive lipid peroxidation-driven unsaturated aldehyde, and glutathione (GSH) and the endogenous histidine-containing dipeptides carnosine (CAR) and anserine (ANS), with the final aim to use conjugated adducts as specific and unequivocal markers of lipid peroxidation. Samples (skeletal muscle homogenates from male rats) were prepared by protein precipitation with 1 vol. of a HClO(4) solution (4.2%; w/v) containing H-Tyr-His-OH as internal standard. The supernatant, diluted (1:1, v/v) in mobile phase, was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water-acetonitrile-heptafluorobutyric acid (9:1:0.01, v/v/v) at a flow rate of 0.2 ml/min, with a run time of 12 min. Detection was on a triple quadrupole mass spectrometer equipped with an ESI interface operating in positive ionization mode. The acquisitions were in multiple reaction monitoring (MRM) mode using the following precursor-->product ion combinations: H-Tyr-His-OH (IS): m/z 319.2--> 156.5+301.6; GS-HNE: m/z 464.3--> 179.1+308.0; CAR-HNE: m/z 383.1--> 110.1+266.6; ANS-HNE: m/z 397.2--> 109.1+126.1. The method was validated over the concentration ranges 1.5-90 (GS-HNE) and 0.4-40 (CAR-HNE, ANS-HNE) nmoles/g wet tissue, and the LLOQ were 1.25 and 0.33 pmoles injected respectively. The intra- and inter-day precisions (CV%) were <7.38% (相似文献   

Stable adducts of nerve agents sarin,soman and cyclosarin with TRIS,TES and related buffer compounds—Characterization by LC-ESI-MS/MS and NMR and implications for analytical chemistry

Buffering compounds like TRIS are frequently used in chemical, biochemical and biomedical applications to control pH in solution. One of the prerequisites of a buffer compound, in addition to sufficient buffering capacity and pH stability over time, is its non-reactivity with other constituents of the solution. This is especially important in the field of analytical chemistry where analytes are to be determined quantitatively. Investigating the enzymatic hydrolysis of G-type nerve agents sarin, soman and cyclosarin in buffered solution we have identified stable buffer adducts of TRIS, TES and other buffer compounds with the nerve agents. We identified the molecular structure of these adducts as phosphonic diesters using 1D ¹H-³¹P HSQC NMR and LC-ESI-MS/MS techniques. Reaction rates with TRIS and TES are fast enough to compete with spontaneous hydrolysis in aqueous solution and to yield substantial amounts (up to 20–40%) of buffer adduct over the course of several hours. A reaction mechanism is proposed in which the amino function of the buffer serves as an intramolecular proton acceptor rendering the buffer hydroxyl groups nucleophilic enough for attack on the phosphorus atom of the agents. Results show that similar buffer adducts are formed with a range of hydroxyl and amino function containing buffers including TES, BES, TRIS, BIS-TRIS, BIS-TRIS propane, Tricine, Bicine, HEPES and triethanol amine. It is recommended to use alternative buffers like MOPS, MES and CHES when working with G-type nerve agents especially at higher concentrations and over prolonged times.     

An Azido-Biotin Reagent for Use in the Isolation of Protein Adducts of Lipid-derived Electrophiles by Streptavidin Catch and Photorelease

HNE (4-hydroxynonenal), a byproduct of lipid peroxidation, reacts with nucleophilic centers on proteins. A terminal alkynyl analog of HNE (alkynyl HNE, aHNE) serves as a surrogate for HNE itself, both compounds reacting with protein amine and thiol functional groups by similar chemistry. Proteins modified with aHNE undergo reaction with a click reagent that bears azido and biotin groups separated by a photocleavable linker. Peptides and proteins modified in this way are affinity purified on streptavidin beads. Photolysis of the beads with a low intensity UV light releases bound biotinylated proteins or peptides, i.e. proteins or peptides modified by aHNE. Two strategies, (a) protein catch and photorelease and (b) peptide catch and photorelease, are employed to enrich adducted proteins or peptide mixtures highly enriched in adducts. Proteomics analysis of the streptavidin-purified peptides by LC-MS/MS permits identification of the adduction site. Identification of 30 separate peptides from human serum albumin by peptide catch and photorelease reveals 18 different aHNE adduction sites on the protein. Protein catch and photorelease shows that both HSA and ApoA1 in human plasma undergo significant modification by aHNE.Polyunsaturated lipids in biological membranes are particularly reactive targets for oxygen radicals (1–3). Lipid peroxidation, the chain reaction of peroxyl radicals that is a consequence of oxidative stress, is thought to be involved in human diseases such as cancer, atherosclerosis, and neurodegenerative disorders (4–8). A variety of electrophilic compounds are byproducts of lipid peroxidation, 4-hydroxynon-2-enal (HNE)¹ being a particularly toxic electrophile (9–12) that forms mutagenic DNA adducts (13–15). HNE and other lipid-derived electrophiles also form protein modifications, and some of these adducts have been characterized on a limited number of proteins and peptides by mass spectrometry (MS) and in tissues by antibody-based methods (16). Until recently, relatively little was known about the target selectivity of oxidant-derived electrophiles in proteins, the relative reactivities of different amino acid targets, and the properties of the adducts. We recently described the application of a post-labeling strategy in which biotin hydrazide was used to biotinylate carbonyl-containing adducts formed by HNE in RKO cells (17). When combined with shotgun proteome analysis of the captured proteins, this approach provided a global perspective on patterns of protein damage by a prototypical lipid electrophile. However, biotin hydrazide labels many carbonyls, thus generating a background inventory derived from endogenous carbonyls, which is difficult to characterize and may mask more subtle patterns of selectivity in protein adduction. Moreover, the biotin hydrazide approach can only capture adducts with a reactive carbonyl group.To deal with these limitations, we have explored labeled electrophile probes and selective adduct capture chemistries (18). We recently reported that 4-hydroxynon-2-en-8-ynal, alkynyl-HNE (aHNE), can be used as an HNE surrogate in whole cells to isolate proteins that are adducted by this electrophile (19). aHNE displays similar toxicity in RKO cells as does HNE, and studies with model peptides and isolated proteins show that HNE and the alkynyl surrogate display similar chemistry in reactions with protein nucleophiles. For example, reaction of aHNE with proteins or peptides followed by sodium borohydride reduction gives Michael and imine adducts as shown in structures 1 and 2. This same chemistry is observed for HNE itself.Reaction of cellular aHNE protein adducts with an azido-biotin reagent followed by capture of the triazole cycloadducts on streptavidin beads permitted a number of adducted proteins to be identified by shotgun proteomics (19). Thus, tryptic digestion of the proteins pulled down by means of the alkyne affinity tag generates mixtures that include adducted peptides such as 3 as well as unmodified peptides. The chemistry associated with the alkynyl electrophile works as planned, but the strategy suffers from two significant drawbacks. First, nonspecific protein binding to the streptavidin beads complicates the identification of adducted proteins and second, biotinylated peptides such as 3 generated in the sequence have MS/MS fragmentation patterns that do not permit the ready identification of the amino acid adduction site on the peptide. The biotin appendage is a major site of positive charge localization in the MS/MS experiment, and the formation of characteristic b and y ions is frequently not sufficient for peptide identification.Open in a separate windowWe report here a strategy that couples the alkynyl electrophile azido-biotin capture for the isolation of adducted protein with a photochemical release of the adduct from streptavidin. This approach reduces the protein nonspecific binding problem because release from the bead requires only a photochemical event, and it permits the identification of specific nucleophilic sites on proteins that are modified by reactive electrophiles. By the application of this strategy to capture both adducted proteins and peptides, we have identified plasma protein targets of the probes and also mapped several nucleophilic sites on the plasma protein ApoA1 that are modified by aHNE.     

Caloric restriction of rhesus monkeys lowers oxidative damage in skeletal muscle.

In laboratory rodents, caloric restriction (CR) retards several age-dependent physiological and biochemical changes in skeletal muscle, including increased steady-state levels of oxidative damage to lipids, DNA, and proteins. We used immunogold electron microscopic (EM) techniques with antibodies raised against 4-hydroxy-2-nonenal (HNE) -modified proteins, dinitrophenol, and nitrotyrosine to quantify and localize the age-dependent accrual of oxidative damage in rhesus monkey vastus lateralis skeletal muscle. Using immunogold EM analysis of muscle from rhesus monkeys ranging in age from 2 to 34 years old, a fourfold maximal increase in levels of HNE-modified proteins was observed. Likewise, carbonyl levels increased approximately twofold with aging. Comparing 17- to 23-year-old normally fed to age-matched monkeys subjected to CR for 10 years, levels of HNE-modified proteins, carbonyls, and nitrotyrosine in skeletal muscle from the CR group were significantly less than control group values. Oxidative damage largely localized to myofibrils, with lesser labeling in other subcellular compartments. Accumulation of lipid peroxidation-derived aldehydes, such as malondialdehyde and 4-hydroxy-2-alkenals, and protein carbonyls were measured biochemically and confirmed the morphological data. Our study is the first to quantify morphologically and localize the age-dependent accrual of oxidative damage in mammalian skeletal muscle and to demonstrate that oxidative damage in primates is lowered by CR.