Computational study of the effects of protein tyrosine nitrations on the catalytic activity of human thymidylate synthase

Tyrosine nitration is a widespread post-translational modification capable of affecting both the function and structure of the host protein molecule. Enzyme thymidylate synthase (TS), a homodimer, is a molecular target for anticancer therapy. Recently purified TS preparations, isolated from mammalian tissues, were found to be nitrated, suggesting this modification to appear endogenously in normal and tumor tissues. Moreover, human TS (hTS) nitration in vitro led to a by twofold lowered catalytic activity following nitration in average of 1 tyrosine residue per monomer (D?browska-Ma? et al. in Org Biomol Chem 10:323–331, 2012), with the modification identified by mass spectrometry at seven different sites (Y33, Y65, Y135, Y213, Y230, Y258 and Y301). In the present paper, combined computational approach, including molecular and essential dynamics and free energy computations, was used to predict the influence on the activity of hTS of nitration of each of the seven tyrosine residues. The simulations were based on the crystal structure of hTS ternary complex with dUMP and Tomudex (PDB code: 1I00), with the Tomudex molecule replaced by the molecule of TS cofactor analogue, tetrahydrofolate. The present results indicate that while with nitration of five out of seven residues (Y33, Y135, Y230, Y258 and Y301), single residue modification appears to have a strong reducing effect on the activity, with the remaining two, Y65 and Y213, no or a weaker influence is apparent. Taken together, these results demonstrate that tyrosine nitrations in the hTS enzyme show clear tendency to influence the structure and dynamics and, in turn, catalytic properties of the host enzyme. These effects are overall distance-dependent.     

Identification of Nitrotyrosine Containing Peptides using Combined Fractional Diagonal Chromatography (COFRADIC) and Off-Line Nano-LC-MALDI

Protein nitration take place on tyrosine residues under oxidative stress conditions and may influence a number of processes including enzyme activity, protein-protein interactions and phospho-tyrosine signalling pathways. Nitrated proteins have been identified in a number of diseases, however, the study of these proteins has been compromised by the lack of good methods for identifying nitrated proteins, their nitration sites and the level of nitration. Here, we present a method for identification of nitrated peptides that allows the site specific assignment of nitration, is easy to use and reproducible, and opens up for the possibility to quantify the level of nitration of specific peptides as function of different oxidative conditions, namely combined fractional diagonal chromatography (COFRADIC) in combination with off-line nano-LC-MALDI. We identify six nitrated peptides from in vitro nitrated bovine serum albumin and propose that automated COFRADIC using nano-LC and off-line MALDI-MS might be a possibility for identification of tyrosine nitrated proteins and the nitration sites in complex samples.     

Investigation of tyrosine nitration in proteins by mass spectrometry.

In vivo nitration of tyrosine residues is a post-translational modification mediated by peroxynitrite that may be involved in a number of diseases. The aim of this study was to evaluate possibilities for site-specific detection of tyrosine nitration by mass spectrometry. Angiotensin II and bovine serum albumin (BSA) nitrated with tetranitromethane (TNM) were used as model compounds. Three strategies were investigated: (i) analysis of single peptides and protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping, (ii) peptide mass mapping by electrospray ionization (ESI) mass spectrometry and (iii) screening for nitration by selective detection of the immonium ion of nitrotyrosine by precursor ion scanning with subsequent sequencing of the modified peptides. The MALDI time-of-flight mass spectrum of nitrated angiotensin II showed an unexpected prompt fragmentation involving the nitro group, in contrast to ESI-MS, where no fragmentation of nitrated angiotensin II was observed. The ESI mass spectra showed that mono- and dinitrated angiotensin II were obtained after treatment with TNM. ESI-MS/MS revealed that the mononitrated angiotensin II was nitrated on the side-chain of tyrosine. The dinitrated angiotensin II contained two nitro groups on the tyrosine residue. Nitration of BSA was confirmed by Western blotting with an antibody against nitrotyrosine and the sites for nitration were investigated by peptide mass mapping after in-gel digestion. Direct mass mapping by ESI revealed that two peptides were nitrated. Precursor ion scanning for the immonium ion for nitrotyrosine revealed two additional partially nitrated peptides. Based on the studies with the two model compounds, we suggest that the investigation of in vivo nitration of tyrosine and identification of nitrated peptides might be performed by precursor ion scanning for the specific immonium ion at m/z 181.06 combined with ESI-MS/MS for identification of the specific nitration sites.     

Investigation of tyrosine nitration and nitrosylation of angiotensin II and bovine serum albumin with electrospray ionization mass spectrometry

Protein tyrosine nitration is one of the important regulatory mechanisms in various cellular phenomena such as cell adhesion, endo/exo-cytosis of cellular materials, and signal transduction. In the present study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) with a linear ion-trap mass spectrometer was applied for identification of nitrated proteins and localization of the modified tyrosine residues. When angiotensin II(DRVYIHPF) was nitrated in vitro with tetranitromethane (TNM), the mass spectrum showed a shift of +45 Da which corresponded to tyrosine nitration. An additional +29 Da mass shift was also detected by ESI-MS. This differed from nitrated peptide analysis with matrix-associated laser desorption/ionization mass spectrometry (MALDI-MS), which showed oxygen neutral loss from the nitrated tyrosine residues upon laser irradiation. Hence the +29 Da mass shift of the nitrated peptide observed by ESI-MS suggested the introduction of an NO group for nitrosylation of tyrosine residues. To confirm this in vitro nitrosylation on the protein level, bovine serum albumin was in vitro nitrated with TNM and analyzed by ESI-MS/MS. As expected, +29 as well as +45 Da mass shifts were detected, and the +29 Da mass shift was found to correspond to the modification on tyrosine residues by NO. Although the chemical mechanism by which this occurs in ESI-MS is not clear, the +29 Da mass shift could be a new potential marker of nitrosylated peptides.     

Analysis of nitrated proteins and tryptic peptides by HPLC-chip-MS/MS: site-specific quantification,nitration degree,and reactivity of tyrosine residues

The reaction products and pathways of protein nitration were studied with bovine serum albumin (BSA) and ovalbumin (OVA) nitrated by liquid tetranitromethane (TNM) or by gaseous nitrogen dioxide and ozone (NO₂ + O₃). Native and nitrated proteins were enzymatically digested with trypsin, and the tryptic peptides were analyzed by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) using a chip cube nano-flow system (Agilent). Upon nitration by TNM, up to ten of 17 tyrosine residues in BSA and up to five of ten tyrosine residues in OVA could be detected in nitrated form. Upon nitration by NO₂ + O₃, only three nitrated tyrosine residues were found in BSA. The nitration degrees of individual nitrotyrosine residues (ND_Y) were determined by site-specific quantification and compared to the total protein nitration degrees (ND) determined by photometric detection of HPLC-DAD. The slopes of the observed linear correlations between ND_Y and ND varied in the range of ~0.02–2.4 for BSA and ~0.2–1.6 for OVA. They provide information about the relative rates of nitration or reaction probabilities for different tyrosine residues. In BSA, the tyrosine residue Y₁₆₁ was by far most reactive against NO₂ + O₃ and one of the four most reactive positions with regard to nitration by TNM. In OVA, all except one tyrosine residue detected in nitrated form exhibited similar reactivities. The observed nitration patterns show how the site selectivity of protein nitration depends on the nitrating agent, reaction conditions, and molecular structure of the protein (primary, secondary, and tertiary).     

Identification of the tyrosine nitration sites in human endothelial nitric oxide synthase by liquid chromatography-mass spectrometry

The formation of nitric oxide (NO) in biological systems has led to the discovery of a number of post- translational protein modifications that can affect biological conditions such as vasodilation. Studies both from our laboratory and others have shown that beside its effect on cGMP generation from soluble guanylate cylcase, NO can produce protein modifications through both S-nitrosylation of cysteine residues. Previously, we have identified the potential S-nitrosylation sites on endothelial NO synthase (eNOS). Thus, the goal of this study was to further increase our understanding of reactive nitrogen protein modifications of eNOS by identifing tyrosine residues within eNOS that are susceptible to nitration in vitro. To accomplish this, nitration was carried out using tetranitromethane followed by tryptic digest of the protein. The resulting tryptic peptides were analyzed by liquid chromatography/mass spectrometry (LC/MS) and the position of nitrated tyrosines in eNOS were identified. The eNOS sequence contains 30 tyrosine residues and our data indicate that multiple tyrosine residues are capable of being nitrated. We could identify 25 of the 30 residues in our tryptic digests and 19 of these were susceptible to nitration. Interstingly, our data identified four tyrosine residues that can be modified by nitration that are located in the region of eNOS responsible for the binding to heat shock protein 90 (Hsp90), which is responsible for ensuring efficient coupling of eNOS.     

A Non‐damaging Method to Analyze the Configuration and Dynamics of Nitrotyrosines in Proteins

Often, deregulation of protein activity and turnover by tyrosine nitration drives cells toward pathogenesis. Hence, understanding how the nitration of a protein affects both its function and stability is of outstanding interest. Nowadays, most of the in vitro analyses of nitrated proteins rely on chemical treatment of native proteins with an excess of a chemical reagent. One such reagent, peroxynitrite, stands out for its biological relevance. However, given the excess of the nitrating reagent, the resulting in vitro modification could differ from the physiological nitration. Here, we determine unequivocally the configuration of distinct nitrated‐tyrosine rings in single‐tyrosine mutants of cytochrome c. We aimed to confirm the nitration position by a non‐destructive method. Thus, we have resorted to ¹H‐¹⁵N heteronuclear single quantum coherence(HSQC) spectra to identify the ³J(N? H) correlation between a ¹⁵N‐tagged nitro group and the adjacent aromatic proton. Once the chemical shift of this proton was determined, we compared the ¹H‐¹³C HSQC spectra of untreated and nitrated samples. All tyrosines were nitrated at ε positions, in agreement to previous analysis by indirect techniques. Notably, the various nitrotyrosine residues show a different dynamic behaviour that is consistent with molecular dynamics computations.     

Mass spectrometric characterization of photooxidative protein modifications in Arabidopsis thaliana thylakoid membranes

Oxidative and nitrosative stress leaves footprints in the plant chloroplast in the form of oxidatively modified proteins. Using a mass spectrometric approach, we identified 126 tyrosine and 12 tryptophan nitration sites in 164 nitrated proteolytic peptides, mainly from photosystem I (PSI), photosystem II (PSII), cytochrome b(6) /f and ATP-synthase complexes and 140 oxidation products of tyrosine, tryptophan, proline, phenylalanine and histidine residues. While a high number of nitration sites were found in proteins from four photosynthetic complexes indicating that the nitration belongs to one of the prominent posttranslational protein modifications in photosynthetic apparatus, amino acid oxidation products were determined mostly in PSII and to a lower extent in PSI. Exposure of plants to light stress resulted in an increased level of tyrosine and tryptophan nitration and tryptophan oxidation in proteins of PSII reaction center and the oxygen-evolving complex, as compared to low light conditions. In contrast, the level of nitration and oxidation of these amino acid residues strongly decreased for all light-harvesting proteins of PSII under the same conditions. Based on these data, we propose that oxidative modifications of proteins by reactive oxygen and nitrogen species might represent an important regulatory mechanism of protein turnover under light stress conditions, especially for PSII and its antenna proteins.     

Identification of nitrated tyrosine residues of protein kinase G-Iα by mass spectrometry

The nitration of tyrosine to 3-nitrotyrosine is an oxidative modification of tyrosine by nitric oxide and is associated with many diseases, and targeting of protein kinase G (PKG)-I represents a potential therapeutic strategy for pulmonary hypertension and chronic pain. The direct assignment of tyrosine residues of PKG-I has remained to be made due to the low sensitivity of the current proteomic approach. In order to assign modified tyrosine residues of PKG-I, we nitrated purified PKG-Iα expressed in insect Sf9 cells by use of peroxynitrite in vitro and analyzed the trypsin-digested fragments by matrix-assisted laser desorption/ionization–time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Among the 21 tyrosine residues of PKG-Iα, 16 tyrosine residues were assigned in 13 fragments; and six tyrosine residues were nitrated, those at Y71, Y141, Y212, Y336, Y345, and Y567, in the peroxynitrite-treated sample. Single mutation of tyrosine residues at Y71, Y212, and Y336 to phenylalanine significantly reduced the nitration of PKG-Iα; and four mutations at Y71, Y141, Y212, and Y336 (Y4F mutant) reduced it additively. PKG-Iα activity was inhibited by peroxynitrite in a concentration-dependent manner from 30 μM to 1 mM, and this inhibition was attenuated in the Y4F mutant. These results demonstrated that PKG-Iα was nitrated at multiple tyrosine residues and that its activity was reduced by nitration of these residues.     

A non-damaging method to analyze the configuration and dynamics of nitrotyrosines in proteins

Often, deregulation of protein activity and turnover by tyrosine nitration drives cells toward pathogenesis. Hence, understanding how the nitration of a protein affects both its function and stability is of outstanding interest. Nowadays, most of the in vitro analyses of nitrated proteins rely on chemical treatment of native proteins with an excess of a chemical reagent. One such reagent, peroxynitrite, stands out for its biological relevance. However, given the excess of the nitrating reagent, the resulting in vitro modification could differ from the physiological nitration. Here, we determine unequivocally the configuration of distinct nitrated-tyrosine rings in single-tyrosine mutants of cytochrome?c. We aimed to confirm the nitration position by a non-destructive method. Thus, we have resorted to (1)H-(15)N heteronuclear single quantum coherence(HSQC) spectra to identify the (3)J(N?H) correlation between a (15)N-tagged nitro group and the adjacent aromatic proton. Once the chemical shift of this proton was determined, we compared the (1)H-(13)C HSQC spectra of untreated and nitrated samples. All tyrosines were nitrated at ε positions, in agreement to previous analysis by indirect techniques. Notably, the various nitrotyrosine residues show a different dynamic behaviour that is consistent with molecular dynamics computations.     

Mass spectrometry analysis of in vitro nitration of a recombinant human IgG1 monoclonal antibody

Nitration of a recombinant human monoclonal antibody was carried out in vitro by incubating the antibody with the nitrating reagent tetranitromethane (TNM). The susceptible sites of nitration were identified using high-performance liquid chromatography/mass spectrometry (HPLC/MS). In general, tyrosine residues in the variable domains of the antibody are more susceptible to nitration, while tyrosine residues in the constant domains are relatively resistant to nitration. However, one tyrosine residue in the CH1 domain and one tyrosine residue in the CH2 domain are highly susceptible to nitration. Interestingly, the susceptible tyrosine residue in the CH2 domain is followed by the conserved asparagine residue that is glycosylated.     

Electron capture dissociation mass spectrometry of tyrosine nitrated peptides

In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification. Here, we have investigated the electron capture dissociation (ECD) and collision-induced dissociation (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains.     

Mechanistic studies of the isomerization of peroxynitrite to nitrate catalyzed by distal histidine metmyoglobin mutants

Hemoproteins are known to react with the strong nitrating and oxidizing agent peroxynitrite according to different mechanisms. In this article, we show that the iron(iii) forms of the sperm whale myoglobin (sw Mb) mutants H64A, H64D, H64L, F43W/H64L, and H64Y/H93G catalyze the isomerization of peroxynitrite to nitrate. The two most efficient catalysts are H64A (k(cat) = (5.8 +/- 0.1) x 10(6) M(-1) s(-1), at pH 7.5 and 20 degrees C) and H64D metMb (k(cat) = (4.8 +/- 0.1) x 10(6) M(-1) s(-1), at pH 7.5 and 20 degrees C). The pH dependence of the values of k(cat) shows that HOONO is the species which reacts with the heme. In the presence of physiologically relevant concentrations of CO(2) (1.2 mM), the decay of peroxynitrite is accelerated by these metMb mutants via the concurring reaction of HOONO with their iron(iii) centers. Studies in the presence of free added tyrosine show that the metMb mutants prevent peroxynitrite-mediated nitration. The efficiency of the different sw metMb mutants correlates with the value of k(cat). Finally, we show that sw WT-metMb is nitrated to a larger extent than horse heart metMb, a result that suggests that the additional Tyr151 is a site of preferential nitration. Again, the extent of nitration of the tyrosine residues of the metMb mutants correlates with the values of k(cat).     

Conversion of 3‐nitrotyrosine to 3‐aminotyrosine residues facilitates mapping of tyrosine nitration in proteins by electrospray ionization–tandem mass spectrometry using electron capture dissociation

Protein tyrosine nitration is associated with oxidative stress and various human diseases. Tandem mass spectrometry has been the method of choice for the identification and localization of this posttranslational modification to understand the underlying mechanisms and functional consequences. Due to the electron predator effect of the nitro group limiting fragmentation of the peptide backbone, electron‐based dissociation has not been applicable, however, to nitrotyrosine‐containing peptides. A straightforward conversion of the nitrotyrosine to the aminotyrosine residues is introduced to address this limitation. When tested with nitrated ubiquitin and human serum albumin as model proteins in top‐down and bottom‐up approaches, respectively, this chemical derivatization enhanced backbone fragmentation of the corresponding nitroproteins and nitropeptides by electron capture dissociation (ECD). Increased sequence coverage has been obtained by combining in the bottom‐up strategy the conversion of nitrotyrosine to aminotyrosine and introducing, in addition to trypsin, a further digesting enzyme of complementary specificity, when protein nitration was mapped by liquid chromatography–electrospray ionization tandem mass spectrometry using both collision‐induced dissociation (CID) and ECD. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of tyrosine nitration in UCH-L1 and GAPDH

Protein tyrosine nitration is a post-translational modification commonly used as a marker of cellular oxidative stress associated with numerous pathophysiological conditions. We focused on ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1) and glyceraldehyde-3-phosphate (GAPDH) which are high-abundant brain proteins that have been identified to be highly susceptible to oxidative modification. Both UCH-L1 and GAPDH have been linked to the pathogenesis of Alzheimer's and Parkinson's disease, however specific nitration sites have not been elucidated. Identification of specific nitration sites and quantitation of endogenous nitrated proteins are important in correlating this modification to disease pathology. In this study, purified UCH-L1 and GAPDH were nitrated in vitro with peroxynitrite and the presence of nitrated proteins was confirmed by anti-3-nitrotyrosine Western blots. Data-dependent LC-MS/MS analysis identified several distinct tyrosine nitration sites in UCH-L1 (Tyr-80) and GAPDH (Tyr-47, Tyr-92, and Tyr-312). Subsequent validation with synthetic peptides was conducted for selected nitropeptides. An LC-MS/MS method was developed for semi-quantitative determination of the synthetic nitropeptides: KGQEVSPKVY(*) (UCH-L1) and mFQY(*) DSTHGKF (GAPDH). The nitropeptides were detectable in the mid-attomole range and the peak area response was linear over three orders of magnitude. Targeted analysis of endogenous UCH-L1 and GAPDH nitration was then conducted in an in vivo second-hand smoke rat model to evaluate the utility of this approach.     

Identification of nitroglycerin‐induced cysteine modifications of pro‐matrix metalloproteinase‐9

Nitroglycerin (NTG), an important cardiovascular agent, has been shown recently to activate matrix metalloproteinase‐9 (MMP‐9) in biological systems, possibly leading to destabilization of atherosclerotic plaques. The chemical mechanism for this activation, particularly on the cysteine switch of the pro‐form of MMP‐9 (proMMP‐9), has not been investigated and was examined here using nano‐flow liquid chromatography coupled to mass spectrometry. In order to obtain high sequence coverage, two orthogonal enzymes (trypsin and GluC) were employed to digest the protein in parallel. Two complementary activation methods, collision‐induced dissociation (CID) and electron‐transfer dissociation (ETD), were employed for the identification of various modifications. A high‐resolution Orbitrap analyzer was used to enable confident identification. Incubation of NTG with proMMP‐9 resulted in the formation of an unstable thionitrate intermediate and oxidation of the cysteine switch to sulfinic and irreversible sulfonic acid derivatives. The unstable thionitrate modification was confirmed by both CID and ETD in the proteolytic peptides produced by both trypsin and GluC. Incubation of proMMP‐9 with diethylenetriamine NONOate (a nitric oxide donor) led to sulfonic acid formation, but no observable sulfinic acid modification. Extensive tyrosine nitration by NTG was observed at Tyr‐262, in close proximity to an oxidized Cys‐256 of proMMP‐9. The intramolecular interaction between these two residues toward NTG‐induced oxidation was examined using a synthesized peptide representing the sequence in this domain, PWCSTTANYDTDDR, and the modification status was compared against an analog in which Cys was substituted by Ala. We observed a thionitrate product, extensive Cys oxidative modifications and enhanced tyrosine nitration with the Cys peptide but not with the Ala analog. Our results indicated that neighboring Cys and Tyr residues can facilitate each other's oxidation in the presence of NTG. Copyright © 2011 John Wiley & Sons, Ltd.     

Enzyme immunoassays for the investigation of protein nitration by air pollutants

Two enzyme immunoassays have been developed, characterised, and applied to investigate protein nitration in birch pollen extract (BPE) and bovine serum albumin (BSA) samples exposed to air pollutants. The monoclonal antibody CAY-189542 against nitrotyrosine (raised against peroxynitrite-treated keyhole limpet hemocyanine) was characterised in an indirect competitive assay (affinity and cross-reactivities) and applied in a new one-sided enzyme immunoassay for nitrated proteins. The one-sided assay was calibrated against a nitrated BSA standard with an average of 14 nitrotyrosine residues per molecule (nitro-(14)-BSA; detection limit 8.3 pmol L(-1)), and the sensitivity of the test was found to be significantly enhanced by a multivalent binding mode of the monoclonal antibody (bonus effect of multivalency). The same antibody and a polyclonal antibody against Bet v 1, the most prominent birch pollen allergen, were used in a new sandwich immunoassay for specific determination of nitrated Bet v 1. This assay was calibrated against a nitrated Bet v 1 standard with an average of 3 nitrotyrosine residues per molecule (nitro-(3)-Bet v 1; detection limit 0.2 nmol L(-1)). Bet v 1 and BSA exposed to polluted urban outdoor air and to synthetic gas mixtures containing NO2 and O3 at atmospherically relevant concentration levels were found to be efficiently nitrated within hours to days. Pronounced correlations of nitro-(14)-BSA equivalent concentrations with exposure time and with nitro-(3)-Bet v 1 equivalent concentrations in nitrated BPE samples were observed. Test experiments indicated that the efficiency of protein nitration was strongly enhanced by reactive species formed upon interaction of NO2 with O3 and H2O (e.g. NO3 and HNO3). Potential implications of protein nitration by air pollutants are outlined and discussed.     

Quantification of nitrotyrosine in nitrated proteins

For kinetic studies of protein nitration reactions, we have developed a method for the quantification of nitrotyrosine residues in protein molecules by liquid chromatography coupled to a diode array detector of ultraviolet-visible absorption. Nitrated bovine serum albumin (BSA) and nitrated ovalbumin (OVA) were synthesized and used as standards for the determination of the protein nitration degree (ND), which is defined as the average number of nitrotyrosine residues divided by the total number of tyrosine residues in a protein molecule. The obtained calibration curves of the ratio of chromatographic peak areas of absorbance at 357 and at 280 nm vs. nitration degree are nearly the same for BSA and OVA (relative deviations <5%). They are near-linear at low ND (< 0.1) and can be described by a second-order polynomial fit up to $ {\hbox{ND}} = 0.5\left( {{R^2} > 0.99} \right) $ {\hbox{ND}} = 0.5\left( {{R^2} > 0.99} \right) . A change of chromatographic column led to changes in absolute peak areas but not in the peak area ratios and related calibration functions, which confirms the robustness of the analytical method. First results of laboratory experiments confirm that the method is applicable for the investigation of the reaction kinetics of protein nitration. The main advantage over alternative methods is that nitration degrees can be efficiently determined without hydrolysis or digestion of the investigated protein molecules.     

Characterization of Tyrosine Nitration and Cysteine Nitrosylation Modifications by Metastable Atom-Activation Dissociation Mass Spectrometry

The fragmentation behavior of nitrated and S-nitrosylated peptides were studied using collision induced dissociation (CID) and metastable atom-activated dissociation mass spectrometry (MAD-MS). Various charge states, such as 1+, 2+, 3+, 2–, of modified and unmodified peptides were exposed to a beam of high kinetic energy helium (He) metastable atoms resulting in extensive backbone fragmentation with significant retention of the post-translation modifications (PTMs). Whereas the high electron affinity of the nitrotyrosine moiety quenches radical chemistry and fragmentation in electron capture dissociation (ECD) and electron transfer dissociation (ETD), MAD does produce numerous backbone cleavages in the vicinity of the modification. Fragment ions of nitrosylated cysteine modifications typically exhibit more abundant neutral losses than nitrated tyrosine modifications because of the extremely labile nature of the nitrosylated cysteine residues. However, compared with CID, MAD produced between 66% and 86% more fragment ions, which preserved the labile –NO modification. MAD was also able to differentiate I/L residues in the modified peptides. MAD is able to induce radical ion chemistry even in the presence of strong radical traps and therefore offers unique advantages to ECD, ETD, and CID for determination of PTMs such as nitrated and S-nitrosylated peptides.     

Analysis of Redox Activity of Proteins on the Carbon Screen Printed Electrodes

Direct redox activity of different proteins was investigated on the surface of carbon screen printed electrodes (SPE). The signal attributed to the electrochemical oxidation of amino acid residues (cysteine (Cys), tryptophan (Trp) and tyrosine (Tyr)) was registered at E_max from 0.6 to 0.7 V (vs. Ag/AgCl). Based on the difference in the redox behavior of L ‐tyrosine and 3‐nitro‐L ‐tyrosine, the selective electrochemical detection of native and nitrated albumins was demonstrated. It was shown that the electrochemical signal correlated with the surface density of electroactive amino acid residues on the protein molecule. A simple electrochemical method for the total protein analysis was proposed.