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.     

A method to produce fully characterized ubiquitin covalently modified by 4-hydroxy-nonenal,glyoxal, methylglyoxal,and malondialdehyde

Reactive carbonyl species (RCS) and the corresponding protein adducts (advanced glycoxidation or lipoxidation end products, i.e. AGEs and ALEs) are now widely studied from different points of view, since they can be considered as biomarkers, pathogenic factors, toxic mediators and drug targets. One of the main limits of the research in this field is the lack of standardized and fully characterized AGEs and ALEs to be used for biological, toxicological, and analytical studies. In this work, we set up a procedure to prepare and fully characterize a set of AGEs and ALEs by incubating ubiquitin – a model protein selected as target for carbonylation – with four different RCS: 4-hydroxy-trans-2-nonenal (HNE), methylglyoxal (MGO), glyoxal (GO), and malondialdehyde (MDA). After 24?h of incubation, the extent of protein carbonylation was estimated using a recently developed quantitative strategy based on high-resolution mass spectrometry. The resulting AGEs and ALEs were fully characterized by both intact protein and bottom-up analyses in terms of: stoichiometry of the total amount of modified protein, elucidation of the structure of the RCS-deriving adducts, and localization of the RCS-modified amino acids. Each RCS exhibited different reactivity toward ubiquitin, as detected by quantifying the extent of protein modification. The order of reactivity was MGO?>?GO?>?HNE?>?MDA. A variety of reaction products was identified and mapped on lysine, arginine, and histidine residues of the protein. In summary, a highly standardized and reproducible method to prepare fully characterized AGEs/ALEs is here presented.     

Isotopic labelling for the characterisation of HNE-sequestering agents in plant-based extracts and its application for the identification of anthocyanidins in black rice with giant embryo

Reactive carbonyl species (RCS) are cytotoxic molecules that originate from lipid peroxidation and sugar oxidation. Natural derivatives can be an attractive source of potential RCS scavenger. However, the lack of analytical methods to screen and identify bioactive compounds contained in complex matrices has hindered their identification. The sequestering actions of various rice extracts on RCS have been determined using ubiquitin and 4-hydroxy-2-nonenal (HNE) as a protein and RCS model, respectively. Black rice with giant embryo extract was found to be the most effective among various rice varieties. The identification of bioactive compounds was then carried out by an isotopic signature profile method using the characteristic isotopic ion cluster generated by the mixture of HNE: ²H₅-HNE mixed at a 1:1 stoichiometric ratio. An in-house database was used to obtain the structures of the possible bioactive components. The identified compounds were further confirmed as HNE sequestering agents through HPLC-UV analysis.     

Actin carbonylation: from a simple marker of protein oxidation to relevant signs of severe functional impairment.

The number of protein-bound carbonyl groups is an established marker of protein oxidation. Recent evidence indicates a significant increase in actin carbonyl content in both Alzheimer's disease brains and ischemic hearts. The enhancement of actin carbonylation, causing the disruption of the actin cytoskeleton and the loss of the barrier function, has also been found in human colonic cells after exposure to hypochlorous acid (HOCl). Here, the effects of oxidation induced by HOCl on purified actin are presented. Results show that HOCl causes a rapidly increasing yield of carbonyl groups. However, when carbonylation becomes evident, some Cys and Met residues have been already oxidized. Covalent intermolecular cross-linking as well as some noncovalent aggregation of carbonylated actin have been found. The covalent cross-linking, unaffected by reducing and denaturing agents, parallels an increase in dityrosine fluorescence. Moreover, HOCl-mediated oxidation induces the progressive disruption of actin filaments and the inhibition of F-actin formation. The molar ratios of HOCl to actin that lead to inhibition of actin polymerization seem to have effect only on cysteines and methionines. The process that involves oxidation of amino acid side chains with formation of a carbonyl group would occur at an extent of oxidative insult higher than that causing the oxidation of some critical amino acid residues. Therefore, the increase in actin content of carbonyl groups found in vivo would indicate drastic oxidative modification leading to drastic functional impairments.     

Traditional reactive carbonyl scavengers do not prevent the carbonylation of brain proteins induced by acute glutathione depletion

Abstract

This study investigated the effect of reactive carbonyl species (RCS)-trapping agents on the formation of protein carbonyls during depletion of brain glutathione (GSH). To this end, rat brain slices were incubated with the GSH-depletor diethyl maleate in the absence or presence of chemically different RCS scavengers (hydralazine, methoxylamine, aminoguanidine, pyridoxamine, carnosine, taurine and z-histidine hydrazide). Despite their strong reactivity towards the most common RCS, none of the scavengers tested, with the exception of hydralazine, prevented protein carbonylation. These findings suggest that the majority of protein-associated carbonyl groups in this oxidative stress paradigm do not derive from stable lipid peroxidation products like malondialdehyde (MDA), acrolein and 4-hydroxynonenal (4-HNE). This conclusion was confirmed by the observation that the amount of MDA-, acrolein- and 4-HNE-protein adducts does not increase upon GSH depletion. Additional studies revealed that the efficacy of hydralazine at preventing carbonylation was due to its ability to reduce oxidative stress, most likely by inhibiting mitochondrial production of superoxide and/or by scavenging lipid free radicals.     

Both oxidative and nitrosative stress are associated with muscle wasting in tumour-bearing rats

Reactive oxygen and nitrogen species (ROS and RNS) have been proposed as mechanisms of cancer-induced cachexia. In this study, we assessed using Western blot analysis the levels of total protein carbonylation (2,4-dinitrophenylhydrazine assay), both malondialdehyde- (MDA-) and 2-hydroxy-4-nonenal- (HNE-) protein adducts, Mn-superoxide dismutase (Mn-SOD), catalase, heme oxygenase-1 (HO-1) and 3-nitrotyrosine formation in gastrocnemius muscles of rats bearing the Yoshida AH-130 hepatoma. In the muscles of the tumour-bearing animals, protein carbonylation as measured by total levels of carbonyl group formation and both HNE and MDA-protein adducts, and protein tyrosine nitration were significantly greater than in control muscles. Protein levels of the antioxidant enzymes Mn-SOD, catalase, and HO-1 were not significantly modified in the rat cachectic muscles compared to controls. The inefficiency of the antioxidant enzymes in neutralizing excessive ROS production may account for elevated markers of protein oxidation and be responsible for the development of both oxidative and nitrosative stress in cancer-induced cachexia.     

Carbonylation of mitochondrial proteins in Drosophila melanogaster during aging

Age-related changes in carbonylation of mitochondrial proteins were determined in mitochondria from the flight muscles of Drosophila melanogaster. Reactivity with antibodies against (i) adducts of dinitrophenyl hydrazone (DNP), commonly assumed to react broadly with derivatized carbonyl groups, (ii) malondialdehyde (MDA), or (iii) hydroxynonenal (HNE), was compared at five different ages of flies. MDA and HNE are carbonyl-containing products of lipid peroxidation, which can form covalent adducts with proteins. Specific objectives were to address the following inter-related issues: (1) what are the sources of adducts involved in protein carbonylation in mitochondria during aging; (2) is carbonylation by different adducts detectable solely by the DNP antibodies, as assumed widely; (3) can the adducts formed by lipid peroxidation products in vivo, be used as markers for monitoring age-associated changes in oxidative damage to proteins. The total amounts of immunoreactive proteins, detected by all three antibodies, were found to increase with age; however, the immunodensity of individual reactive bands and the magnitude of the increases were variable, and unrelated to the relative abundance of a protein. While some protein bands were strongly immunopositive for all three antibodies, others were quite selective. The amounts of high molecular weight cross-linked proteins (>200kDa) increased with age. In general, the anti-HNE antibody reacted with more protein bands compared to the anti-MDA or -DNP antibody. The results suggest that sources of the carbonyl-containing protein adducts vary and no single antibody reacts with all of them. Overall, the results indicate that HNE shows robust age-associated increases in adductation with mitochondrial proteins, and is a good marker for monitoring protein oxidative damage during aging.     

Lipid peroxidation and protein oxidation in patients affected by Hodgkin's lymphoma

A dysregulation of the redox homoeostasis has been reported in various neoplastic disorders. Malondialdehyde/4-hydroxy-2,3-nonenal (MDA/HNE) and protein carbonyl groups represent in vivo indexes of lipid peroxidation and protein oxidation, respectively, suitable to investigate radical-mediated physio-pathological conditions. We evaluated MDA/HNE and protein carbonyl groups in sera of untreated Hodgkin's lymphoma (HL) patients in advanced disease stages, in order to quantify the oxidative stress. HL patients displayed significantly higher levels of both MDA/HNE and protein carbonyl groups as compared with healthy controls. This is the first evidence that a strong increase in HL is one of the most common haematological malignancies, representing approximately 30% of all lymphomas in the circulating protein carbonyl content in HL. These findings may contribute to a better definition of the redox homoeostasis dysregulation in HL.     

The Drosophila carbonyl reductase sniffer is an efficient 4-oxonon-2-enal (4ONE) reductase

Studies with the fruit-fly Drosophila melanogaster demonstrated that the enzyme sniffer prevented oxidative stress-induced neurodegeneration. Mutant flies overexpressing sniffer had significantly extended life spans in a 99.5% oxygen atmosphere compared to wild-type flies. However, the molecular mechanism of this protection remained unclear. Sequence analysis and database searches identified sniffer as a member of the short-chain dehydrogenase/reductase superfamily with a 27.4% identity to the human enzyme carbonyl reductase type I (CBR1). As CBR1 catalyzes the reduction of the lipid peroxidation products 4HNE and 4ONE, we tested whether sniffer is able to metabolize these lipid derived aldehydes by carbonyl reduction. To produce recombinant enzyme, the coding sequence of sniffer was amplified from a cDNA-library, cloned into a bacterial expression vector and the His-tagged protein was purified by Ni-chelate chromatography. We found that sniffer catalyzed the NADPH-dependent carbonyl reduction of 4ONE (K(m)=24±2 μM, k(cat)=500±10 min(-1), k(cat)/K(m)=350 s(-1) mM(-1)) but not that of 4HNE. The reaction product of 4ONE reduction by sniffer was mainly 4HNE as shown by HPLC- and GC/MS analysis. Since 4HNE, though still a potent electrophile, is less neurotoxic and protein reactive than 4ONE, one mechanism by which sniffer exerts its neuroprotective effects in Drosophila after oxidative stress may be enzymatic reduction of 4ONE.     

Myeloperoxidase-dependent Lipid Peroxidation Promotes the Oxidative Modification of Cytosolic Proteins in Phagocytic Neutrophils

Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification, we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A 4-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NADPH oxidase and myeloperoxidase activity and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. The addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation.     

Tandem mass spectrometry for the detection of plant pathogenic fungi and the effects of database composition on protein inferences

LC-MS/MS has demonstrated potential for detecting plant pathogens. Unlike PCR or ELISA, LC-MS/MS does not require pathogen-specific reagents for the detection of pathogen-specific proteins and peptides. However, the MS/MS approach we and others have explored does require a protein sequence reference database and database-search software to interpret tandem mass spectra. To evaluate the limitations of database composition on pathogen identification, we analyzed proteins from cultured Ustilago maydis, Phytophthora sojae, Fusarium graminearum, and Rhizoctonia solani by LC-MS/MS. When the search database did not contain sequences for a target pathogen, or contained sequences to related pathogens, target pathogen spectra were reliably matched to protein sequences from nontarget organisms, giving an illusion that proteins from nontarget organisms were identified. Our analysis demonstrates that when database-search software is used as part of the identification process, a paradox exists whereby additional sequences needed to detect a wide variety of possible organisms may lead to more cross-species protein matches and misidentification of pathogens.     

Inhibition of neutral sphingomyelinase decreases elevated levels of nitrative and oxidative stress markers in liver ischemia–reperfusion injury

Oxidative stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS)-2 have been shown in the pathogenesis of liver ischemia–reperfusion (IR) injury. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression therefore this study determined the role of selective N-SMase inhibition on nitrative and oxidative stress markers following liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Nitrative and oxidative stress markers were determined by evaluating NOS2 expression, protein nitration, nitrite/nitrate levels, 4-hydroxynonenal (HNE) formation, protein carbonyl levels and xanthine oxidase/xanthine dehydrogenase (XO/XDH) activity. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reaction monitoring method using ultra-fast liquid chromatography coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared to controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. NOS2 expression, nitrite/nitrate levels and protein nitration were significantly greater in IR injury and decreased with N-SMase inhibition. Treatment with a selective N-SMase inhibitor significantly decreased HNE formation, protein carbonyl levels and the hepatic conversion of XO. Data confirm the role of nitrative and oxidative injury in IR and highlight the protective effect of selective N-SMase inhibition. Future studies evaluating agents blocking N-SMase activity can facilitate the development of treatment strategies to alleviate oxidative injury in liver I/R injury.     

Mining phosphopeptide signals in liquid chromatography-mass spectrometry data for protein phosphorylation analysis

Protein phosphorylation is a key post-translational modification that governs biological processes. Despite the fact that a number of analytical strategies have been exploited for the characterization of protein phosphorylation, the identification of protein phosphorylation sites is still challenging. We proposed here an alternative approach to mine phosphopeptide signals generated from a mixture of proteins when liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is involved. The approach combined dephosphorylation reaction, accurate mass measurements from a quadrupole/time-of-flight mass spectrometer, and a computing algorithm to differentiate possible phosphopeptide signals obtained from the LC-MS analyses by taking advantage of the mass shift generated by alkaline phosphatase treatment. The retention times and m/z values of these selected LC-MS signals were used to facilitate subsequent LC-MS/MS experiments for phosphorylation site determination. Unlike commonly used neutral loss scan experiments for phosphopeptide detection, this strategy may not bias against tyrosine-phosphorylated peptides. We have demonstrated the applicability of this strategy to sequence more, in comparison with conventional data-dependent LC-MS/MS experiments, phosphopeptides in a mixture of alpha- and beta-caseins. The analytical scheme was applied to characterize the nasopharyngeal carcinoma (NPC) cellular phosphoproteome and yielded 221 distinct phosphorylation sites. Our data presented in this paper demonstrated the merits of computation in mining phosphopeptide signals from a complex mass spectrometric data set.     

MSSimulator: Simulation of mass spectrometry data

Mass spectrometry coupled to liquid chromatography (LC-MS and LC-MS/MS) is commonly used to analyze the protein content of biological samples in large scale studies, enabling quantitation and identification of proteins and peptides using a wide range of experimental protocols, algorithms, and statistical models to analyze the data. Currently it is difficult to compare the plethora of algorithms for these tasks. So far, curated benchmark data exists for peptide identification algorithms but data that represents a ground truth for the evaluation of LC-MS data is limited. Hence there have been attempts to simulate such data in a controlled fashion to evaluate and compare algorithms. We present MSSimulator, a simulation software for LC-MS and LC-MS/MS experiments. Starting from a list of proteins from a FASTA file, the simulation will perform in-silico digestion, retention time prediction, ionization filtering, and raw signal simulation (including MS/MS), while providing many options to change the properties of the resulting data like elution profile shape, resolution and sampling rate. Several protocols for SILAC, iTRAQ or MS(E) are available, in addition to the usual label-free approach, making MSSimulator the most comprehensive simulator for LC-MS and LC-MS/MS data.     

Quantitation of protein carbonylation by dot blot

Protein carbonylation is the most commonly used measure of oxidative modification of proteins. It is frequently measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent, 2,4-dinitrophenylhydrazine. We developed an immunochemical dot blot method for quantitation of protein carbonylation in homogenates or purified proteins. Dimethyl sulfoxide was employed as the solvent because it very efficiently extracts proteins from tissues and keeps them soluble. It also readily dissolves 2,4-dinitrophenylhydrazine and wets polyvinylidene difluoride (PVDF) membranes. The detection limit is 0.19 ± 0.04 pmol of carbonyl, and 60 ng of protein is sufficient to measure protein carbonyl content. This level of sensitivity allowed measurement of protein carbonylation in individual Drosophila.     

Human carbonyl reductase catalyzes reduction of 4-oxonon-2-enal

4-Oxonon-2-enal (4ONE) was demonstrated to be a product of lipid peroxidation, and previous studies found that it was highly reactive toward DNA and protein. The present study sought to determine whether carbonyl reductase (CR) catalyzes reduction of 4ONE, representing a potential pathway for metabolism of the lipid peroxidation product. Recombinant CR was cloned from a human liver cDNA library, expressed in Escherichia coli, and purified by metal chelate chromatography. Both 4ONE and its glutathione conjugate were found to be substrates for CR, and kinetic parameters were calculated. TLC analysis of reaction products revealed the presence of three compounds, two of which were identified as 4-hydroxynon-2-enal (4HNE) and 1-hydroxynon-2-en-4-one (1HNO). GC/MS analysis confirmed 4HNE and 1HNO and identified the unknown reaction product as 4-oxononanal (4ONA). Analysis of oxime derivatives of the reaction products via LC/MS confirmed the unknown as 4ONA. The time course for CR-mediated, NADPH-dependent 4ONE reduction and appearance of 4HNE and 1HNO was determined using HPLC, demonstrating 4HNE to be a major product and 1HNO and 4ONA to be minor products. Simulated structures of 4ONE in the active site of CR/NADPH calculated via docking experiments predict the ketone positioned as primary hydride acceptor. Results of the present study demonstrate that 4ONE is a substrate for CR/NADPH and the enzyme may represent a pathway for biotransformation of the lipid. Furthermore, these findings reveal that CR catalyzes hydride transfer selectively to the ketone but also to the aldehyde and C=C of 4ONE, resulting in 4HNE, 1HNO, and 4ONA, respectively.     

Inactivation of human liver bile acid CoA:amino acid N-acyltransferase by the electrophilic lipid, 4-hydroxynonenal

The hepatic enzyme bile acid CoA:amino acid N-acyltransferase (BAT) catalyzes the formation of amino acid-conjugated bile acids. In the present study, protein carbonylation of BAT, consistent with modification by reactive oxygen species and their products, was increased in hepatic homogenates of apolipoprotein E knock-out mice. 4-Hydroxynonenal (4HNE), an electrophilic lipid generated by oxidation of polyunsaturated long-chain fatty acids, typically reacts with the amino acids Cys, His, Lys, and Arg to form adducts, some of which (Michael adducts) preserve the aldehyde (i.e., carbonyl) moiety. Because two of these amino acids (Cys and His) are members of the catalytic triad of human BAT, it was proposed that 4HNE would cause inactivation of this enzyme. As expected, human BAT (1.6 microM) was inactivated by 4HNE in a dose-dependent manner. To establish the sites of 4HNE's reaction with BAT, peptides from proteolysis of 4HNE-treated, recombinant human BAT were analyzed by peptide mass fingerprinting and by electrospray ionization-tandem mass spectrometry using a hybrid linear ion trap Fourier transform-ion cyclotron resonance mass spectrometer. The data revealed that the active-site His (His362) dose-dependently formed a 4HNE adduct, contributing to loss of activity, although 4HNE adducts on other residues may also contribute.     

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.     

Quantification of trans-4-hydroxy-2-nonenal enantiomers and metabolites by LC-ESI-MS/MS

Lipid peroxidation is a causal factor in multiple diseases including Alzheimer's disease, atherosclerosis, and alcoholic liver disease. One of the most studied products of lipid peroxidation, trans-4-hydroxy-2-nonenal (HNE), has multiple cell signaling and cytotoxic effects. In this work, we developed an LC-MS/MS method for the quantitation of HNE enantiomers, the metabolite trans-4-hydroxy-2-nonenoic acid, and HNE-glutathione adducts in a single chromatographic run. In this method, (R)-HNE and (S)-HNE are derivatized by (S)-carbidopa to form diastereomers that are separated by a reversed-phase column. This method was successfully validated and tested using respiring rat brain mitochondria that enantioselectively metabolize HNE. Metabolic profiles of HNE biotransformation, including the enantiomeric disposition of HNE, will provide useful biomarker data regarding lipid peroxidation in disease states.     

Shotgun cross-linking analysis for studying quaternary and tertiary protein structures

We developed a new approach that employs a novel computer algorithm for the sensitive and high-throughput analysis of tertiary and quaternary interaction sites from chemically cross-linked proteins or multi-protein complexes. First, we directly analyze the digests of the chemically cross-linked proteins using only high-accuracy LC-MS/MS data. We analyze these data using a computer algorithm, we term X!Link, to find cross-links between two peptides. Our algorithm is rapid, taking only a few seconds to analyze approximately 5000 MS/MS spectra. We applied this algorithm to analyze cross-linked sites generated chemically using the amino specific reagent, BS3, in both cytochrome c and the mitochondrial division dynamin mutant, Dnm1G385D, which exists as a stable homodimer. From cytochrome c, a well-established test protein, we identified a total of 31 cross-links, 21 interpeptide and 10 intrapeptide cross-links, in 257 MS/MS spectra from a single LC-MS/MS data set. The high sensitivity of this technique is indicated by the fact that all 19 lysines in cytochrome c were detected as a cross-link product and 33% of all the Lys pairs within 20 A were also observed as a cross-link. Analysis of the cross-linked dimeric form of Dnm1G385D identified a total of 46 cross-links, 38 interpeptide and 8 intrapeptide cross-links, in 98 MS/MS spectra in a single LC-MS/MS data set. These results represent the most abundant cross-links identified in a single protein or protein dimer to date. Statistical analysis suggests a 1% false discovery rate after optimization of filtering parameters. Further analysis of the cross-links identified using our approach indicates that careful manual inspection is important for the correct assignment of cross-linking sites when multiple cross-linkable sites or several similar sequences exist. In summary, we have developed a sensitive MS-based approach to identify peptide-peptide cross-links that does not require isotopic labeling or comparison with non-cross-linked controls, making it faster and simpler than current methodologies.