Katalin Prokai-Tatrai

Identification of carbonylation sites in apomyoglobin after exposure to 4-hydroxy-2-nonenal by solid-phase enrichment and liquid chromatography-electrospray ionization tandem mass spectrometry.

Identification of protein carbonylation because of covalent attachment of a lipid peroxidation end-product was performed by combining proteolytic digestion followed by solid-phase hydrazide enrichment and liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using both collision-induced dissociation (CID) and electron capture dissociation (ECD). To evaluate this approach, we selected apomyoglobin and 4-hydroxy-2-nonenal (4-HNE) as a model protein and a representative end-product of lipid peroxidation, respectively. Although the characteristic elimination of 4-HNE (156 Da) in CID was found to serve as a signature tag for the modified peptides, generation of nearly complete fragment ion series because of efficient peptide backbone cleavage (in most cases over 75%) and the capability to retain the labile 4-HNE moiety of the tryptic peptides significantly aided the elucidation of primary structural information and assignment of exact carbonylation sites in the protein, when ECD was employed. We have concluded that solid-phase enrichment with both CID- and ECD-MS/MS are advantageous during an in-depth interrogation and unequivocal localization of 4-HNE-induced carbonylation of apomyoglobin that occurs via Michael addition to its histidine residues.

Selective Chemoprecipitation and Subsequent Release of Tagged Species for the Analysis of Nitropeptides by Liquid Chromatography–Tandem Mass Spectrometry

Tyrosine nitration is a low-abundance post-translational protein modification that requires appropriate enrichment techniques to enable proteomic analyses. We report a simple yet highly specific method to enrich nitropeptides by chemoprecipitation involving only two straightforward chemical modifications of the nitropeptides before capturing the obtained derivatives with a strategically designed solid-phase active ester reagent. Specifically, capping of the aliphatic amines in the peptides is done first by reductive methylation to preserve the charge state of peptides for electrospray ionization mass spectrometric analysis, followed by reduction of nitrotyrosines to the corresponding aminotyrosines. These peptides are then immobilized on the solid-phase active ester reagent, whereas other peptides carrying no free amino groups are separated from the immobilized species by thoroughly washing the beads from which the tagged peptide derivatives can easily be released by acid-catalyzed hydrolysis at room temperature. The benefits of selective enrichment from a matrix of unmodified peptides for liquid chromatography-tandem mass spectrometry are demonstrated on three synthetic nitropeptides that are nitrated fragments of biologically relevant proteins. Identification of several in vitro nitrated human plasma proteins, also implicated under various pathological processes, by database searches from the enriched and tagged tryptic nitropeptides is presented as a practical application. We also show that converting the nitro-group to the small 4-formylbenzoylamido tag does not significantly alter fragmentation properties upon collision-induced dissociation compared with those of the native nitropeptides, and at the same time this derivatization actually improves electron capture dissociation due to conversion of the electron-predator nitro-group to this novel tag.

Separation of dansylated 17β-estradiol, 17α-estradiol and estrone on a single HPLC column for simultaneous quantitation by LC-MS/MS

AbstractWe show here that baseline separation of dansylated estrone, 17β-estradiol, and 17α-estradiol can be done, contrary to previous reports, within a short run time on a single RP-LC analytical column packed with particles bonded with phenyl-hexyl stationary phase. The chromatographic method coupled with isotope dilution tandem MS offers a simple assay enabling the simultaneous analysis of these analytes. The method employs 13C-labeled estrogens as internal standards to eliminate potential matrix effects arising from the use of deuterated estrogens. The assay also offers adequate accuracy and sensitivity to be useful for biological samples. The practical applicability of the validated method is demonstrated by the quantitative analyses of in vivo samples obtained from rats treated with Premarin®. FigureQuantification of estrogens from rat samples by LC–MS/MS

The prodrug DHED selectively delivers 17β-estradiol to the brain for treating estrogen-responsive disorders

The bioprecursor prodrug 10β, 17β-dihydroxyestra-1,4-dien-3-one (DHED) allows 17β-estradiol to form in the female brain for neuroprotection and to treat neurological and psychiatric symptoms of menopause. Estrogen prodrug protects the brain Although estrogen is considered to heal the brain of many neurological and psychiatric symptoms, targeting the hormone to the brain only—and avoiding activity in other tissues—is no trivial feat. Prokai et al. discovered a “prodrug,” called DHED (10β, 17β-dihydroxyestra-1,4-dien-3-one), that is selectively converted to one main estrogen, 17β-estradiol, only in the brain. Similar to 17β-estradiol itself, DHED protected menopausal female rodents from neurological symptoms of estrogen deprivation, and also provided neuroprotection to rats after stroke, but without the negative systemic (specifically uterotrophic and cancerous) effects of the free hormone. With an efficacious and safe profile in vivo, this prodrug has the opportunity to provide positive estrogenic effects on the brain and relieve symptoms in patients with a broad range of central nervous system diseases. Many neurological and psychiatric maladies originate from the deprivation of the human brain from estrogens. However, current hormone therapies cannot be used safely to treat these conditions commonly associated with menopause because of detrimental side effects in the periphery. The latter also prevents the use of the hormone for neuroprotection. We show that a small-molecule bioprecursor prodrug, 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED), converts to 17β-estradiol in the brain after systemic administration but remains inert in the rest of the body. The localized and rapid formation of estrogen from the prodrug was revealed by a series of in vivo bioanalytical assays and through in vivo imaging in rodents. DHED treatment efficiently alleviated symptoms that originated from brain estrogen deficiency in animal models of surgical menopause and provided neuroprotection in a rat stroke model. Concomitantly, we determined that 17β-estradiol formed in the brain from DHED elicited changes in gene expression and neuronal morphology identical to those obtained after direct 17β-estradiol treatment. Together, complementary functional and mechanistic data show that our approach is highly relevant therapeutically, because administration of the prodrug selectively produces estrogen in the brain independently from the route of administration and treatment regimen. Therefore, peripheral responses associated with the use of systemic estrogens, such as stimulation of the uterus and estrogen-responsive tumor growth, were absent. Collectively, our brain-selective prodrug approach may safely provide estrogen neuroprotection and medicate neurological and psychiatric symptoms developing from estrogen deficiency, particularly those encountered after surgical menopause, without the adverse side effects of current hormone therapies.

Capture of the volatile carbonyl metabolite of flecainide on 2,4-dinitrophenylhydrazine cartridge for quantitation by stable-isotope dilution mass spectrometry coupled with chromatography.

Carbonyl compounds are common byproducts of many metabolic processes. These volatile chemicals are usually derivatized before mass spectrometric analysis to enhance the sensitivity of their detections. The classically used reagent for this purpose is 2,4-dinitrophenylhydrazine (DNPH) that forms the corresponding hydrazones. When DNPH is immobilized on specific cartridges it permits solvent-free collection and simultaneous derivatization of aldehydes and ketones from gaseous samples. The utility of this approach was tested by assembling a simple apparatus for the in vitro generation of trifluoroacetaldehyde (TFAA) and its subsequent capture on the attached DNPH cartridge. TFAA was generated via cytochrome P450-catalyzed dealkylation of flecainide, an antiarrhythmic agent, in pooled human liver microsomes. Stable-isotope dilution mass spectrometry coupled with GC and LC using negative chemical ionization (NCI) and electrospray ionization (ESI) was evaluated for quantitative analyses. To eliminate isotope effects observed with the use of deuterium-labeled DNPH, we selected its (15)N(4)-labeled analog to synthesize the appropriate TFAA adduct, as internal standard. Quantitation by GC-NCI-MS using selected-ion monitoring outperformed LC-ESI-MS methods considering limits of detection and linearity of the assays. The microsomal metabolism of 1.5 μmol of flecainide for 1.5h resulted in 2.6 ± 0.5 μg TFAA-DNPH, corresponding to 9.3 ± 1.7 nmol TFAA, captured by the cartridge.

Prodrugs of Thyrotropin-Releasing Hormone and Related Peptides as Central Nervous System Agents

Prodrug design for brain delivery of small- and medium-sized neuropeptides was reviewed, focusing on thyrotropin-releasing hormone and structurally related peptides as examples. We have summarized our most important advances in methodology, as well as assessed the benefits and limitations of bioreversible chemical manipulation techniques to achieve targeting of the parent molecules into the central nervous system. The value of prodrug-amenable analogues as potential drug-like central nervous systems agents was highlighted.

Relative quantitation of protein nitration by liquid chromatography–mass spectrometry using isotope-coded dimethyl labeling and chemoprecipitation

Protein nitration has been recognized as an important biomarker for nitroxidative stress associated with various diseases. While identification of protein targets for nitration is important, its quantitative profiling also is necessary to understand the biological impact of this low-abundance posttranslational modification. We have previously reported an efficient and straightforward enrichment method for nitropeptides to reduce sample complexity and permit unambiguous site-specific identifications by LC-MS analyses. This approach relies on two chemical derivatization steps: specifically reductive methylation of aliphatic amines and, then, conversion of nitrotyrosines to the corresponding aminotyrosines before their selective capture by a solid-phase reagent we introduced previously. Hence, the method inherently offers the opportunity for relative quantitation of nitropeptides by using isotopic variants of formaldehyde for reductive methylation. This simple method was tested via LC-MS analyses of differently N-methylated nitropeptides and nitroubiquitin as a model nitroprotein enriched from human serum albumin digest and from human plasma, respectively.

Prodrug Design for Brain Delivery of Small- and Medium-Sized Neuropeptides

The blood-brain barrier (BBB) represents multiple barriers for drug delivery from the circulation. Peptides potentially useful to treat maladies of the brain are especially limited in their ability to cross the BBB due to several shortcomings. Specific delivery strategies have been conceived to outwit the BBB to target neuropeptides into the brain. It should be noted, however, that no unified method is possible for true brain-targeting of these fascinating biomolecules due to their structural features, properties, and intricate interplays among factors governing their entrance into and retention within the brain. In most brain-targeting prodrug approaches, a lipophilic and bioreversible moiety(ies) is covalently attached to the peptide that results in the complete loss of the innate biological activity of the parent peptide (prodrugs are inactive per definition) but significantly improves brain uptake and metabolic stability in the plasma and the interstitial fluid. Once the peptide prodrug has crossed the BBB, specific enzymes liberate the parent agent from its prodrug in the brain. To illustrate the applicability of the prodrug strategy for brain delivery of small neuropeptides, pGlu-Glu-Pro-NH(2), [Glu(2)TRH], a thyrotropin-releasing hormone (TRH) analogue with a vast array of central activities, was chosen as an example. An ester prodrug provided significantly improved brain delivery compared to the unmodified parent peptide. The synthesis, in vitro and in vivo evaluations of this prodrug as specific examples are given for typical exploratory prodrug validation.

Phenolic Compounds Protect Cultured Hippocampal Neurons against Ethanol-Withdrawal Induced Oxidative Stress

Ethanol withdrawal is linked to elevated oxidative damage to neurons. Here we report our findings on the contribution of phenolic antioxidants (17β-estradiol, p-octyl-phenol and 2,6-di-tert-butyl-4-methylphenol) to counterbalance sudden ethanol withdrawal-initiated oxidative events in hippocampus-derived cultured HT-22 cells. We showed that ethanol withdrawal for 4 h after 24-h ethanol treatment provoked greater levels of oxidative damage than the preceding ethanol exposure. Phenolic antioxidant treatment either during ethanol exposure or ethanol withdrawal only, however, dose-dependently reversed cellular oxidative damage, as demonstrated by the significantly enhanced cell viability, reduced malondialdehyde production and protein carbonylation, compared to untreated cells. Interestingly, the antioxidant treatment schedule had no significant impact on the observed neuroprotection. In addition, the efficacy of the three phenolic compounds was practically equipotent in protecting HT-22 cells in spite of predictions based on an in silico study and a cell free assay of lipid peroxidation. This finding implies that free-radical scavenging may not be the sole factor responsible for the observed neuroprotection and warrants further studies to establish, whether the HT-22 line is indeed a suitable model for in vitro screening of antioxidants against EW-related neuronal damage.

Comparison of estrogen-derived ortho-quinone and para-quinol concerning induction of oxidative stress

Ortho-quinones formed from catechol estrogens are considered prooxidants due to the production of superoxide radical anions through redox cycling via semiquinones. Para-quinols have been identified as novel metabolites of and as the major products of hydroxyl-radical scavenging by estrogens. Cycling of these compounds has also been discovered, because they are converted back to the parent estrogen via reductive aromatization in vitro and in vivo. We hypothesized that, unlike ortho-quinones, para-quinols do not induce oxidative stress due to this cycling. Like the estrogen itself, the 17beta-estradiol-derived para-quinol (10beta,17beta-dihydroxyestra-1,4-diene-3-one) did not induce oxidative stress, as the rate of hydrogen peroxide production during the incubations of the compounds in various tissue homogenates was not significantly different from that of the control experiments performed without the addition of a test compound. We also confirmed that the estrogen metabolite estra-1,5(10)-dien-3,4,17-trione (estrone 3,4-quinone) was a profound prooxidant due to redox cycling, especially in uterine tissue. Therefore, we concluded that para-quinols do not induce oxidative stress.