The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice.

BACKGROUND: Ara h 2 and Ara h 6, co-purified together in a 13-25 kD fraction (Ara h 2/6; 20 kD fraction) on gel filtration chromatography, account for the majority of effector activity in a crude peanut extract (CPE) when assayed with RBL SX-38 cells sensitized with IgE from human peanut allergic sera. OBJECTIVES: To determine if Ara h 2/6 are the primary peanut allergens responsible for allergic reactions in vivo and to determine if Ara h 2/6 would be sufficient to prevent allergic reactions to a complete CPE. METHODS: An oral sensitization mouse model of peanut allergy was used to assess the activity of Ara h 2/6 (20 kD) and CPE without the 20 kD fraction (CPE w/o 20 kD) for allergic provocation challenge and immunotherapy. The activity of these preparations was also tested in an assay of histamine release from human basophils in whole blood. RESULTS: Compared with mice challenged with control CPE, mice challenged with CPE w/o 20 kD experienced reduced symptoms (P < 0.05) and a smaller decrease in body temperature (P < 0.01). Results with the basophil histamine release assay corroborated these findings (P < 0.01). The mouse model was also used to administer Ara h 2/6 (20 kD) in an immunotherapy protocol, in which peanut-allergic mice treated with the 20 kD fraction experienced significantly reduced symptoms, changes in body temperature, and mast cell protease (MMCP-1) release compared with placebo (P < 0.01 for all parameters). Importantly, immunotherapy with the 20 kD fraction was just as effective as treatment with CPE, whereas CPE w/o 20 kD was significantly less effective for higher dose peanut challenges. CONCLUSIONS AND CLINICAL RELEVANCE: Ara h 2/6 are the most potent peanut allergens in vivo and can be used to desensitize peanut-allergic mice. These results have potential implications for clinical research in the areas of diagnosis and immunotherapy for peanut allergy.

Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants.

RATIONALE: An important property of allergens is their ability to cross-link IgE and activate mast cells and basophils. The effector activity of peanut allergens has not been well characterized. METHODS: Crude extracts of fresh peanut flour were fractionated by gel filtration. Effector function was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from individual sera and from pools of sera of peanut-allergic donors. RESULTS: Following gel filtration, 75 +/- 7% of the applied protein and 76 +/- 16% (n=3) of the applied activity (assayed with a pool of 11 sera) were recovered in the resultant fractions. The majority (85 +/- 2%; n=3) of the recovered activity resided in a fraction with a theoretical average molecular weight of approximately 20 kDa and a range of 13-25 kDa. When all the individual fractions were recombined, the measured activity was similar to that of the original extract [140 +/- 43% when measured with a pool of serum (n=2) and 66 +/- 7% when measured with individual sera (n=4)]; when all individual fractions excluding the 20 kDa fraction were recombined, the measured activity was only 8 +/- 2% (n=2) of the original extract when assayed with the serum pool and 10 +/- 4% (n=3) when assayed with the individual sera. Two-dimensional gel electrophoresis of this biologically active fraction revealed >60 protein spots. Analysis of 50 of the most prominent spots by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and of the full mixture by automated tandem mass spectrometry coupled to online capillary liquid chromatography revealed that >97% of the protein mass consisted of Ara h 2.0101, Ara h 2.0201, Ara h 6 isoforms, and variants of these proteins. CONCLUSIONS: Ara h 2 and Ara h 6 account for the majority of the effector activity found in a crude peanut extract.

Contribution of Ara h 2 to peanut-specific, immunoglobulin E-mediated, cell activation.

BACKGROUND: Ara h 2 is a potent peanut allergen but its contribution to the ability of a crude peanut extract (CPE) to cross-link IgE and activate mast cells has not been rigorously evaluated. OBJECTIVE: To measure the contribution that Ara h 2 makes to the effector function of a CPE. METHODS: Ara h 2 was specifically removed from a CPE as demonstrated by immunoblots, 2D gels, and an inhibitory ELISA. Functional assays of sham-treated and Ara h 2-depleted CPEs were performed with RBL SX-38 cells sensitized with IgE from highly peanut-allergic subjects and with naturally sensitized basophils. RESULTS: Depletion of approximately 99% of the Ara h 2 from the CPE led to an increase in the concentration of the CPE necessary to give 50% of maximal degranulation (EC50) of the SX-38 cells following sensitization with sera that contain anti-Ara h 2 IgE. Assays with a pool of 10 sera showed a small but significant increase in the EC50 following depletion of Ara h 2 (1.65+/-0.15-fold; P<0.05) and assays of seven individual sera showed a similar increase in the average EC50 (1.7+/-0.2-fold; P<0.02). The percent of the anti-peanut IgE that binds Ara h 2 correlated with an increase in the EC50 of the CPE following depletion of Ara h 2 (r=0.83; P<0.02). On the other hand, data from three of these patients studied with a basophil histamine release assay did not show a significant effect of depletion of Ara h 2. CONCLUSION: Based on its ability to cross-link IgE effectively, Ara h 2 is clearly an important peanut allergen. Its ability to cross-link IgE effectively from a specific serum is related to the proportion of anti-Ara h 2 in that serum but Ara h 2 does not account for a majority of the effector activity of the CPE for any of the sera studied.

Proteomic analysis of multiple sclerosis cerebrospinal fluid.

Two-dimensional gel electrophoresis and peptide mass fingerprinting were used to identify proteins in cerebrospinal fluid (CSF) pooled from three patients with multiple sclerosis (MS) and in CSF pooled from three patients with non-MS inflammatory central nervous system (CNS) disorders. Resolution of CSF proteins on three pH gradients (3-10, 4-7 and 6-11) enabled identification of a total of 430 spots in the MS CSF proteome that represented 61 distinct proteins. The gels containing MS CSF revealed 103 protein spots that were not seen on control gels. All but four of these 103 spots were proteins known to be present in normal human CSF. The four exceptions were: CRTAC-IB (cartilage acidic protein), tetranectin (a plasminogen-binding protein), SPARC-like protein (a calcium binding cell signalling glycoprotein), and autotaxin t (a phosphodiesterase). It remains unknown whether these four proteins are related to the cause and pathogenesis of MS.

A review of approaches to the analysis of 3-nitrotyrosine.

Our understanding of in vivo tyrosine nitration has been confounded by problems associated with the analytical approaches that have been employed to quantify 3-nitroyrosine (3-NT). Trace analysis is a demanding task under the best of circumstances, but 3-NT offers some special concerns. This review examines some of these concerns and discusses approaches to ensuring that carefully validated analytical data are generated.

Characterization of a mechanism-based inhibitor of NAD(P)H:quinone oxidoreductase 1 by biochemical, X-ray crystallographic, and mass spectrometric approaches.

We report the characterization of 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936) as a mechanism-based inhibitor of NQO1. Inactivation of NQO1 by ES936 was time- and concentration-dependent and required the presence of a pyridine nucleotide cofactor consistent with a need for metabolic activation. That ES936 was an efficient inhibitor was demonstrated in these studies by the low partition ratio (1.40 +/- 0.03). The orientation of ES936 in the active site of NQO1 was examined by X-ray crystallography and found to be opposite to that observed for other indolequinones acting as substrates. ES936 was oriented in such a manner that, after enzymatic reduction and loss of a nitrophenol leaving group, a reactive iminium species was located in close proximity to nucleophilic His 162 and Tyr 127 and Tyr 129 residues in the active site. To determine if ES936 was covalently modifying NQO1, ES936-treated protein was analyzed by electrospray ionization liquid chromatography/mass spectrometry (ESI-LC/MS). The control NQO1 protein had a mass of 30864 +/- 6 Da (n = 20, theoretical, 30868.6 Da) which increased by 217 Da after ES936 treatment (31081 +/- 7 Da, n = 20) in the presence of NADH. The shift in mass was consistent with adduction of NQO1 by the reactive iminium derived from ES936 (M + 218 Da). Chymotryptic digestion of the protein followed by LC/MS analysis located a tetrapeptide spanning amino acids 126-129 which was adducted with the reactive iminium species derived from ES936. LC/MS/MS analysis of the peptide fragment confirmed adduction of either Tyr 127 or Tyr 129 residues. This work demonstrates that ES936 is a potent mechanism-based inhibitor of NQO1 and may be a useful tool in defining the role of NQO1 in cellular systems and in vivo.

Oral estrogen antagonizes the metabolic actions of growth hormone in growth hormone-deficient women.

We have determined whether oral estrogen reduces the biological effects of growth hormone (GH) in GH-deficient (GHD) women compared with transdermal estrogen treatment. In two separate studies, eight GHD women randomly received either oral or transdermal estrogen for 8 wk before crossing over to the alternate route of administration. The first study assessed the effects of incremental doses of GH (0.5, 1.0, 2.0 IU/day for 1 wk each) on insulin-like growth factor I (IGF-I) levels during each estrogen treatment phase. The second study assessed the effects of GH (2 IU/day) on lipid oxidation and on protein metabolism using the whole body leucine turnover technique. Mean IGF-I level was significantly lower during oral estrogen treatment (P < 0.05) and rose dose dependently during GH administration by a lesser magnitude (P < 0.05) compared with transdermal treatment. Postprandial lipid oxidation was significantly lower with oral estrogen treatment, both before (P < 0.05) and during (P < 0.05) GH administration, compared with transdermal treatment. Protein synthesis was lower during oral estrogen both before and during GH administration (P < 0.05). Oral estrogen antagonizes several of the metabolic actions of GH. It may aggravate body composition abnormalities already present in GHD women and attenuate the beneficial effects of GH therapy. Estrogen replacement in GHD women should be administered by a nonoral route.

Measurements of protein carbonyls, ortho- and meta-tyrosine and oxidative phosphorylation complex activity in mitochondria from young and old rats.

Mitochondrial bioenergetic function is often reported to decline with age and the accumulation of oxidative damage is thought to contribute. However, there are considerable uncertainties about the amount and significance of mitochondrial oxidative damage in aging. We hypothesized that, as radical production in mitochondria is greater than the rest of the cell, protein oxidative damage should accumulate more in mitochondria than the cytoplasm, and that this relative accumulation should increase with age. To test these hypotheses we measured the accumulation of three markers of protein oxidative damage in liver, brain, and heart from young and old rats. Ortho- and meta-tyrosine levels in protein hydrolysates were measured by a gas chromatography/mass spectrometry assay, and protein carbonyl content was determined by ELISA. Using these assays we found no evidence for increased protein oxidative damage in mitochondria relative to the cytosol. Most increases found in protein oxidative damage on aging were modest for all three tissues and there was no consistent pattern of increased oxidative damage in mitochondrial proteins on aging. Mitochondrial oxidative phosphorylation complex activities were also assessed revealing 39-42% decreases in F0F1--ATP synthase activity in liver and heart on aging, but not in other oxidative phosphorylation complexes. These findings have implications for the contribution of mitochondrial oxidative damage and dysfunction to aging.

Fluorometric and mass spectrometric analysis of nonenzymatic glycosylated albumin.

Albumin is the major transport protein in blood and intramolecular movement contributes to this function. Nonenzymatic glycosylation (NEG) of albumin occurs in diabetes and, in this study, fluorometric methods were used to determine the effect of increasing levels of NEG upon intramolecular movement in human serum albumin. Low levels of NEG significantly reduced and left-shifted Trp fluorescence, reduced quenching by acrylamide and inhibited penetration of bis-ANS, while these changes became only modestly more pronounced at higher levels of NEG. Mass spectrometry of tryptic and CNBr NEG-HSA fragments identified potential glycosylation sites and demonstrated only late glycosylation of the C- and N-terminal regions of the protein. Similar changes in diabetes may contribute to altered transport function in these patients.

Nicotine and cotinine adducts of a melanin intermediate demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Pigmentation is a major factor in the incorporation of many drugs into hair. In an attempt to elucidate potential mechanisms of drug-melanin interaction, melanin was synthesized in vitro in the presence of nicotine, which we have shown to have a substantial interaction with melanin, and cotinine, a primary nicotine metabolite. L-DOPA, a precursor of eumelanin, was oxidized and oligomerized with tyrosinase. Nicotine, cotinine, and/or their deuterated analogues were added to the oligomerization reaction mixture in a 10:1 L-DOPA:drug ratio. A black precipitate formed within 60 min. Aliquots were removed from the incubation mixture at 60, 120, and 360 min. MALDI-TOF MS determinations were carried out on each sample to provide a mean and standard error for the masses of interest. Internal calibration allowed accurate mass measurement of the products. A careful comparison of the spectra of samples prepared both with and without drug indicated the presence of masses corresponding to the protonated drug, melanin oligomers, and nicotine or continine adducts of the monomeric melanin intermediate dopaquinone (DOPAQ). Additional support for the presence of drug-melanin adducts was provided by employing deuterated analogues of nicotine and L-DOPA in the reaction and observing that the masses shifted accordingly. Structures of the adducts were further confirmed by select ion gating and postsource decay analysis.

Quantification of 3-nitrotyrosine in biological tissues and fluids: generating valid results by eliminating artifactual formation.

Reactive nitrogen species such as peroxynitrite can nitrate specific amino acids, whether free or protein bound, and 3-nitrotyrosine is believed to be one marker of this reaction. To examine the significance of this pathway in biological systems we have developed an accurate, sensitive, and specific assay for 3-nitrotyrosine based on combined liquid chromatography tandem mass spectrometry. Our approach allowed simultaneous analysis of both tyrosine and 3-nitrotyrosine and employs isotopomer standards (i.e., [15N1, 13C9]-tyrosine and [13C6]-3nitrotyrosine). Calibration curves were linear (r2 = 0.999) across the range 0.5-100 pg/microL (i.e., 2.2-442 fmol/microL), and the detection limit for standard samples was 0.5 pg/microL (2.2 fmol/microL, or 10 fmol on column; S/N = 5) or 1 pg/microL (4.4 fmol/microL) for extracted (biological) samples. As a component of this study we have undertaken an extensive investigation of artifactual formation of 3-nitrotyrosine under conditions that exist during sample extraction and derivatization. Our studies show that under appropriate conditions (low pH, elevated temperatures, and in the presence of a vast excess of the two substrates, tyrosine and the nitrate anion), 3-nitrotyrosine can readily be formed as an artifact.

A covalent thymine-tyrosine adduct involved in DNA-protein crosslinks: synthesis, characterization, and quantification.

A thymine-tyrosine adduct, (3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl]-L-tyrosine), was synthesized using a simple, single-step condensation between 5-(hydroxymethyl)uracil and L-tyrosine. This approach provides access to useful quantities (mg-g) of analytically pure reference material, and with minor modification, to stable isotope-labeled analogues (isotopomers). With reference material and a suitable internal standard available, isotope-dilution liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/MS/MS) was used to assay the adduct in a model system purged of oxygen, i.e., a gamma-irradiated N2O-saturated aqueous solution of thymine and tyrosine. The convenient synthetic route to standards and the method for quantification reported here will prove useful in assessing the significance of the adduct in biological systems. These studies also highlight the potential for artefactual adduct formation if the appropriate substrates are present under acidic conditions.

Trifluoroacetic anhydride-catalyzed nitration of toluene as an approach to the specific analysis of nitrate by gas chromatography-mass spectrometry.

The nitration of aromatic compounds by electrophilic substitution is often utilized in analyses of nitrate concentrations in physiological samples by gas chromatographic methods. Problems associated with the use of concentrated sulfuric acid, which is normally used to catalyze this reaction, led us to investigate an alternative method. We describe here a facile GC/MS assay for nitrate in plasma or urine samples which takes advantage of the ability of trifluoroacetic anhydride (TFAA) to catalyze the nitration of aromatics. Toluene, utilized as both reaction solvent and electrophile, was shown to react with nitrate in the presence of TFAA to quantitatively produce the three nitrotoluene isomers (ratio o-:m:p-, approx 57:3:40). Following the incorporation of 15N-labeled nitrate as internal standard, nitrotoluene was quantified using GC/MS by analysis of the selected the ion pairs m/z 120 and 121 (M+ -OH) for the o-isomer or m/z 137 and 138 (molecular ion, M+) for the p-isomer. The limit of detection for nitrate after TFAA-catalyzed conversion to nitrotoluene was less than 100 fmol on column (s/n; 40:1). The TFAA-based GC/MS assay was compared with that utilizing the usual catalyst, concentrated sulfuric acid. With the exception of samples containing nitroarginine analogues, good correlation was found for urine or plasma samples analyzed using either a standard sulfuric acid-catalyzed method or the TFAA-catalyzed procedure. Nitroarginine analogues, which can be present in samples following their use as nitric oxide synthase inhibitors, did not decompose under the conditions of the TFAA-catalyzed assay and, hence, do not give rise to significant interference with nitrate analysis in this procedure. In contrast, catalytic sulfuric acid caused nitroarginine analogues to decompose (essentially quantitatively) and cause spuriously high nitrate levels in samples. The use of TFAA as a catalyst for the nitration of toluene enables a facile and sensitive GC/MS analysis for nitrate which offers improved safety and sample integrity.

Amino Acid analysis of peptides and proteins on the femtomole scale by gas chromatography/mass spectrometry.

Amino acid analysis (AAA) is a useful aid in protein chemistry, but its routine application is limited by a modest limit of detection. Typically, 10 pmol of material is required, but even at this level the reproducibility can be poor. We have employed isotope dilution gas chromatography electron capture negative ionization mass spectrometry (GC/ECNI/MS) to provide accurate and reliable data on less than 100 fmol of material. Precision and accuracy are good, and all 20 non-hydrolyzed amino acids can be determined in this manner. The protein is hydrolyzed (HCl), and then a cocktail, composed of all 20 amino acids as stable isotope-labeled forms (i.e., (13)C and (2)H), is added. The mixture of protein-derived and stable isotope-labeled amino acids is then converted to volatile electron-capturing derivatives with a multistep approach employing heptafluorobutyric anhydride (HFBA), pentafluorobenzyl bromide (PFBBr), and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). These derivatives are then injected directly into the GC/MS system. Groups of selected ions, characteristic of each derivatized amino acid, are thereafter monitored at appropriate time intervals. The ratios of the ion current for the selected ions for the native amino acid and its labeled form are determined and converted to absolute amounts of the native amino acids in the protein hydrolyzate by reference to standard samples prepared at the time of the analysis.

Peroxynitrite-mediated nitration of peptides: characterization of the products by electrospray and combined gas chromatography-mass spectrometry.

Peroxynitrite (ONOO-) can react with a wide range of biomolecules resulting in peroxidation, oxidation, and/or nitration and as a consequence cause their inactivation. In this study mass spectrometry (MS) combined with both liquid (LC) and gas chromatography (GC) has been employed to identify the products formed following ONOO- treatment of three peptides at physiological pH: leucine-enkephalin (YGGFL), V3 loop (GPGRAF), and LVV-hemorphin7 (LVVYPWTQRF). LC-MS analysis of leucine-enkephalin following ONOO treatment indicated the formation of products corresponding in mass to mono- and dinitrated forms of the starting material. LC-MS-MS and GC-MS analyses revealed no evidence for the formation of nitrophenylalanine; however, both 3-nitrotyrosine and 3,5-dinitrotyrosine were observed and characterized. GC-MS analysis of hydrolyzed peptides following ONOO- treatment confirmed the presence of nitrated and dinitrated tyrosine. However, when a 20-fold molar excess of ONOO- was reacted with leucine-enkephalin, only about half of the tyrosine originally present in the peptide could be accounted for in the acid hydrolysate. The main product was 3-nitrotyrosine which represented ca. 50% of the original tyrosine; traces of 3,5-dinitrotyrosine (ca. 3% of the original tyrosine) were also present.

gamma-tocopherol traps mutagenic electrophiles such as NO(X) and complements alpha-tocopherol: physiological implications.

Peroxynitrite, a powerful mutagenic oxidant and nitrating species, is formed by the near diffusion-limited reaction of .NO and O2.- during activation of phagocytes. Chronic inflammation induced by phagocytes is a major contributor to cancer and other degenerative diseases. We examined how gamma-tocopherol (gammaT), the principal form of vitamin E in the United States diet, and alpha-tocopherol (alphaT), the major form in supplements, protect against peroxynitrite-induced lipid oxidation. Lipid hydroperoxide formation in liposomes (but not isolated low-density lipoprotein) exposed to peroxynitrite or the .NO and O2.- generator SIN-1 (3-morpholinosydnonimine) was inhibited more effectively by gammaT than alphaT. More importantly, nitration of gammaT at the nucleophilic 5-position, which proceeded in both liposomes and human low density lipoprotein at yields of approximately 50% and approximately 75%, respectively, was not affected by the presence of alphaT. These results suggest that despite alphaT's action as an antioxidant gammaT is required to effectively remove the peroxynitrite-derived nitrating species. We postulate that gammaT acts in vivo as a trap for membrane-soluble electrophilic nitrogen oxides and other electrophilic mutagens, forming stable carbon-centered adducts through the nucleophilic 5-position, which is blocked in alphaT. Because large doses of dietary alphaT displace gammaT in plasma and other tissues, the current wisdom of vitamin E supplementation with primarily alphaT should be reconsidered.

Mass spectrometric identification and quantification of hemorphins extracted from human adrenal and pheochromocytoma tissue.

The hemorphins are a family of recently identified opioid receptor binding peptides derived from the proteolytic processing of the beta, gamma, delta, and epsilon chains of hemoglobin. They have previously been identified at high concentration in human pituitary glands and in the CSF of patients with cerebral bleeding. Hemorphins are potent inhibitors of angiotensin converting enzyme and therefore possibly have a role to play in blood pressure regulation. We report the presence of four hemorphin peptides in extracts of normal adrenal tissue and in pheochromocytoma tumors. The hemorphins were quantified and structurally characterized using mass spectrometry. High concentrations of hemorphins were found in all samples, comparable with the levels reported in the literature for pituitary and brain tissue.

Conserved motifs as the basis for recognition of homologous proteins across species boundaries using peptide-mass fingerprinting.

Two-dimensional gel electrophoresis of any biological system presently resolves a plethora of highly purified proteins for which no function or identity has been determined. Theoretical and experimental data were used to demonstrate that peptide-mass fingerprinting (PMF) could aid in the recognition of conserved motifs across species boundaries, and thereby assist in attributing putative function to some of these molecules. Amino acids residue substitutions produced by biological diversity and phylogenetic distance combine to highlight regions of functional significance within proteins. Using 10 prokaryotic and two eukaryotic elongation factors (EF), up to 25 peptide fragments (> 800 Da) per molecule were compared across species boundaries within a 12 x 12 contingency table (66 cross-species comparisons), based upon the degree of molecular mass and amino acid sequence identity. Total amino acid sequence identity ranged from 29.4-80.9% for these molecules. Peptide fragments with homologous sequence across three or more EF were defined as containing, or being near to, conserved functional motifs. Twelve such fragments (> 800 Da) were found in this group of proteins. In addition, an 808.9 Da peptide of unknown functional significance was seen to occur in three of the 12 molecules studied and in another three EF-Tu molecules. At the 83% (five of six residues) identity level, this fragment was found in a further 35 EF-Tu molecules and in 14 unrelated proteins. Further investigation should reveal a role for this fragment (motif) in structural integrity or protein function. A FASTA search conducted on a peptide fragment containing a conserved GTP-binding motif (GHVDHGK) of EF-Tu from Euglena gracilis was used as an example to putatively attribute partial function to three hypothetical proteins derived from DNA sequencing initiatives.