Mass spectrometric analysis of rat cerebrospinal fluid proteins following exposure to the neurotoxicant carbonyl sulfide.

RATIONALE: Using a proteomic-based approach we have investigated possible altered expression of a range of cerebral spinal fluid (CSF) proteins following exposure to the neurotoxicant carbonyl sulfide (COS). CSF is ideal for the investigation of markers of brain injury or disease since it is secreted from several central nervous system structures and changes in the CSF composition may reflect brain insult and many pathological processes. METHODS: Animals were placed in exposure chambers and were exposed to 0 ppm or 500 ppm COS for 1, 2 or 3 days, 6 h per day. After the last inhalation exposure, 50-70 µL CSF sample was obtained by lumbar puncture. CSF samples were analyzed by electrospray ionization mass spectrometry (ESI-MS) on either a Premier quadrupole time-of-flight (QTOF) or an Agilent 6340 ion trap and by matrix-assisted laser desorption/ionization (MALDI)-MS on a 4800 MALDI-TOF/TOF analyzer. RESULTS: The dynamic range of abundance of the identified proteins spanned over more than three orders of magnitude. The four most abundant proteins identified (albumin, cystatin C, serotransferrin, transthyretin) are major proteins that are present in both CSF and blood at high levels but the fifth most abundant protein identified (prostaglandin H2D isomerase) is the second most abundant protein in human CSF and is secreted and synthesized in the rat central nervous system. No significant differences were observed between COS-treated CSF samples and the control CSF samples because of blood contamination. CONCLUSIONS: Quantitative MS protein analyses of rat CSF is limited by the low sample volumes that can practicably be obtained from rats and the low protein concentrations in rat CSF. Results of this work suggest a clear need for CSF collection that would minimize blood contamination. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.

Identification of Maillard reaction products on peanut allergens that influence binding to the receptor for advanced glycation end products.

BACKGROUND: Recent immunological data demonstrated that dendritic cells preferentially recognize advanced glycation end product (AGE)-modified proteins, upregulate expression of the receptor for AGE (RAGE), and consequently bias the immune response toward allergy. METHODS: Peanut extract was characterized by mass spectrometry (MS) to elucidate the specific residues and specific AGE modifications found in raw and roasted peanuts and on rAra h 1 that was artificially glycated by incubation with glucose or xylose. The binding of the RAGE-V1C1 domain to peanut allergens was assessed by PAGE and Western analysis with anti-Ara h 1, 2, and 3 antibodies. IgE binding to rAra h 1 was also assessed using the same methods. RESULTS: AGE modifications were found on Ara h 1 and Ara h 3 in both raw and roasted peanut extract. No AGE modifications were found on Ara h 2. Mass spectrometry and Western blot analysis demonstrated that RAGE binds selectively to Ara h 1 and Ara h 3 derived from peanut extract, whereas the analysis failed to demonstrate Ara h 2 binding to RAGE. rAra h 1 with no AGE modifications did not bind RAGE; however, after AGE modification with xylose, rAra h 1 bound to RAGE. CONCLUSIONS: AGE modifications to Ara h 1 and Ara h 3 can be found in both raw and roasted peanuts. Receptor for AGE was demonstrated to selectively interact with AGE-modified rAra h 1. If sensitization to peanut allergens occurs in dendritic cells via RAGE interactions, these cells are likely interacting with modified Ara h 1 and Ara h 3, but not Ara h 2.

Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR.

RXR is a nuclear receptor that plays a central role in cell signaling by pairing with a host of other receptors. Previously, 9-cis-retinoic acid (9cRA) was defined as a potent RXR activator. Here we describe a unique RXR effector identified from organic extracts of bovine serum by following RXR-dependent transcriptional activity. Structural analyses of material in active fractions pointed to the saturated diterpenoid phytanic acid, which induced RXR-dependent transcription at concentrations between 4 and 64 microM. Although 200 times more potent than phytanic acid, 9cRA was undetectable in equivalent amounts of extract and cannot be present at a concentration that could account for the activity. Phytanic acid, another phytol metabolite, was synthesized and stimulated RXR with a potency and efficacy similar to phytanic acid. These metabolites specifically displaced [3H]-9cRA from RXR with Ki values of 4 microM, indicating that their transcriptional effects are mediated by direct receptor interactions. Phytol metabolites are compelling candidates for physiological effectors, because their RXR binding affinities and activation potencies match their micromolar circulating concentrations. Given their exclusive dietary origin, these chlorophyll metabolites may represent essential nutrients that coordinate cellular metabolism through RXR-dependent signaling pathways.

Tandem mass spectrometry for the structural determination of backbone-modified peptides.

A variety of backbone-modified peptides were desorbed by fast atom bombardment and collisionally activated. These peptide modifications involve the replacement of a normal [CONH] peptide linkage with such groups as thiomethylene ether (CH2S), thioamide (CSNH), methyleneamine (CH2NH), and thiomethylene sulfoxide (CH2SO) moieties. Modified linear peptides decompose to give fragmentations characteristic of the modifications as well as typical peptide bond fragments. The presence of a replacement group in cyclic peptides can induce new fragmentations. The presence of other functional groups, such as an exocyclic N-terminal residue, however, can dominate the observed fragmentations. Upon collisional activation, unmodified linear peptides fragment to give N-terminal ions as the most abundant daughter ions. In comparison, ψ[CH2NH] and ψ[CH2S ] modified linear peptides decompose to give prominent C-terminal sequence ions. The ψ[CH2SO] modified linear peptides, however, fragment into both N- and C-terminal ions of high relative abundance. Depending on the modification, daughter ions or internal fragment ions are observed that are characteristic of the amide bond replacement. Useful structural information can therefore be obtained.

Continuous flow fast atom bombardment with packed microcolumns: A comparison of precolumn versus coaxial matrix delivery.

The effect of adding glycerol to the mobile phase on the chromatographic separation of peptides has been investigated using a continuous flow fast atom bombardment (CFFAB) interface coupled with commercial packed microcolumns (25 cm × 320 µm.i.d.). In a comparative study using a UV detector, it was found that chromatographic peak broadening progressively increased with increasing percentage of glycerol in the mobile phase. In the liquid chromatographic FAB mass spectrometric analysis, this effect is compounded by the dynamic mixing of the column effluent on the probe. Improvements of 25-155% in the overall separation efficiencies were obtained by introducing the matrix independently to the probe tip via a coaxial arrangement. Application of this coaxial CFFAB is demonstrated by the analysis of peptide mixtures and tryptic digests.

Identification of radical adducts formed in the reactions of unsaturated fatty acids with soybean lipoxygenase using continuous flow fast atom bombardment with tandem mass spectrometry.

Structures of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) radical adducts formed in the reactions of soybean lipoxygenase with linoleic acid, linolenic acid, and arachidonic acid were determined using continuous flow fast atom bombardment (CF-FAB) combined with tandem mass spectrometry. The radical adducts of these lipoxygenase-dependent reactions were: n-octanoic acid radical, 12,13-dihydroxylinoleic acid radical, 12,13-epoxylinoleic acid radical, and n-pentyl radical from linoleic acid; n-octanoic acid radical, ethyl radical, and cis/trans and/or positional isomers (1- and 3-pentenyl) of pentenyl radical from linolenic acid; and 14,15-epoxyarachidonic acid radical and n-pentyl radical from arachidonic acid. Of these radical adducts, the n-octanoic acid radical from linoleic and linolenic acid, the ethyl radical from linolenic acid, and the 12,13-dihydroxylinoleic acid radical are identified for the first time in the reactions of soybean lipoxygenase. Thus the CF-FAB combined with tandem mass spectrometry employed here, by which both radical adducts and their fragment ions can be detected, is shown to be a powerful tool in the structural identification of free radicals.

Preliminary comparison of precursor scans and liquid chromatography-tandem mass spectrometry on a hybrid quadrupole time-of-flight mass spectrometer.

Recent mass spectrometry instrumentation developments include the appearance of novel hybrid tandem instrumentation, Q-TOF, consisting of a quadrupole mass analyzer (MS1) and a time-of-flight (TOF) analyzer. The TOF analyzer is not scanned, but collects all fragment ions entering the analyzer at a given time. Thus, the typical precursor scan experiment cannot be performed. Instead, a full MS-MS spectrum can be acquired for each mass passed by MS1. Appropriate data manipulation, i.e. extracted ion current chromatograms, can correlate specific fragment ion formation to the parent ion. Precursor scanning and LC-MS-MS are compared on a Q-TOF instrument for the determination of protein modifications, including acetylation and phosphorylation. Model peptides used for phosphopeptide detection were generated from a mixture of beta-casein. Model acetylated peptides were generated from a mixture of acetylated substance P1-9 and substance P1-11. The results were then applied to a more complex mixture, a digest of HIV-p24. Results indicate that precursor scanning is useful for screening, but that LC-MS-MS has a sensitivity advantage and is less susceptible to suppression effects. LC-MS-MS, therefore, appears to be better for the detection of trace components in complex mixtures.

Tris(2-chloroethyl) phosphate pharmacokinetics in the Fischer 344 rat: a comparison of conventional methods and in vivo microdialysis coupled with tandem mass spectrometry.

The pharmacokinetics of tris(2-chloroethyl) phosphate (TRCP, 20 mg/kg, iv) were investigated in awake male and female and anesthetized male Fischer 344 (F344) rats by conventional (CONV) sampling/detection methods (blood withdrawal with sample workup and analysis for TRCP). TRCP pharmacokinetics were also investigated in anesthetized male F344 rats using a new sampling/detection technique, in vivo microdialysis coupled with tandem mass spectrometry (MD/MS/MS). The concentration of free TRCP in plasma versus time profiles were analyzed using noncompartmental methods to estimate pharmacokinetic parameters. Comparisons of mean parameter estimates were made for (1) awake males versus females in CONV studies (t test, no significant differences, p < or = 0.05) and (2) awake and anesthetized males in CONV studies and anesthetized males in MD/MS/MS studies. There were significant differences (Scheffe's test) for the three groups of male rats, most notably the free TRCP concentration in plasma at early time points in CONV versus MD/MS/MS studies. The contributions of an indwelling jugular cannula, the blood sampling regimen, and the in vitro MD/MS/MS standard calibration curve were investigated. It appears that quantitation of TRCP by mass spectrometry using an in vitro standard calibration is responsible for the difference.

Coaxial continuous-flow fast-atom bombardment vs electrospray ionization: a sensitivity comparison on a magnetic sector instrument.

The performance of an electrospray (ESI) source was evaluated and compared to that of a coaxial continuous-flow fast-atom bombardment source on a magnetic mass spectrometer using ten different peptides over the mass range of 400 to 3500 Da and using a variety of scanning modes. Results show that sensitivities using the two ionization techniques are similar, with limits-of-detection in the attomole to low femtomole range. In addition, proteins can be routinely detected using ESI at femtomole levels. The observance of the noncovalent complex of RNase A and cytidine 2'-monophosphate yields evidence that these complexes can be studied using instruments that operate at high accelerating voltages.

Determination of bioactive peptides using capillary zone electrophoresis/mass spectrometry.

Mixtures of bioactive peptides have been analyzed by capillary zone electrophoresis/mass spectrometry (CZE/MS) using an on-line coaxial continuous-flow fast atom bombardment interface. High separation efficiencies (up to 410,000 theoretical plates) were obtained from low femtomole levels of peptides. The analysis of basic peptides was accomplished by using aminopropyl-silylated CZE columns to minimize zone broadening due to adsorption effects. CZE/MS/MS data were acquired from femtomole levels of peptides in electrophoretic real time.

Coaxial continuous flow fast atom bombardment for higher-molecular-weight peptides: comparison with static fast atom bombardment and electrospray ionization.

A comparison of coaxial continuous flow fast atom bombardment (FAB) with static FAB and with electrospray ionization (ESI) for the analysis of 'high'-mass peptides (Mr = 3000-4000) is presented. Sensitivities of the peptides by coaxial continuous flow FAB is nearly an order of magnitude better than by static FAB. Single-scan spectra with good signal-to-noise can be obtained from as little as 200 fmol (by flow injection analysis). Detection limits by ESI mass spectrometry were found to be equivalent to 20 times higher than by coaxial continuous flow FAB on a per mole basis, but 4-20 times lower on a concentration basis, owing to the greater flow per unit time employed in the ESI mass spectrometric experiments.

Nanoscale packed-capillary liquid chromatography coupled with mass spectrometry using a coaxial continuous-flow fast atom bombardment interface.

Nanoscale packed-capillary liquid chromatography (LC) columns have been coupled with mass spectrometry (MS) using a coaxial continuous-flow fast atom bombardment interface. The combined system has been applied to the analysis of mixtures of peptides, including synthetic mixtures of bioactive peptides and tryptic digests of proteins. Nanoscale packed-capillary columns offer two principal advantages for LC/MS analysis--high chromatographic separation efficiencies and low mobile-phase flow rates. The high separation efficiencies facilitate the separation of complex mixtures, and the low mobile-phase flow rates reduce problems with coupling the LC effluent with the high-vacuum, high-voltage environment of sector MS ion sources. The columns used in this work were 50- or 75-micron i.d., 1-2 m long, packed with 10-micron C18 particles, using mobile-phase flow rates of 50-350 nL/min.

Mapping of the 5'-2-deoxyribose-5-phosphate lyase active site in DNA polymerase beta by mass spectrometry.

The mechanism of the 5'-2-deoxyribose-5-phosphate lyase reaction catalyzed by mammalian DNA beta-polymerase (beta-pol) was investigated using a cross-linking methodology in combination with mass spectrometric analyses. The approach included proteolysis of the covalently cross-linked protein-DNA complex with trypsin, followed by isolation, peptide mapping, and mass spectrometric and tandem mass spectrometric analyses. The 8-kDa domain of beta-pol was covalently cross-linked to a 5'-2-deoxyribose-5-phosphate-containing DNA substrate by sodium borohydride reduction. Using tandem mass spectrometry, the location of the DNA adduct on the 8-kDa domain was unequivocally determined to be at the Lys(72) residue. No additional amino acid residues were found as minor cross-linked species. These data allow assignment of Lys(72) as the sole Schiff base nucleophile in the 8-kDa domain of beta-pol. These results provide the first direct evidence in support of a catalytic mechanism involving nucleophilic attack by Lys(72) at the abasic site.

Nature of the inhibition of horseradish peroxidase and mitochondrial cytochrome c oxidase by cyanyl radical.

Previous studies established that the cyanyl radical ((*)CN), detected as 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(*)CN by the electron spin resonance (ESR) spin-trapping technique, can be generated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H(2)O(2)) and by mitochondrial cytochrome c oxidase (CcO) in the absence of H(2)O(2). To investigate the mechanism of inhibition by cyanyl radical, we isolated and characterized the iron protoporphyrin IX and heme a from the reactions of CN(-) with HRP and CcO, respectively. The purified heme from the reaction mixture of HRP/H(2)O(2)/KCN was unambiguously identified as cyanoheme by the observation of the protonated molecule, (M + H)(+), of m/z = 642.9 in the matrix-assisted laser desorption/ionization (MALDI) mass spectrum. The proton NMR spectrum of the bipyridyl ferrous cyanoheme complex revealed that one of the four meso protons was missing and had been replaced with a cyanyl group, indicating that the single, heme-derived product was meso-cyanoheme. The holoenzyme of HRP from the reconstitution of meso-cyanoheme with the apoenzyme of HRP (apoHRP) showed no detectable catalytic activity. The Soret peak of cyanoheme-reconstituted apoHRP was shifted to 411 nm from the 403 nm peak of native HRP. In contrast, the heme a isolated from partially or fully inhibited CcO did not show any change in the structure of the protoporphyrin IX as indicated by its MALDI mass spectrum, which showed an (M + H)(+) of m/z = 853.6, and by its pyridine hemochromogen spectrum. However, a protein-centered radical on the CcO can be detected in the reaction of CcO with cyanide and was identified as the thiyl radical(s) based on inhibition of its formation by N-ethylmaleimide pretreatment, suggesting that the protein matrix rather than protoporphyrin IX was attacked by the cyanyl radical. In addition to the difference in heme structures between HRP and CcO, the available crystallographic data also suggested that the distinct heme environments may contribute to the different inhibition mechanisms of HRP and CcO by cyanyl radical.

Molecular characterization of a tetramolecular complex between dsDNA and a DNA-binding leucine zipper peptide dimer by mass spectrometry.

The characterization of sequence-specific noncovalent complexes of the GCN4 peptides and dsDNA using mass spectrometry is reported. The GCN4 peptides belong to a class of proteins which bind to sequence-specific dsDNA and are important in the regulation of gene transcription in yeast. These proteins contain a bZIP structural motif which consists of a basic DNA-binding domain and a leucine zipper dimerization domain. The protein dimers specifically bind double-stranded DNA containing the binding element 5'-ATGA(C/G)TCAT-3' to form a tetramolecular noncovalent complex. Using electrospray ionization, we report the detection of such a specific tetramolecular complex using mass spectrometry. Under conditions necessary for observation of the tetramolecular complex, no ions were detected for the GCN4 peptide dimer or the GCN4 monomer with dsDNA. These observations indicate that the specific interaction of the dsDNA with the protein dimer stabilizes the biologically significant noncovalent complex in the gas phase. Complexes were observed for various lengths of both blunt-ended and cohesive-ended double-stranded DNA containing the specific recognition sequence. The binding specificity of the complex was verified with the use of control DNA not containing the recognition sequence and control peptides not known to bind DNA specifically. Additionally, combining limited proteolysis of GCN4 peptide-DNA complexes with mass spectrometric determination of the products compared to identical experiments with noncomplexed peptides was used to probe interactions of specific amino acids with the DNA. The ability to observe these complexes by mass spectrometry and to probe the specific interactions involved opens the door for utilizing this analytical technique to other structural biological problems including the study of transcription processes and determining the specific binding regions between dsDNA and proteins.

On-line coupling of in vivo microdialysis with tandem mass spectrometry.

The capability of interfacing in vivo microdialysis with mass spectrometry has been demonstrated. The goal of this research was to demonstrate the feasibility of real-time analysis in biological systems using microdialysis in combination with tandem mass spectrometry (MS/MS). Microdialysis sampling was accomplished by surgically implanting a small microdialysis probe into a tissue or area of interest. Molecules diffuse through the membrane of the microdialysis probe due to concentration differences. These molecules are collected in a sample loop and analyzed by tandem mass spectrometry. Sequential injections can be made in as little as 2 min. This capability is advantageous in the study of molecules with very rapid elimination rates. Tris(2-chloroethyl)phosphate (TRCP) was used as a model compound in the development of this analytical technique. As an example of an application of the microdialysis/MS/MS technique, plasma concentration vs time curves were obtained and compared with the plasma concentration profiles obtained using conventional studies. For the microdialysis/MS/MS studies, the average slope from three animals was -0.086 min-1. In comparison, the average slope from four animals from the conventional studies was -0.035 min-1.

Hormone-mediated dephosphorylation of specific histone H1 isoforms.

We have previously shown a connection between histone H1 phosphorylation and the transcriptional competence of the hormone inducible mouse mammary tumor virus (MMTV) promoter. Prolonged exposure of mouse cells to dexamethasone concurrently dephosphorylated histone H1 and rendered the MMTV promoter refractory to hormonal stimulation and, therefore, transcriptionally unresponsive. Using electrospray mass spectrometry, we demonstrate here that prolonged dexamethasone treatment differentially effects a subset of the six somatic H1 isoforms in mouse cells. H1 isoforms H1.0, H1.1, and H1.2 are non-responsive to hormone whereas prolonged dexamethasone treatment effectively dephosphorylated the H1.3, H1.4, and H1.5 isoforms. The protein kinase inhibitor staurosporine, shown to dephosphorylate histone H1 and down-regulate MMTV in cultured cells, appears only to completely dephosphorylate the H1.3 isoform. These results suggest that dephosphorylation of specific histone H1 isoforms may contribute to the previously observed decrease in transcriptional competence of the MMTV promoter through the modulation of chromatin structure. In a broader sense, this work advances the hypothesis that post-translational modifications of individual histone H1 isoforms directly influence the transcriptional activation/repression of specific genes.

Characterization of cytochrome c free radical reactions with peptides by mass spectrometry.

The reactions of horse heart cytochrome c, hydrogen peroxide, and the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid with a series of polypeptides were investigated using mass spectrometry. The mass spectra obtained from these reactions revealed that after a free radical has been generated on the heme-containing protein horse heart cytochrome c, it can be transferred to other biomolecules. In addition, the number of free radicals transferred to the target molecule could be determined. Recipient peptides/proteins that contained a tyrosine and/or tryptophan amino acid residue were most susceptible to free radical transfer. Using tandem mass spectrometry, the location of the 3,5-dibromo-4-nitrosobenzenesulfonic acid radical adduct on the nonapeptide RWIILGLNK was unequivocally determined to be at the tryptophan residue. We also demonstrated that the presence of an antioxidant in the reaction mixture not only inhibits free radical formation on horse heart cytochrome c, but also interferes with the transfer of the free radical, once it has been formed on cytochrome c.

Applications of fast atom bombardment and tandem mass spectrometry.

The application of fast atom bombardment combined with tandem mass spectrometry to the structure elucidation of carcinogen-modified oligonucleotides, glutathione, cysteine and N-acetyl cysteine conjugates of exogenous toxins and chemically modified peptides is described.