Differentiation of Vitis vinifera varieties by MALDI-MS analysis of the grape seed proteins

Until now the study of pathogenic related proteins in grape juice and wine, performed by ESI-MS, LC/ESI-MS, and MALDI/MS, has been proposed for differentiation of varieties. In fact, chitinases and thaumatin-like proteins persist through the vinification process and cause hazes and sediments in bottled wines. An additional instrument, potentially suitable for the grape varieties differentiation, has been developed by MALDI/MS for the grape seed protein analysis. The hydrosoluble protein profiles of seeds extract from three different Vitis vinifera grape (red and white) varieties were analyzed and compared. In order to evaluate the environmental conditions and harvest effects, the seed protein profiles of one grape variety from different locations and harvests were studied.     

Development of a method for simultaneous determination of eflucimibe and its three major metabolites in rat plasma by liquid chromatography/electrospray tandem mass spectrometry: a preliminary study

A high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry (HPLC/ESI-MS/MS) method has been developed for the simultaneous determination of eflucimibe, a powerful acyl-coenzyme A cholesterol O-acyltransferase (ACAT) inhibitor, and its main metabolites, in plasma. The ESI and MS/MS parameters were investigated and optimised for each of the four compounds in the positive ion mode. Plasma samples were deproteinised by precipitation with acetonitrile and directly analysed by HPLC/ESI-MS/MS in less than 4 min. Quantitation was performed in the multiple reaction monitoring (MRM) mode for highest sensitivity, selecting the protonated molecules [M+H](+) as precursor ions. The method was demonstrated to be specific and sensitive, and a linear response was observed within a 1-25 ng/mL concentration range. Correlation coefficients (r(2)) greater than 0.9960 were obtained by least-squares regression, and limits of detection down to 0.2 ng/mL were calculated. Therefore, this HPLC/ESI-MS/MS method appears to be an efficient tool, able to provide valuable information for a pharmacological purpose.     

Relative binding affinities of molecular capsules investigated by ESI-mass spectrometry

ESI-MS(/MS) has been used as a method which allows the fast, unambiguous and sensitive simultaneous detection and relative stability approximation of supramolecular assemblies in mixtures. In spite of the obvious fundamental differences between solution and gas phase, ESI-MS in the case of self-assembled molecular capsules has been shown to produce very similar results to single binding experiments monitored by NMR titrations as well as conformational searches performed by Monte-Carlo simulations. MS/MS experiments reveal the same relative order of gas phase stabilities as previously found in solution. Moreover, proton transfer reactions which lead to new molecular capsules, are not detectable in the time-averaged NMR spectrum. However, the newly produced species are found in the complex mixtures by ESI-MS and can be conveniently characterized by subsequent MS/MS experiments: in a collision-induced dissociation the single half-spheres are easily discovered and structurally assigned. Thus, ESI-MS has worked as a valuable tool for the rapid screening of complex supramolecular mixtures and in combination with MS/MS experiments elucidated both the path of unexpected side reactions as well as the thermodynamic gas-phase stabilities of all components in the mixture.     

Rapid protein identification using a disposable on-line clean-up/concentrating device and electrospray ionization mass spectrometry

A simple, low-cost, expedient method has been developed for identification of proteins isolated from two-dimensional (2D) gels. The method described uses a disposable on-line clean-up device, a syringe infusion pump and electrospray ionization mass spectrometry (ESI-MS). The on-line clean-up and concentrating device is a tapered capillary column filled with 1.5 cm of 5 microm C18 particles. The short column was easily prepared and was connected directly to the ESI source through a low-flow ESI sprayer. Peptides resulting from enzymatic digestion of proteins were eluted from the short column isocratically using a syringe infusion pump and analyzed by ESI-MS. This simple set-up was found useful in the analysis of proteins isolated from 2D gels. Compared to the more conventional micro-liquid chromatography/tandem mass spectrometry (microLC/MS/MS), this method can identify proteins rapidly without the need for an HPLC pump and removes the problem of cross-contamination caused by system carryover. These advantages make the method described competitive with conventional LC/MS even though the latter method gives slightly expanded sequence coverage.     

Analysis of iron-phytosiderophore complexes in soil related samples: LC-ESI-MS/MS versus CE-MS

Phytosiderophores (PS) form stable complexes with various transition metals. These ligands are exuded by the roots of graminacous plants as a mechanism for mobilizing and acquiring soil iron. To investigate iron mobilization and transport, a novel LC method in combination with ESI-MS/MS for the determination of three Fe(III)-complexes with mugineic acid (MA), 2'-epi-MA and 2'-deoxymugineic acid (DMA) has been developed. Liquid chromatographic separation was realized using a silica-based mixed-mode reversed phase/weak-anion exchange type stationary phase and a 50 mM ammonium acetate buffer, pH 6.5. Baseline separation of the two complex diastereomers Fe(III)-MA and Fe(III)-epi-MA could be achieved. ESI-MS/MS detection allowed for simultaneous quantification of the complexes and the free ligands. Limits of detection were determined to be 0.001 and 0.05 μM for DMA and Fe(III)-DMA, respectively. The analytical figures of merit of the novel method were evaluated and compared with a CE-ESI-MS method that we had published earlier. The LC-ESI-MS/MS method has been successfully applied to real samples derived from preliminary extraction experiments.     

The mechanism of Tröger's base formation probed by electrospray ionization mass spectrometry

Using direct infusion electrospray ionization mass and tandem mass spectrometric experiments [ESI-MS(/MS)], we have performed on-line monitoring of some reactions used to form Tr?ger's bases. Key intermediates, either as cationic species or as protonated forms of neutral species, have been intercepted and characterized. The role of urotropine as the methylene source in these reactions has also been accessed. Reaction pathways shown by ESI-MS(/MS) have been probed by gas-phase ion/molecule reactions, and an expanded mechanism for Tr?ger's base formation based on the mass spectrometric data has been elaborated.     

Analysis of phospholipids by NACE with on-line ESI-MS

A hyphenated method of nonaqueous capillary electrophoresis coupled to electrospray ionization mass spectrometry (NACE-ESI-MS) is described for the simultaneous analysis of phospholipids. The best results were obtained with a mixed solution of methanol/ACN (40:60 v/v) containing 20 mM ammonium acetate and 0.5% acetic acid, under the applied voltage of 30 kV and capillary temperature of 25 degrees C. ESI-MS measurements were performed in the negative mode with methanol/ACN (40:60 v/v) containing 50 mM ammonium acetate as sheath liquid at a flow rate of 2 microL/min. Different phospholipid classes have been successfully separated within 16 min, and the molecular species of every single class have been identified by using MS(2) or MS(3), which generates characteristic fragments through CID. The developed method has been applied to analyze the phospholipids extracted from rat peritoneal surface and the molecular species of phospholipid classes are presented.     

Determination of 1,1-Dimethylhydrazine and its Transformation Products in Soil by Zwitterionic Hydrophilic Interaction Liquid Chromatography/Tandem Mass Spectrometry

1,1-Dimethylhydrazine is widely used as a fuel by some classes of carrier rockets. Being an extremely toxic and reactive substance, it gives a number of hazardous transformation products and poses a serious threat to the ecological state of the launch sites and territories used for landing of spent rocket parts. On the basis of studies of the retention of analytes on the sulfobetaine zwitterionic stationary phase, the HILIC–ESI-MS/MS method for simultaneous and rapid determination of unsymmetrical dimethylhydrazine and six major products of its transformation (methylhydrazine, N-nitrosodimethylamine, N,N-dimethylformamide, 1-methyl-1,2,4-1H-triazole, 1,1,4,4-tetramethyl-2-tetrazene, 1,1-dimethylguanidine) was developed. The achieved detection limits for the analytes were 0.02–7 μg L^?1 and, for most compounds, they are significantly lower compared to the existing IC–MS/MS method. Direct combination of HILIC–MS/MS with preliminary pressurized extraction with acetonitrile allowed analysis of peat bog soils contaminated with rocket fuel within 40 min, including all sample preparation steps. The developed method was successfully tested on a sample of real soil from the falling place of the spent carrier rocket stage.     

Quantitative analysis of a quaternary ammonium drug: ipratropium bromide by LC/ESI-MS(n) in horse plasma and urine

A quantitative method, using LC/ESI-MS(n) with a quadrupole linear ion trap mass analyzer, has been developed for the analysis of ipratropium cation in horse plasma and urine. The method applies solid-phase extraction with WCX cartridges for plasma and MM2 cartridges for urine, prior to analysis by LC/ESI-MS(n). The efficiency of extraction combined with the sensitivity and the selectivity of MS(n) allows for the quantification of ipratropium cation at picogram per milliliter levels. The analytical capabilities of the method have been successfully checked by the quantitative analysis of ipratropium cation in post-administration samples collected from horses treated by nebulization.     

Development of capillary zone electrophoresis-electrospray ionization-mass spectrometry for the determination of lamotrigine in human plasma

A method of coupling capillary zone electrophoresis (CZE) with electrospray ionization-mass spectrometry (ESI-MS) detection has been developed for monitoring an antiepileptic drug, lamotrigine (LTG) in human plasma. The CZE-MS was developed in three stages: (i) CZE separation and ESI-MS detection of LTG and tyramine (TRM, internal standard) were simultaneously optimized by studying the influence of CZE background electrolyte (BGE) pH, BGE ionic strength, and nebulizer pressure of the MS sprayer; (ii) sheath liquid parameters, such as pH, ionic strength, organic modifier content, and flow rate of the sheath liquid, were systematically varied under optimum CZE-MS conditions developed in the first stage; (iii) MS sprayer chamber parameters (drying gas temperature and drying gas flow rate) were varied for the best MS detection of LTG. The developed assay was finally applied for the determination of LTG in plasma samples. The linear range of LTG in plasma sample assay was between 0.1-5.0 microg/mL with a limit of detection as low as 0.05 microg/mL and run time less than 6 min. Finally, the concentration-time profile of LTG in human plasma sample was found to correlate well when CZE-ESI-MS was compared to a more established method of high-performance liquid chromatography with ultraviolet detection.     

Evaluation of a method based on liquid chromatography/electrospray tandem mass spectrometry for analyzing eight triazolic and pyrimidine fungicides in extracts of processed fruits and vegetables

The feasibility of using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) for determining 8 fungicides (triadimenol, penconazole, propiconazole, hexaconazole, cyproconazole, myclobutanil, fenarimol, and bitertanol) in extracts of tomato puree and lemon juice concentrate has been evaluated. A miniaturized extraction-partition procedure requiring small amounts of nonchlorinated solvents has been used. The extracts (5 microL) were analyzed by LC/ESI-MS/MS without any previous cleanup step. Chromatographic determination has been performed using a C18 column and isocratic elution. Seventeen MS/MS transitions of precursor ions were monitored simultaneously (2 or 3 for each pesticide). The excellent selectivity and good linearity of the LC/MS/MS method allowed quantitation and identification at low levels (limits of quantitation <0.010 mg/kg), even in difficult matrixes, with a run time of only 1.5 min.     

Novel phosphoryl derivatization method for peptide sequencing by electrospray ionization mass spectrometry

A one-step phosphoryl derivatization method has been used in a peptide sequencing procedure for electrospray ionization tandem mass spectrometry (ESI-MS/MS). The sodiated derivatized peptides exhibit very simple dissociation patterns, in which two kinds of fragment ions, [b(n) + OH + Na]+ and [a(n) + Na]+, are formed. Since the amino acid residues are lost sequentially from the C-terminus, peptide sequences can be identified easily. The fragmentation efficiency of peptides increased as a result of the phosphorylation, and also provided peaks of useful intensity at lower m/z. A peptide with lysine at the C-terminus was derivatized and analyzed by ESI-MS/MS. Similar mass spectra, from which the sequence could be read out, were obtained. This is a novel derivatization method yielding neutral derivatives that should be suitable for peptide sequencing by LC/ESI-MS/MS.     

Top-down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry

A system of microchip capillary electrophoresis/electrospray ionization mass spectrometry (microchip-CE/ESI-MS) for rapid characterization of proteins has been developed. Capillary electrophoresis (CE) enables rapid analysis of a sample present in very small quantity, such as at femtomole levels, at high resolution. Faster CE/MS analysis is expected by downsizing the normal capillary to the microchip (microchip) capillary. Although rapidity and high resolution are advantages of CE separation, electroosmotic flow (EOF) instability caused by the interaction between proteins and the microchannel surface results in low reproducibility in the analysis of basic proteins under neutral pH conditions. By coating the microchannel surface with a basic polymer, polyE-323, basic proteins, which have pI values of over 7.5, could be separated and detected by microchip-CE/MS on quadrupole (Q) and time-of-flight (TOF) hybrid instruments. By increasing the cone and collision voltages during the analysis by microchip-CE/ESI-MS of a small protein, some product ions, which contain the sequence information, could also be obtained, i.e., 'top-down' analysis of the protein could be accomplished with this microchip-CE/MS system. To our knowledge, this is the first report of 'top-down' analysis of a protein by microchip-CE/MS. Since it requires a much shorter time and a smaller sample amount for analysis than the conventional liquid chromatography (LC)/ESI-MS method, microchip-CE/MS promises to be suitable for the high-throughput characterization of proteins.     

Matrix-assisted laser desorption/ionization directed nano-electrospray ionization tandem mass spectrometric analysis for protein identification

In those cases where the information obtained by peptide mass fingerprinting or matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) is not sufficient for unambiguous protein identification, nano-electrospray ionization (nano-ESI) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis must be performed. The sensitivity of nano-ESI/MS, however, is lower than that of MALDI-MS, especially at very low analyte concentrations and/or in the presence of contaminants, such as salt and detergents. Moreover, to perform ESI-MS/MS, the peptide masses of the precursor ions must be known. The approach described in this paper, MALDI-directed nano-ESI-MS/MS, makes use of information obtained from the more sensitive MALDI-MS experiments in order to direct subsequent nano-ESI-MS/MS experiments. Peptide molecular ions found in the MALDI-MS analysis are then selected, as their (+2) precursor ions, for nano-ESI-MS/MS sequencing, even though these ions cannot be detected in the ESI-MS spectra. This method, originally proposed by Tempst et al. (Anal. Chem. 2000, 72: 777-790), has been extended to provide better sensitivity and shorter analysis times; also, a comparison with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been performed. These experiments, performed using quadrupole time-of-flight instruments equipped with commercially available nano-ESI sources, have allowed the unambiguous identification of in-gel digested proteins at levels below their ESI-MS detection limits, even in the presence of salts and detergents.     

Characterization of isomeric cationic porphyrins with β-pyrrolic substituents by electrospray mass spectrometry: The singular behavior of a potential virus photoinactivator

Electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) have been used to differentiate the 2- and 4-methylpyridyl isomers of free-base and metallated cationic beta-vinylpyridylporphyrins. The analysis by ESI-MS/MS of the deuterated analogs and semiempirical calculations of structural and electronic parameters were also undertaken. The two free-base isomers are easily differentiated by ESI-MS/MS but the presence of a metallic center renders differentiation of the metallated isomers less effective. The data acquired show that of all the studied compounds, the free-base 2-methylpyridyl isomer, which was operative in the in vitro photoinactivation of Herpes simples virus, has a different gas-phase behavior. Local distortion of the macrocycle due to the presence of the beta-vinylpyridyl substituent occurs for all the compounds, but a different electron density distribution can account for the observed gas-phase behavior of this potential virus photoinactivator.     

Microfluidic technologies for MALDI-MS in proteomics

The field of microfluidics continues to offer great promise as an enabling technology for advanced analytical tools. For biomolecular analysis, there is often a critical need to couple on-chip microfluidic sample manipulation with back-end MS. Though interfacing microfluidics to MS has been most often reported through the use of direct ESI-MS, there are compelling reasons for coupling microfluidics to MALDI-MS as an alternative to ESI-MS for both online and offline analysis. The intent of this review is to provide a summary of recent developments in the integration of microfluidic systems with MALDI-MS, with an emphasis on applications in proteomics. Key points are summarized, followed by a review of relevant technologies and a discussion of outlook for the field.     

Ring contraction reactions of 2,3-dihydro-4H-1,3-oxazin-4-ones under electrospray ionization conditions

The mass spectral behavior of 2,3-dihydro-4H-1,3-oxazin-4-ones has been investigated using electrospray ionization multi-stage mass spectrometry (ESI-MS(n)). All compounds showed a predominant retro-Diels-Alder (RDA) fragmentation pathway, and a novel ring contraction reaction by loss of isocynates was also found. The fragmentation mechanisms proposed for 4H-1,3-oxazin-4-ones are supported by ESI-MS/MS/MS spectra.     

A new method for the identification and the structural characterisation of carotenoid esters in freshwater microorganisms by liquid chromatography/electrospray ionisation tandem mass spectrometry

Liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) has been employed to identify carotenoid esters present in raw organic extracts of pigmented freshwater microalgae and to gain structural information on these compounds. In particular, acyl carotenoid derivatives of Haematococcus pluvialis and Euglena sanguinea have been characterised by tandem mass spectrometry (MS/MS) in a quadrupole ion trap. ESI-MS/MS allows recognition of the presence of carotenoid esters in complicated mixtures without any initial chromatographic work-up and without the need to use UV-Vis photo-diode array (PDA) detectors. Product ion scans of the [M + Na]+ ion lead to known neutral losses of the C7H8 and C8H10 residues from the conjugated polyene moiety of the carotenoid unit, that permit the unambiguous identification of the carotenoid itself. These structurally relevant ions are not observed in positive or negative ion APCI (atmospheric pressure chemical ionisation) mass spectra. Moreover, the several product ions observed in positive and/or negative ion ESI-MS/MS not only are a diagnostic signature of the main structural features of the acyl chains such as length, position and unsaturation, but also display the nominal mass of the parent xanthophyll. Our methodology has been validated (i) by using esters of astaxanthin obtained from off-line purification of the H. pluvialis extracts and structurally elucidated through proton nuclear magnetic resonance (1H-NMR) spectroscopy and (ii) by product analysis of esters by alkaline hydrolysis. The characterisation of the unknown carotenoid esters of E. sanguinea is a demonstration of the capabilities of this methodology.     

Porous graphitic carbon chromatography/tandem mass spectrometric determination of cytarabine in mouse plasma

A high-performance liquid chromatography (HPLC) system using a porous graphitic carbon (PGC) stationary phase interfaced with an electrospray ionization (ESI) source and a tandem mass spectrometer (MS/MS) for the analysis of cytarabine (ara-C) in mouse plasma samples has been developed in support of a pharmacodynamic study. The graphitized carbon column was adopted for the separation of ara-C and endogenous peaks from mouse plasma samples under the reversed-phase phase mode in liquid chromatography. The retention characteristics of the PGC column and the ionization efficiencies of all analytes based on the experimental factors such as the composition of mobile phases were investigated. The potential of ionization suppression resulting from the endogenous biological matrices on the PGC column during HPLC/ESI-MS/MS was investigated using post-column infusion. The concentrations of ara-C in mouse plasma obtained by using PGC-HPLC/MS/MS and ion-pairing HPLC/MS/MS were found to be in good agreement in terms of analytical accuracy.     

Growth hormone abuse in the horse: preliminary assessment of a mass spectrometric procedure for IGF-1 identification and quantitation.

Previous studies have shown that insulin-like growth factor 1 (IGF-1) is a promising marker for the detection of growth hormone (GH) abuse in the horse. The significant increases observed with GH administration in comparison to natural levels imply the possibility of setting a threshold level for IGF-1 that would be indicative of GH abuse. Although an immunoradiometric assay (IRMA) has been identified as a reliable screening method, a more specific IGF-1 quantification method needs to be developed for the prosecution of GH abuse by horseracing authorities. This study describes such an HPLC electrospray mass spectrometry (LC/ESI-MS) method that was developed and then assessed for the specific analysis of IGF-1 at the low levels encountered in serum. The structural identity of IGF-1 was confirmed by endoproteinase Asp-N digestion followed by LC/MS and LC/MS/MS characterisation. This was followed by quantification of IGF-1 as the intact molecule against an internal standard.