Mass spectrometry study on snake venoms and enzymes

In this paper, three kinds of snake venoms and lour kinds of enzymes (phospholipase A(2), fibrinolytic enzyme, arginine esterase and L-amino acid oxidase) isolated from the snake venom were analyzed. As the snake venom was different, the MALDI/TOF/MS showed difference, The MALDI/TOF/MS determination results could be affected Ly the concentrations of snake venom enzymes, And the mechanisms of desorption and ionization was also given in this study, By using MALDI/TOF/MS we obtained the accurate molecular weights and homogeneities of the enzymes. The apparent characteristics of the positive MALDI/TOF/MS of enzymes composed by two subunits were also given out, The results showed that MALDI/TOF/MS is an effective analytic method for discovering new components from snake venom complexes. And it is reliable to use this method to determine the molecular weights and purifies of protein molecules.

Use of nitrocellulose films for affinity-directed mass spectrometry for the analysis of antibody/antigen interactions

Combination of affinity extraction procedures with mass spectrometric analyses is termed affinity-directed mass spectrometry, a technique that has gained broad interest in immunology and is extended here with several improvements from methods used in previous studies. A monoclonal antibody was immobilized on a nitrocellulose (NC) membrane, allowing the corresponding antigen to be selectively captured from a complex solution for analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This method was also used to rapidly determine the approximate binding region responsible for the antibody/antigen interaction. The tryptic fragments of antigen protein in buffer were applied to the antibody immobilized on NC film and allowed to interact. The NC film was then washed to remove salts and other unbound components, and subjected to analysis by MALDI-TOFMS. Using interferon-alpha (2a) and anti-interferon-alpha (2a) monoclonal antibody IgG as a model system, we successfully extracted the antigen protein and determined the approximate binding region for the antigen/antibody interaction (i.e., the tryptic fragment responsible). Copyright (C) 2001 John Wiley & Sons, Ltd.

Matrixassisted laser desorption/ionization mass spectral study of saccharides

Matrixassisted laser desorption/ionization (MALDI) mass spectra of saccharides with three common matrixes were studied here. It is shown that Na+ and K+ play important roles in the determination of oligasaccharide molecule weight and the molecular weights (MW) of glucans(dextran) with MW more than 10 000 is determined successfully with the help of column chromatography, By comparing the effects of three matrixes in saccharide analysis and comparing positive-ion and negative-ion matrixassisted laser desorption/ionization mass spectra of oligo- and polysaccharides, the most suitable matrix for saccharide analysis, 2,5-dihydroxylbezonic acid (DHB), is selected and the ion formation processes of saccharides under matrix-assisted laser adsorption/ionization condition are clarified.

Study on dimeric ion of toluene in the gas phase by tandem mass spectrometry

The reaction character of m/z183 and 184 ions generated from ion -molecule reaction of toluene under self-chemical ionization was studied using Collision-Induced Dissociation (CID). The results Show that the m/z183 and 184 ions have several transition state structures; such as diphenyl methane derivative, alpha-bond structure formed between toluene and tropylium, pi-complex formed between toluene radical ion and toluene and pi-complex consisted of benzyl ion and toluene.

Determination of free fatty acids in soil and bryophyte plants by precolumn derivatization via HPLC with fluorescence detection and tandem mass spectrometry (HPLC-MS/MS)

A sensitive method for the determination of 30 kinds of free fatty acids (FFAs, C-1-C-30) with 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f] 9,10-phenan- threne (TSPP) as labeling reagent and using high performance liquid chromatography with fluorescence detection and identification by online postcolumn mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion mode (HPLC/MS/APCI) has been developed. TSPP could easily and quickly label FFAs in the presence of K2CO3 catalyst at 90 degrees C for 30 min in N,N-dimethylformamide (DMF) solvent, and maximal labeling yields close to 100% were observed with a 5-fold excess of molar reagent. Derivatives were stable enough to be efficiently analyzed by high performance liquid chromatography. TSPP was introduced into fatty acid molecules and effectively augmented MS ionization of fatty acid derivatives and led to regular MS and MS/MS information. The collision induced cleavage of protonated molecular ions formed specific fragment ions at m/z [MH](+)(molecular ion), m/z [M'+CH2CH2](+)(M' was molecular mass of the corresponding FFA) and m/z 295.0 (the, mass of protonated molecular core structure of TSPP). Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C-8 column (4.6 x 150 mm, 5 mu m, Agilent) with a good baseline resolution in combination with a gradient elution. Linear ranges of 30 FFAs are 2.441 x 10(-3) to 20 mu mol/L, detection limits are 3.24 similar to 36.97 fmol (injection volume 10 mu L, at a signal-to-noise ratio of 3, S/N 3:1). The mean interday precision ranged from 93.4 to 106.2% with the largest mean coefficients of variation (R.S.D.) < 7,5%. The mean intraday precision for all standards was < 6.4% of the expected concentration. Excellent linear responses were observed with correlation coefficients of > 0.9991. Good compositional data could be obtained from the analysis of extracted fatty acids from as little as 200 mg of bryophyte plant samples.Therefore, the facile TSPP derivatization coupled with HPLC/MS/APCI analysis allowed the development of a highly sensitive method for the quantitation of trace levels of short and long chain fatty acids from biological and natural environmental samples.

Comparison of comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry and gas chromatography-mass spectrometry for the analysis of tobacco essential oils

A sample of tobacco essential oil was analyzed using gas chromatography-mass spectrometry (GUMS)and comprehensive two-dimensional gas chromatography coupled to a time-of-flight mass spectrometry (GC x GC/TOFMS), respectively. In the GUMS analysis, serially coupled columns were used. By comparing the GUMS results with GC x GC/TOFMS result,,, many more components in the essential oil could be found within the two-dimensional separation space of GC x GC. The quantitative determination of components in the essential oil was performed by GC x GC with flame ionization detection (FID), using a method of multiple internal standards calibration, (c) 2005 Elsevier B.V. All rights reserved.

Quantitative determination of compounds in tobacco essential oils by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry

A quantitative analysis of the individual compounds in tobacco essential oils is performed by comprehensive two-dimensional gas chromatography (GC x GC) combined with flame ionization detector (FID). A time-of-flight mass spectrometer (TOF/MS) was coupled to GC x GC for the identification of the resolved peaks. The response of a flame ionization detector to different compound classes was calibrated using multiple internal standards. In total, 172 compounds were identified with good match and 61 compounds with high probability value were reliably quantified. For comparative purposes, the essential oil sample was also quantified by one-dimensional gas chromatography-mass spectrometry (GC/MS) with multiple internal standards method. The results showed that there was close agreement between the two analysis methods when the peak purity and match quality in one-dimensional GC/MS are high enough. (c) 2005 Elsevier B.V. All rights reserved.

Mass spectrometry-based thermal shift assay for protein-ligand binding analysis.

Described here is a mass spectrometry-based screening assay for the detection of protein-ligand binding interactions in multicomponent protein mixtures. The assay utilizes an oxidation labeling protocol that involves using hydrogen peroxide to selectively oxidize methionine residues in proteins in order to probe the solvent accessibility of these residues as a function of temperature. The extent to which methionine residues in a protein are oxidized after specified reaction times at a range of temperatures is determined in a MALDI analysis of the intact proteins and/or an LC-MS analysis of tryptic peptide fragments generated after the oxidation reaction is quenched. Ultimately, the mass spectral data is used to construct thermal denaturation curves for the detected proteins. In this proof-of-principle work, the protocol is applied to a four-protein model mixture comprised of ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). The new protocol's ability to detect protein-ligand binding interactions by comparing thermal denaturation data obtained in the absence and in the presence of ligand is demonstrated using cyclosporin A (CsA) as a test ligand. The known binding interaction between CsA and CypA was detected using both the MALDI- and LC-MS-based readouts described here.

Identification of an unknown b-agonist in feed by liquid chromatography/bioassay/quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement.

A new approach to the search for residues of unknown growth promoting agents such as anabolic steroids and -agonists in feed is presented. Following primary extraction and clean-up, samples are separated using gradient liquid chromatography (LC). The effluent is split towards two identical 96-well fraction collectors and an optional electrospray quadrupole time-of-flight mass spectrometry (QTOFMS) system for accurate mass measurement. One 96-well plate is used for a bioassay (enzyme-immuno assay, receptor assay) and will detect the bioactivity and position of the relevant peak in the chromatogram. The positive well in the second 96-well plate is used for identification by LC/QTOFMS/MS. The value of this LC/bioassay/QTOFMS/MS methodology is highlighted by the finding and structure elucidation of a new -agonist in a feed extract.

Rapid confirmatory method for the determination of sixteen synthetic growth promoters and bisphenol A in bovine milk using dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry

A rapid liquid chromatographic-tandem mass spectrometric (LC-MS/MS) multi-residue method for the simultaneous quantitation and identification of sixteen synthetic growth promoters and bisphenol A in bovine milk has been developed and validated. Sample preparation was straightforward, efficient and economically advantageous. Milk was extracted with acetonitrile followed by phase separation with NaCl. After centrifugation, the extract was purified by dispersive solid-phase extraction with C18 sorbent material. The compounds were analysed by reversed-phase LC-MS/MS using both positive and negative ionization and operated in multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for unambiguous confirmation. Total chromatographic run time was less than 10 min for each sample. The method was validated at a level of 1 mu g L-1. A wide variety of deuterated internal standards were used to improve method performance. The accuracy and precision of the method were satisfactory for all analytes. The confirmative quantitative liquid chromatographic tandem mass spectrometric (LC-MS/MS) method was validated according to Commission Decision 2002/657/EC. The decision limit (CC alpha) and the detection capability (CC beta) were found to be below the chosen validation level of 1 mu g L-1 for all compounds. (C) 2010 Elsevier B.V. All rights reserved.

Mass spectrometry-based metabolomics applied to the chemical safety of food

Mass spectrometry (MS)-based metabolomics is emerging as an important field of research in many scientific areas, including chemical safety of food. A particular strength of this approach is its potential to reveal some physiological effects induced by complex mixtures of chemicals present at trace concentrations. The limitations of other analytical approaches currently employed to detect low-dose and mixture effects of chemicals make detection very problematic. Besides this basic technical challenge, numerous analytical choices have to be made at each step of a metabolomics study, and each step can have a direct impact on the final results obtained and their interpretation (i.e. sample preparation, sample introduction, ionization, signal acquisition, data processing, and data analysis). As the application of metabolomics to chemical analysis of food is still in its infancy, no consensus has yet been reached on defining many of these important parameters. In this context, the aim of the present study is to review all these aspects of MS-based approaches to metabolomics, and to give a comprehensive, critical overview of the current state of the art, possible pitfalls, and future challenges and trends linked to this emerging field. (C) 2010 Elsevier Ltd. All rights reserved.

Optimised extraction of heterocyclic aromatic amines from blood using hollow fibre membrane liquid-phase microextraction and triple quadrupole mass spectrometry

Heterocyclic aromatic amines (HCA) are carcinogenic mutagens formed during cooking of proteinaceous foods, particularly meat. To assist in the ongoing search for biomarkers of HCA exposure in blood, a method is described for the extraction from human plasma of the most abundant HCAs: 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) (and its isomer 7,8-DiMeIQx), using Hollow Fibre Membrane Liquid-Phase Microextraction. This technique employs 2.5 cm lengths of porous polypropylene fibres impregnated with organic solvent to facilitate simultaneous extraction from an alkaline aqueous sample into a low volume acidic acceptor phase. This low cost protocol is extensively optimised for fibre length, extraction time, sample pH and volume. Detection is by UPLC-MS/MS using positive mode electrospray ionisation with a 3.4 min runtime, with optimum peak shape, sensitivity and baseline separation being achieved at pH 9.5. To our knowledge this is the first description of HCA chromatography under alkaline conditions. Application of fixed ion ratio tolerances for confirmation of analyte identity is discussed. Assay precision is between 4.5 and 8.8% while lower limits of detection between 2 and 5 pg/mL are below the concentrations postulated for acid-labile HCA-protein adducts in blood.

Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine.

Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R (2) > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.

Rapid high-performance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography-mass spectrometry.

Furosemide (FD: Lasix) is a loop diuretic which strongly increases both urine flow and electrolyte urinary excretion. Healthy volunteers were administered 40 mg orally (dissolved in water) and concentrations of FD were determined in serum and urine for up to 6 h for eight subjects, who absorbed water at a rate of 400 ml/h. Quantification was performed by HPLC with fluorescence detection (excitation at 233 nm, emission at 389 nm) with a limit of detection of 5 ng/ml for a 300-microliters sample. The elution of FD was completed within 4 min using a gradient of acetonitrile concentration rising from 30 to 50% in 0.08 M phosphoric acid. The delay to the peak serum concentration ranged from 60 to 120 min. FD was still easily measurable in the sera from all subjects 6 h after administration. In urine, the excretion rates reached their maximum between 1 and 3 h. The total amount of FD excreted in the urine averaged 11.2 mg (range 7.6-14.0 mg), with a mean urine volume of 3024 ml (range 2620-3596 ml). Moreover, the urine density was lower than 1.010 (recommended as an upper limit in doping analysis to screen diuretics) only for 2 h. An additional volunteer was administered 40 mg of FD and his urine was collected over a longer period. FD was still detectable 48 h after intake. Gas chromatography-mass spectrometry with different types of ionization was used to confirm the occurrence of FD after permethylation of the extract. Negative-ion chemical ionization, with ammonia as reactant gas, was found to be the most sensitive method of detection.

A simple, robust and rapid approach to detect carbapenemases in Gram-negative isolates by MALDI-TOF mass spectrometry: validation with triple quadripole tandem mass spectrometry, microarray and PCR.

Carbapenemases should be accurately and rapidly detected, given their possible epidemiological spread and their impact on treatment options. Here, we developed a simple, easy and rapid matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-based assay to detect carbapenemases and compared this innovative test with four other diagnostic approaches on 47 clinical isolates. Tandem mass spectrometry (MS-MS) was also used to determine accurately the amount of antibiotic present in the supernatant after 1 h of incubation and both MALDI-TOF and MS-MS approaches exhibited a 100% sensitivity and a 100% specificity. By comparison, molecular genetic techniques (Check-MDR Carba PCR and Check-MDR CT103 microarray) showed a 90.5% sensitivity and a 100% specificity, as two strains of Aeromonas were not detected because their chromosomal carbapenemase is not targeted by probes used in both kits. Altogether, this innovative MALDI-TOF-based approach that uses a stable 10-μg disk of ertapenem was highly efficient in detecting carbapenemase, with a sensitivity higher than that of PCR and microarray.