Sulforaphane interaction with amyloid beta 1-40 peptide studied by electrospray ionization mass spectrometry.

RATIONALE: Aggregation of amyloid beta 1-40 (Aß) in the brain causes Alzheimer's disease (AD) and several small molecules are known to inhibit the aggregation process. Sulforaphane (SFN) is a natural isothiocyanate which is known to prevent various neurodegenerative processes. However, its interaction with Aß is yet to be explored. Such studies could provide new mechanistic insights for its neuroprotective properties. METHODS: Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and in-source fragmentation experiments were performed on an Orbitrap mass spectrometer. The solution of Aß and SFN was incubated and analyzed by mass spectrometry. Isotopic distribution patterns, accurate mass values and theoretical product ions were used to analyze the mass spectrometry data. The nature of binding of SFN and its binding sites with Aß were evaluated by LC/MS and trypsin digestion experiments. RESULTS: ESI-MS analysis of the incubated solution of Aß and SFN showed a 1:1 complex of [Aß+SFN]. LC/MS analysis revealed that the solution contains three different [Aß+SFN] complexes due to covalent binding of SFN to Aß at three different sites. The in-source fragmentation experiments revealed that SFN is binding to free NH(2) groups (N-terminal amino acid and lysines) in Aß. Trypsin digestion experiments further confirmed the SFN binding sites in Aß. CONCLUSIONS: The interaction of SFN, an anticancer agent, with Aß was studied using ESI-MS. SFN is found to bind covalently and specifically with the free NH(2) group of N-terminal aspartic acid and the ε-amino group of lysine at positions 16 and 28. Aggregation assay studies showed a lesser inclination of Aß to aggregate when SFN is present. Hence the present study helps in understanding the mechanism of the action of SFN on the Aß peptide.

Rapid screening of N-oxides of chemical warfare agents degradation products by ESI-tandem mass spectrometry.

Rapid detection and identification of chemical warfare agents and related precursors/degradation products in various environmental matrices is of paramount importance for verification of standards set by the chemical weapons convention (CWC). Nitrogen mustards, N,N-dialkylaminoethyl-2-chlorides, N,N-dialkylaminoethanols, N-alkyldiethanolamines, and triethanolamine, which are listed CWC scheduled chemicals, are prone to undergo N-oxidation in environmental matrices or during decontamination process. Thus, screening of the oxidized products of these compounds is also an important task in the verification process because the presence of these products reveals alleged use of nitrogen mustards or precursors of VX compounds. The N-oxides of aminoethanols and aminoethylchlorides easily produce [M + H](+) ions under electrospray ionization conditions, and their collision-induced dissociation spectra include a specific neutral loss of 48 u (OH + CH2OH) and 66 u (OH + CH2Cl), respectively. Based on this specific fragmentation, a rapid screening method was developed for screening of the N-oxides by applying neutral loss scan technique. The method was validated and the applicability of the method was demonstrated by analyzing positive and negative samples. The method was useful in the detection of N-oxides of aminoethanols and aminoethylchlorides in environmental matrices at trace levels (LOD, up to 500 ppb), even in the presence of complex masking agents, without the use of time-consuming sample preparation methods and chromatographic steps. This method is advantageous for the off-site verification program and also for participation in official proficiency tests conducted by the Organization for the Prohibition of Chemical Weapons (OPCW), the Netherlands. The structure of N-oxides can be confirmed by the MS/MS experiments on the detected peaks. A liquid chromatography-mass spectrometry (LC-MS) method was developed for the separation of isomeric N-oxides of aminoethanols and aminoethylchlorides using a C18 Hilic column. Critical isomeric compounds can be confirmed by LC-MS/MS experiments, after detecting the N-oxides from the neutral loss scanning method.

Electrospray ionization mass spectral studies of N,N-dialkylaminoethane-2-sulphonic acids.

Oxidative reactions of VX type compounds and N,N-dialkylaminoethane-2-thiols that are precursors for VX compounds produce N,N-dialkylaminoethane-2-sulphonic acids, N(R(1))(R(2))-CH(2)-CH(2)SO(3)H (where R(1) and R(2) = methyl, ethyl, n-propyl and isopropyl, 1-10), as the degradation products, and these degradation products are considered as markers for the detection of chemicals listed in the schedules of Chemical Weapons Convention (CWC) chemicals. Off-site detection of such degradation products in aqueous samples is an important task in the verification of CWC-related chemicals. Here we report a simple method involving the direct analysis of aqueous samples using positive and/or negative ion electrospray ionization (ESI) for the screening, detection and identification of N,N-dialkylaminoethane-2-sulphonic acids, avoiding sample preparation and chromatographic steps. The positive ion ESI mass spectra of all the compounds result in abundant [M+Na](+) ions, and the negative ion spectra show abundant [M-H](-) ions to confirm their molecular weight. The collision-induced dissociation spectra of [M+Na](+) and [M-H](-) give characteristic product ions by which it is easy to detect and identify all the studied N,N-dialkylaminoethane-2-sulphonic acids including those of isomeric compounds. The method is successfully applied to detect the spiked chemical, N,N-diisopropylaminoethane-2-sulphonic acid, present in a water sample received in a proficiency test.

In situ nucleophilic substitution reaction of N,N-dialkylaminoethyl-2-chlorides monitored by gas chromatography/mass spectrometry.

The detection and identification of degradation products of scheduled chemicals, which are characteristic markers of Chemical Warfare agents (CWAs), plays a key role in verification analysis. Identification of such non-scheduled but specific markers of CWAs helps in deciphering the kind of agent that was present in the sample submitted for off-site analysis. This paper describes the stability of N,N-dialkylaminoethyl-2-chlorides, which are precursors for highly toxic chemicals like VX, in different solvents. These compounds are stable in chloroform, acetonitrile, hexane and dichloromethane but tend to undergo in situ nucleophilic substitution reaction in the presence of alcohols giving the corresponding alkyl ether. The study shows that N,N-dialkylaminoethyl alkyl ethers can be used as markers of N,N-dialkylaminoethyl-2-chlorides. A detailed degradation study of these compounds in the presence of alcohols was carried out and it was found that the reaction follows pseudo-first order kinetics. Electron ionization mass spectral data for the methyl ethers of all the compounds are briefly discussed.

Electrospray ionisation mass spectral studies on hydrolysed products of sulfur mustards.

Off-site detection of the hydrolysed products of sulfur mustards in aqueous samples is an important task in the verification of Chemical Weapons Convention (CWC)-related chemicals. The hydrolysed products of sulfur mustards are studied under positive and negative electrospray ionisation (ESI) conditions using an additive with a view to detecting them at trace levels. In the presence of cations (Li(+), Na(+), K(+) and NH(4) (+)), the positive ion ESI mass spectra of all the compounds include the corresponding cationised species; however, only the [M+NH(4)](+) ions form [M+H](+) ions upon decomposition. The tandem mass (MS/MS) spectra of [M+H](+) ions from all the hydrolysed products of the sulfur mustard homologues were distinct and allowed these compounds to be characterised unambiguously. Similarly, the negative ion ESI mass spectra of all the compounds show prominent adducts with added anions (F(-), Cl(-), Br(-), and I(-)), but the [M-H](-) ion can only be generated by decomposition of an [M+F](-) ion. The MS/MS spectra of the [M-H](-) ions from all the compounds result in a common product ion at m/z 77. A precursor ion scan of m/z 77 is shown to be useful in the rapid screening of these compounds in aqueous samples at trace levels, even in the presence of complex masking agents, without the use of time-consuming sample preparation and chromatography steps. An MS/MS method developed to measure the detection limits of the hydrolysed products of sulfur mustards found these to be in the range of 10-500 ppb.

Amplified fragment length polymorphism and metabolomic profiles of hairy roots of Psoralea corylifolia L.

A reproducible protocol for establishment of hairy root cultures of Psoralea corylifolia L. was developed using Agrobacterium rhizogenes strain ATCC 15834. The hairy root clones exhibited typical sigmoid growth curves. Genomic and metabolomic profiles of hairy root clones along with that of untransformed control were analysed. Hairy root clones, Ps I and Ps II, showed significant differences in their amplified fragment length polymorphism (AFLP) profiles as compared to that of control, besides exhibiting Ri T-DNA-specific bands. These results amply indicate the stable integration of Ri T-DNA into the genomes of these clones. Further, the variations observed between clones in the AFLP profiles suggest the variable lengths and independent nature of Ri T-DNA integrations into their genomes. An isoflavonoid, formononetin, and its glycoside were present only in the hairy root clones while they were absent in the untransformed control. Variations observed in the metabolite profiles of these clones may be attributed to the random T-DNA integrations and associated changes caused by them in the recipient genomes. GC/MS analyses revealed the production of three and six clone-specific compounds in Ps I and Ps II, respectively, suggesting that the clones are dissimilar in their secondary metabolism. HPLC/UV-MS analyses disclosed substantial increases in the total isoflavonoids produced in Ps I (184%) and Ps II (94%) compared to untransformed control.

Mass spectral analysis of chloropicrin under negative ion chemical ionization conditions.

A chemical ionization (CI) method is developed for the first time to obtain molecular weight information for chloropicrin (CP), which is used as a chemical warfare agent and as an insecticide. The study includes a detailed investigation on the behavior of CP under electron impact (EI) and CI. Reagent gases of different nature, i.e., methane, isobutane, ammonia, hydrogen, and carbon dioxide, were used for CI analysis. Negative ion mode is found more sensitive than positive ion mode for the EI/CI mass spectrometric analysis of CP, but none of the methods provided molecular weight information, except negative ion CI using ammonia as the reagent gas (NICI (NH3)). The NICI (NH3) showed formation of the quasi-molecular ion, [M + H]-, in addition to other adduct ions. The [M + H]- abundance critically depends on the source temperature, reagent gas pressure, and concentration of the analyte, and it can be 13% under optimized conditions by which CP can be confirmed unambiguously. This method meets the criteria used in official proficiency tests conducted by OPCW for confirming the molecular weight of the unknowns.