Rapid Communications in Mass Spectrometry Wiley Online Library : Rapid Communications in Mass Spectrometryhttp://dx.doi.org/10.1002%2F%28ISSN%291097-0231John Wiley & Sons, Incen© John Wiley & Sons, Ltd.0951-41981097-02312012-03-30T00:00:00-05:0030 March 2012266583718On the polymerization of hexavalent uranium. An electrospray mass spectrometry studyhttp://dx.doi.org/10.1002%2Frcm.6128On the polymerization of hexavalent uranium. An electrospray mass spectrometry studyM. SteppertC. WaltherM. FussS. Büchner2012-03-30T00:00:00-05:00doi:10.1002/rcm.6128John Wiley & Sons, Inc.10.1002/rcm.6128http://dx.doi.org/10.1002%2Frcm.6128Research Article583591

Polymerization in hexavalent uranium solutions was measured by electrospray ionization time-of-flight mass spectrometry in three different acidic media at pH values from 3 to 5.3 in order to detect all hydrolysis species present in solution. The aqueous solutions were directly measured without further dilution in organic solvents. At high uranyl concentrations ([U(VI)] = 10− 3 M) artifacts were observed due to the presence of more than one solution species per formed microdroplet. Those artifacts were composed of ions and neutral species being present in the same droplet. However, by analyzing the detected species carefully, the origin of the artifacts could be traced back to the physically meaningful species. Still, only general trends of the hydrolysis behavior can be deduced from the measurements at [U(VI)] = 1 ⋅ 10− 3 M. The solutions at [U(VI)] = 5 ⋅ 10− 5 M did not show any comparable artifact formation. The detected species distributions resemble the expected trends calculated from the equilibrium constants published in the Nuclear Energy Agency Thermodynamic Database (NEA-TDB). The neutral (UO2)(CO3)0 species present in solution causes, if located in the same microdroplet as a charged species, the apparent formation of dimeric and trimeric ternary hydroxo carbonate complexes at pH 5.3. As the uncharged species is not repelled from the ionic species, it might remain in the same droplet during the droplet fission process. By dividing those detected species into the uncharged (UO2)(CO3)0 and a second ionic species, the relative abundances of the solution species can be corrected, leading to a good agreement with the predictions of the published equilibrium constants. In addition to the well-known trimer, we report the direct mass spectrometric detection of the dimeric inline image species. Copyright © 2012 John Wiley & Sons, Ltd.

]]>Polymerization in hexavalent uranium solutions was measured by electrospray ionization time-of-flight mass spectrometry in three different acidic media at pH values from 3 to 5.3 in order to detect all hydrolysis species present in solution. The aqueous solutions were directly measured without further dilution in organic solvents. At high uranyl concentrations ([U(VI)] = 10− 3 M) artifacts were observed due to the presence of more than one solution species per formed microdroplet. Those artifacts were composed of ions and neutral species being present in the same droplet. However, by analyzing the detected species carefully, the origin of the artifacts could be traced back to the physically meaningful species. Still, only general trends of the hydrolysis behavior can be deduced from the measurements at [U(VI)] = 1 ⋅ 10− 3 M. The solutions at [U(VI)] = 5 ⋅ 10− 5 M did not show any comparable artifact formation. The detected species distributions resemble the expected trends calculated from the equilibrium constants published in the Nuclear Energy Agency Thermodynamic Database (NEA-TDB). The neutral (UO2)(CO3)0 species present in solution causes, if located in the same microdroplet as a charged species, the apparent formation of dimeric and trimeric ternary hydroxo carbonate complexes at pH 5.3. As the uncharged species is not repelled from the ionic species, it might remain in the same droplet during the droplet fission process. By dividing those detected species into the uncharged (UO2)(CO3)0 and a second ionic species, the relative abundances of the solution species can be corrected, leading to a good agreement with the predictions of the published equilibrium constants. In addition to the well-known trimer, we report the direct mass spectrometric detection of the dimeric (UO2)2(OH)22+ species. Copyright © 2012 John Wiley & Sons, Ltd.(S)-(−)-N-(Pentafluorobenzylcarbamoyl)prolyl chloride: a chiral derivatisation reagent designed for gas chromatography/negative ion chemical ionisation mass spectrometry of amino compoundshttp://dx.doi.org/10.1002%2Frcm.6146(S)-(−)-N-(Pentafluorobenzylcarbamoyl)prolyl chloride: a chiral derivatisation reagent designed for gas chromatography/negative ion chemical ionisation mass spectrometry of amino compoundsHans J. LeisWerner Windischhofer2012-03-30T00:00:00-05:00doi:10.1002/rcm.6146John Wiley & Sons, Inc.10.1002/rcm.6146http://dx.doi.org/10.1002%2Frcm.6146Research Article592598

RATIONALE

The synthesis of a novel chiral derivatisation reagent, (S)-(−)-N-(pentafluorobenzylcarbamoyl)prolyl chloride is described which is preferably useful for negative-ion chemical ionisation mass spectrometry.

METHODS

Preparation of the reagent followed a general strategy used to prepare enantioselective reagents based on the N-substitution of L-proline. Pentafluorobenzyl chloroformate smoothly reacted with L-proline to give the desired derivatisation reagent after conversion into the acyl chloride. The product was sufficiently pure to be used in the following steps without any additional purification.

RESULTS

The reagent was tested against selected chiral and non-chiral analytical targets. Chromatographic enantioseparation was at least equal to the commonly used (S)-(−)-N-(heptafluorobutyryl)prolyl derivatives. The derivatives exhibit excellent mass spectral properties under negative ion chemical ionisation, i.e. reduced fragmentation and thus high ion current for the targeted m/z during analysis. With electron ionisation, the fragmentation that occurs is mainly directed by the introduced group. Enantioseparation with gas chromatography/negative-ion chemical ionisation mass spectrometry of the derivatives was demonstrated for the enantiomers of amphetamine, α-aminocaprylic acid methyl ester and threo-methylphenidate.

CONCLUSIONS

The new derivatisation reagent shows highly improved mass spectral properties for negative-ion chemical ionisation mass spectrometry and is thus suitable for sensitive chiral detection of amino compounds. The reagent extends the applicability of dissociative resonance electron capture using pentafluorobenzyl derivatives to chiral analysis. Copyright © 2012 John Wiley & Sons, Ltd.

]]>RATIONALEThe synthesis of a novel chiral derivatisation reagent, (S)-(−)-N-(pentafluorobenzylcarbamoyl)prolyl chloride is described which is preferably useful for negative-ion chemical ionisation mass spectrometry.METHODSPreparation of the reagent followed a general strategy used to prepare enantioselective reagents based on the N-substitution of L-proline. Pentafluorobenzyl chloroformate smoothly reacted with L-proline to give the desired derivatisation reagent after conversion into the acyl chloride. The product was sufficiently pure to be used in the following steps without any additional purification.RESULTSThe reagent was tested against selected chiral and non-chiral analytical targets. Chromatographic enantioseparation was at least equal to the commonly used (S)-(−)-N-(heptafluorobutyryl)prolyl derivatives. The derivatives exhibit excellent mass spectral properties under negative ion chemical ionisation, i.e. reduced fragmentation and thus high ion current for the targeted m/z during analysis. With electron ionisation, the fragmentation that occurs is mainly directed by the introduced group. Enantioseparation with gas chromatography/negative-ion chemical ionisation mass spectrometry of the derivatives was demonstrated for the enantiomers of amphetamine, α-aminocaprylic acid methyl ester and threo-methylphenidate.CONCLUSIONSThe new derivatisation reagent shows highly improved mass spectral properties for negative-ion chemical ionisation mass spectrometry and is thus suitable for sensitive chiral detection of amino compounds. The reagent extends the applicability of dissociative resonance electron capture using pentafluorobenzyl derivatives to chiral analysis. Copyright © 2012 John Wiley & Sons, Ltd.Development of a liquid chromatography/atmospheric pressure photo-ionization high-resolution mass spectrometry analytical method for the simultaneous determination of polybrominated diphenyl ethers and their metabolites: application to BDE-47 metabolism in human hepatocyteshttp://dx.doi.org/10.1002%2Frcm.6136Development of a liquid chromatography/atmospheric pressure photo-ionization high-resolution mass spectrometry analytical method for the simultaneous determination of polybrominated diphenyl ethers and their metabolites: application to BDE-47 metabolism in human hepatocytesCharlotte MarteauSylvie ChevolleauIsabelle JouaninElisabeth PerduGeorges De SousaRoger RahmaniJean-Philippe AntignacBruno LeBizecDaniel ZalkoLaurent Debrauwer2012-03-30T00:00:00-05:00doi:10.1002/rcm.6136John Wiley & Sons, Inc.10.1002/rcm.6136http://dx.doi.org/10.1002%2Frcm.6136Research Article599610

Polybrominated diphenyl ethers (PBDEs) are flame retardants widely used in electronic and domestic goods. These persistent pollutants are present in the environment and in humans, and their toxicological properties are of growing concern. PBDEs can be metabolised into compounds suspected to be responsible for their toxicity. These metabolites have been characterised quite well in rodents and fish, but available information in humans remains scarce. For their identification, an efficient method for the simultaneous analysis of PBDEs, hydroxylated PBDEs (OH-PBDEs), and other PBDE metabolites in a single run was needed and has been developed in this work. Atmospheric pressure ionisation modes were compared, and Atmospheric Pressure Photo-Ionization (APPI) was selected. After careful setting of APPI parameters such as dopant and operating temperature, the optimised method was based on APPI ionization coupled to High-Resolution Mass Spectrometry operating in the full scan mode at a resolution of 60 000. This provided excellent sensitivity and specificity, allowing the discrimination of signals which could not be resolved on a triple quadrupole used as a reference. The full-scan high-resolution acquisition mode allowed monitoring of both parent PBDEs and their metabolites, including hydroxylated PBDEs, with detection limits ranging from 0.1 pg to 4.5 pg injected on-column based on the investigated standard compounds. The method was applied to the study of BDE-47 metabolism after incubation with human primary cultures of hepatocytes, and proved to be efficient not only for monitoring the parent compound and expected hydroxylated metabolites, but also for the identification of other non-targeted metabolites. In addition to hydroxy-BDE-47, several conjugated metabolites could be located, and the formation of a dihydrodiol derivative was evidenced for the first time in the case of PBDEs in this work. Copyright © 2012 John Wiley & Sons, Ltd.

]]>Polybrominated diphenyl ethers (PBDEs) are flame retardants widely used in electronic and domestic goods. These persistent pollutants are present in the environment and in humans, and their toxicological properties are of growing concern. PBDEs can be metabolised into compounds suspected to be responsible for their toxicity. These metabolites have been characterised quite well in rodents and fish, but available information in humans remains scarce. For their identification, an efficient method for the simultaneous analysis of PBDEs, hydroxylated PBDEs (OH-PBDEs), and other PBDE metabolites in a single run was needed and has been developed in this work. Atmospheric pressure ionisation modes were compared, and Atmospheric Pressure Photo-Ionization (APPI) was selected. After careful setting of APPI parameters such as dopant and operating temperature, the optimised method was based on APPI ionization coupled to High-Resolution Mass Spectrometry operating in the full scan mode at a resolution of 60 000. This provided excellent sensitivity and specificity, allowing the discrimination of signals which could not be resolved on a triple quadrupole used as a reference. The full-scan high-resolution acquisition mode allowed monitoring of both parent PBDEs and their metabolites, including hydroxylated PBDEs, with detection limits ranging from 0.1 pg to 4.5 pg injected on-column based on the investigated standard compounds. The method was applied to the study of BDE-47 metabolism after incubation with human primary cultures of hepatocytes, and proved to be efficient not only for monitoring the parent compound and expected hydroxylated metabolites, but also for the identification of other non-targeted metabolites. In addition to hydroxy-BDE-47, several conjugated metabolites could be located, and the formation of a dihydrodiol derivative was evidenced for the first time in the case of PBDEs in this work. Copyright © 2012 John Wiley & Sons, Ltd.Encoding substrates with mass tags to resolve stereospecific reactions using Nimzymehttp://dx.doi.org/10.1002%2Frcm.6134Encoding substrates with mass tags to resolve stereospecific reactions using NimzymeKai DengKevin W. GeorgeWolfgang ReindlJay D. KeaslingPaul D. AdamsTaek Soon LeeAnup K. SinghTrent R. Northen2012-03-30T00:00:00-05:00doi:10.1002/rcm.6134John Wiley & Sons, Inc.10.1002/rcm.6134http://dx.doi.org/10.1002%2Frcm.6134Research Article611615

RATIONALE

The nanostructure-initiator mass spectrometry based enzyme assay (Nimzyme) provides a rapid method for screening glycan modifying reactions. However, this approach cannot resolve stereospecific reactions which are common in glycobiology and are typically assayed using lower-throughput methods (gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis) often in conjunction with stable isotopically labeled reactants. However, in many applications, library size necessitates the development of higher-throughput screening approaches of stereospecific reactions from crude sample preparations. Therefore, here we test the approach of utilizing Nimzyme linkers with unique masses to encode substrate identity such that this assay can resolve stereospecific reactions.

METHODS

We utilize the nanostructure-initiator mass spectrometry (NIMS) enzyme assay in conjuction with an accurate mass tagging approach where each reactant is tagged with a unique perfluoronated tail. Mass spectrometric analysis was conducted using conventional MALDI-TOF instrumentation.

RESULTS

Stereospecific reaction pathways of three stereoisomers (maltose, lactose and cellobiose) to afford the same product glucose were resolved simutaneously due to the presence of unique fluorous tags on both reactants and products. Not only purified enzymes, but also crude cell lysates can be used in this assay.

CONCLUSIONS

The Nimzyme assay with accurate mass tagging provides a rapid method for screening for targeted stereospecific reactions using mass spectrometry and may be useful for high-throughput screening and functional annotation of a wide range of glycan-modifying enzymes. Copyright © 2012 John Wiley & Sons, Ltd.

]]>RATIONALEThe nanostructure-initiator mass spectrometry based enzyme assay (Nimzyme) provides a rapid method for screening glycan modifying reactions. However, this approach cannot resolve stereospecific reactions which are common in glycobiology and are typically assayed using lower-throughput methods (gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis) often in conjunction with stable isotopically labeled reactants. However, in many applications, library size necessitates the development of higher-throughput screening approaches of stereospecific reactions from crude sample preparations. Therefore, here we test the approach of utilizing Nimzyme linkers with unique masses to encode substrate identity such that this assay can resolve stereospecific reactions.METHODSWe utilize the nanostructure-initiator mass spectrometry (NIMS) enzyme assay in conjuction with an accurate mass tagging approach where each reactant is tagged with a unique perfluoronated tail. Mass spectrometric analysis was conducted using conventional MALDI-TOF instrumentation.RESULTSStereospecific reaction pathways of three stereoisomers (maltose, lactose and cellobiose) to afford the same product glucose were resolved simutaneously due to the presence of unique fluorous tags on both reactants and products. Not only purified enzymes, but also crude cell lysates can be used in this assay.CONCLUSIONSThe Nimzyme assay with accurate mass tagging provides a rapid method for screening for targeted stereospecific reactions using mass spectrometry and may be useful for high-throughput screening and functional annotation of a wide range of glycan-modifying enzymes. Copyright © 2012 John Wiley & Sons, Ltd.Study on the reactive transient α-λ3-iodanyl-acetophenone complex in the iodine(III)/PhI(I) catalytic cycle of iodobenzene-catalyzed α-acetoxylation reaction of acetophenone by electrospray ionization tandem mass spectrometryhttp://dx.doi.org/10.1002%2Frcm.6145Study on the reactive transient α-λ3-iodanyl-acetophenone complex in the iodine(III)/PhI(I) catalytic cycle of iodobenzene-catalyzed α-acetoxylation reaction of acetophenone by electrospray ionization tandem mass spectrometryHao-Yang WangJuan ZhouYin-Long Guo2012-03-30T00:00:00-05:00doi:10.1002/rcm.6145John Wiley & Sons, Inc.10.1002/rcm.6145http://dx.doi.org/10.1002%2Frcm.6145Research Article616620

RATIONALE

Hypervalent iodine compounds are important and widely used oxidants in organic chemistry. In 2005, Ochiai reported the PhI-catalyzed α-acetoxylation reaction of acetophenone by the oxidation of PhI with m-chloroperbenzoic acid (m-CPBA) in acetic acid. However, until now, the most critical reactive α-λ3-iodine alkyl acetophenone intermediate (3) had not been isolated or directly detected.

METHODS

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to intercept and characterize the transient reactive α-λ3-iodine alkyl acetophenone intermediate in the reaction solution.

RESULTS

The trivalent iodine species was detected when PhI and m-CPBA in acetic acid were mixed, which indicated the facile oxidation of a catalytic amount of PhI(I) to the iodine(III) species by m-CPBA. Most importantly, 3·H+ was observed at m/z 383 from the reaction solution and this ion gave the protonated α-acetoxylation product 4·H+ at m/z 179 in MS/MS by an intramolecular reductive elimination of PhI.

CONCLUSIONS

These ESI-MS/MS studies showed the existence of the reactive α-λ3-iodine alkyl acetophenone intermediate 3 in the catalytic cycle. Moreover, the gas-phase reactivity of 3·H+ was consistent with the proposed solution-phase reactivity of the α-λ3-iodine alkyl acetophenone intermediate 3, thus confirming the reaction mechanism proposed by Ochiai. Copyright © 2012 John Wiley & Sons, Ltd.

]]>RATIONALEHypervalent iodine compounds are important and widely used oxidants in organic chemistry. In 2005, Ochiai reported the PhI-catalyzed α-acetoxylation reaction of acetophenone by the oxidation of PhI with m-chloroperbenzoic acid (m-CPBA) in acetic acid. However, until now, the most critical reactive α-λ3-iodine alkyl acetophenone intermediate (3) had not been isolated or directly detected.METHODSElectrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to intercept and characterize the transient reactive α-λ3-iodine alkyl acetophenone intermediate in the reaction solution.RESULTSThe trivalent iodine species was detected when PhI and m-CPBA in acetic acid were mixed, which indicated the facile oxidation of a catalytic amount of PhI(I) to the iodine(III) species by m-CPBA. Most importantly, 3·H+ was observed at m/z 383 from the reaction solution and this ion gave the protonated α-acetoxylation product 4·H+ at m/z 179 in MS/MS by an intramolecular reductive elimination of PhI.CONCLUSIONSThese ESI-MS/MS studies showed the existence of the reactive α-λ3-iodine alkyl acetophenone intermediate 3 in the catalytic cycle. Moreover, the gas-phase reactivity of 3·H+ was consistent with the proposed solution-phase reactivity of the α-λ3-iodine alkyl acetophenone intermediate 3, thus confirming the reaction mechanism proposed by Ochiai. Copyright © 2012 John Wiley & Sons, Ltd.Analysis of cancer cell lipids using matrix-assisted laser desorption/ionization 15-T Fourier transform ion cyclotron resonance mass spectrometryhttp://dx.doi.org/10.1002%2Frcm.6140Analysis of cancer cell lipids using matrix-assisted laser desorption/ionization 15-T Fourier transform ion cyclotron resonance mass spectrometryHyo-Jik YangKyu Hwan ParkDong Wan LimHyun Sik KimJeongkwon Kim2012-03-30T00:00:00-05:00doi:10.1002/rcm.6140John Wiley & Sons, Inc.10.1002/rcm.6140http://dx.doi.org/10.1002%2Frcm.6140Research Article621630

A combination of methodologies using the extremely high mass accuracy and resolution of 15-T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) was introduced for the identification of intact cancer cell phospholipids. Lipids from a malignant glioma cell line were initially analyzed at a resolution of >200 000 and identified by setting the mass tolerance to ±1 mDa using matrix-assisted laser desorption/ionization (MALDI) 15-T FT-ICR MS in positive ion mode. In most cases, a database search of potential lipid candidates using the exact masses of the lipids yielded only one possible chemical composition. Extremely high mass accuracy (<0.1 ppm) was then attained by using previously identified lipids as internal standards. This, combined with an extremely high resolution (>800 000), yielded well-resolved isotopic fine structures allowing for the identification of lipids by MALDI 15-T FT-ICR MS without using tandem mass spectrometric (MS/MS) analysis. Using this method, a total of 38 unique lipids were successfully identified. Copyright © 2012 John Wiley & Sons, Ltd.

]]>A combination of methodologies using the extremely high mass accuracy and resolution of 15-T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) was introduced for the identification of intact cancer cell phospholipids. Lipids from a malignant glioma cell line were initially analyzed at a resolution of >200 000 and identified by setting the mass tolerance to ±1 mDa using matrix-assisted laser desorption/ionization (MALDI) 15-T FT-ICR MS in positive ion mode. In most cases, a database search of potential lipid candidates using the exact masses of the lipids yielded only one possible chemical composition. Extremely high mass accuracy (<0.1 ppm) was then attained by using previously identified lipids as internal standards. This, combined with an extremely high resolution (>800 000), yielded well-resolved isotopic fine structures allowing for the identification of lipids by MALDI 15-T FT-ICR MS without using tandem mass spectrometric (MS/MS) analysis. Using this method, a total of 38 unique lipids were successfully identified. Copyright © 2012 John Wiley & Sons, Ltd.Substituent effects on the gas-phase fragmentation reactions of protonated peptides containing benzylamine-derivatized lysyl residueshttp://dx.doi.org/10.1002%2Frcm.6141Substituent effects on the gas-phase fragmentation reactions of protonated peptides containing benzylamine-derivatized lysyl residuesE. S. SimonP. G. PapouliasP. C. Andrews2012-03-30T00:00:00-05:00doi:10.1002/rcm.6141John Wiley & Sons, Inc.10.1002/rcm.6141http://dx.doi.org/10.1002%2Frcm.6141Research Article631638

Motivated by the need for chemical strategies designed to tune peptide fragmentation to selective cleavage reactions, benzyl ring substituent influence on the relative formation of carbocation elimination (CCE) products from peptides with benzylamine-derivatized lysyl residues has been examined using collision-induced dissociation (CID) tandem mass spectrometry. Unsubstituted benzylamine-derivatized peptides yield a mixture of products derived from amide backbone cleavage and CCE. The latter involves side-chain cleavage of the derivatized lysyl residue to form a benzylic carbocation [C7H7]+ and an intact peptide product ion [(MHn)n+ – (C7H7)+](n-1)+. The CCE pathway is contingent upon protonation of the secondary ε-amino group (Nε) of the derivatized lysyl residue. Using the Hammett methodology to evaluate the electronic contributions of benzyl ring substituents on chemical reactivity, a direct correlation was observed between changes in the CCE product ion intensity ratios (relative to backbone fragmentation) and the Hammett substituent constants, σ, of the corresponding substituents. There was no correlation between the substituent-influenced gas-phase proton affinity of Nε and the relative ratios of CCE product ions. However, a strong correlation was observed between the π orbital interaction energies (ΔEint) of the eliminated benzylic carbocation and the logarithm of the relative ratios, indicating the predominant factor in the CCE pathway is the substituent effect on the level of hyperconjugation and resonance stability of the eliminated benzylic carbocation. This work effectively demonstrates the applicability of σ (and ΔEint) as substituent selection parameters for the design of benzyl-based peptide-reactive reagents which tune CCE product formation as desired for specific applications. Copyright © 2012 John Wiley & Sons, Ltd.

]]>Motivated by the need for chemical strategies designed to tune peptide fragmentation to selective cleavage reactions, benzyl ring substituent influence on the relative formation of carbocation elimination (CCE) products from peptides with benzylamine-derivatized lysyl residues has been examined using collision-induced dissociation (CID) tandem mass spectrometry. Unsubstituted benzylamine-derivatized peptides yield a mixture of products derived from amide backbone cleavage and CCE. The latter involves side-chain cleavage of the derivatized lysyl residue to form a benzylic carbocation [C7H7]+ and an intact peptide product ion [(MHn)n+ – (C7H7)+](n-1)+. The CCE pathway is contingent upon protonation of the secondary ε-amino group (Nε) of the derivatized lysyl residue. Using the Hammett methodology to evaluate the electronic contributions of benzyl ring substituents on chemical reactivity, a direct correlation was observed between changes in the CCE product ion intensity ratios (relative to backbone fragmentation) and the Hammett substituent constants, σ, of the corresponding substituents. There was no correlation between the substituent-influenced gas-phase proton affinity of Nε and the relative ratios of CCE product ions. However, a strong correlation was observed between the π orbital interaction energies (ΔEint) of the eliminated benzylic carbocation and the logarithm of the relative ratios, indicating the predominant factor in the CCE pathway is the substituent effect on the level of hyperconjugation and resonance stability of the eliminated benzylic carbocation. This work effectively demonstrates the applicability of σ (and ΔEint) as substituent selection parameters for the design of benzyl-based peptide-reactive reagents which tune CCE product formation as desired for specific applications. Copyright © 2012 John Wiley & Sons, Ltd.ISO-CADICA: Isotopic – continuous, automated dissolved inorganic carbon analyserhttp://dx.doi.org/10.1002%2Frcm.6143ISO-CADICA: Isotopic – continuous, automated dissolved inorganic carbon analyserAdrian M. BassMichael I. BirdNiels C. MunksgaardChristopher M. Wurster2012-03-30T00:00:00-05:00doi:10.1002/rcm.6143John Wiley & Sons, Inc.10.1002/rcm.6143http://dx.doi.org/10.1002%2Frcm.6143Research Article639644

RATIONALE

Quantifying the processes that control dissolved inorganic carbon (DIC) dynamics in aquatic systems is essential for progress in ecosystem carbon budgeting. The development of a methodology that allows high-resolution temporal data collection over prolonged periods is essential and is described in this study.

METHODS

A novel sampling instrument that sequentially acidifies aliquots of water and utilises gas-permeable ePTFE tubing to measure the dissolved inorganic carbon (DIC) concentration and δ13CDIC values at sub-hourly intervals by Cavity Ring-down spectrometry (CRDS) is described.

RESULTS

The minimum sensitivity of the isotopic, continuous, automated dissolved inorganic carbon analyser (ISO-CADICA) system is 0.01 mM with an accuracy of 0.008 mM. The analytical uncertainty in δ13CDIC values is proportional to the concentration of DIC in the sample. Where the DIC concentration is greater than 0.3 mM the analytical uncertainty is ±0.1 ‰ and below 0.2 mM stability is < ± 0.3 ‰. The isotopic effects of air temperature, water temperature and CO2 concentrations were found to either be negligible or correctable. Field trials measuring diel variation in δ13CDIC values of coral reef associated sea water revealed significant, short-term temporal changes and illustrated the necessity of this technique.

CONCLUSIONS

Currently, collecting and analysing large numbers of samples for δ13CDIC measurements is not trivial, but essential for accurate carbon models, particularly on small scales. The ISO-CADICA enables on-site, high-resolution determination of DIC concentration and δ13CDIC values with no need for sample storage and laboratory analysis. The initial tests indicate that this system can offer accuracy approaching that of traditional IRMS analysis. Copyright © 2012 John Wiley & Sons, Ltd.

]]>RATIONALEQuantifying the processes that control dissolved inorganic carbon (DIC) dynamics in aquatic systems is essential for progress in ecosystem carbon budgeting. The development of a methodology that allows high-resolution temporal data collection over prolonged periods is essential and is described in this study.METHODSA novel sampling instrument that sequentially acidifies aliquots of water and utilises gas-permeable ePTFE tubing to measure the dissolved inorganic carbon (DIC) concentration and δ13CDIC values at sub-hourly intervals by Cavity Ring-down spectrometry (CRDS) is described.RESULTSThe minimum sensitivity of the isotopic, continuous, automated dissolved inorganic carbon analyser (ISO-CADICA) system is 0.01 mM with an accuracy of 0.008 mM. The analytical uncertainty in δ13CDIC values is proportional to the concentration of DIC in the sample. Where the DIC concentration is greater than 0.3 mM the analytical uncertainty is ±0.1 ‰ and below 0.2 mM stability is < ± 0.3 ‰. The isotopic effects of air temperature, water temperature and CO2 concentrations were found to either be negligible or correctable. Field trials measuring diel variation in δ13CDIC values of coral reef associated sea water revealed significant, short-term temporal changes and illustrated the necessity of this technique.CONCLUSIONSCurrently, collecting and analysing large numbers of samples for δ13CDIC measurements is not trivial, but essential for accurate carbon models, particularly on small scales. The ISO-CADICA enables on-site, high-resolution determination of DIC concentration and δ13CDIC values with no need for sample storage and laboratory analysis. The initial tests indicate that this system can offer accuracy approaching that of traditional IRMS analysis. Copyright © 2012 John Wiley & Sons, Ltd.Dried blood spot (DBS) sample collection for determination of the oxidative stress biomarker 8-epi-PGF2α in humans using liquid chromatography/tandem mass spectrometryhttp://dx.doi.org/10.1002%2Frcm.6149Dried blood spot (DBS) sample collection for determination of the oxidative stress biomarker 8-epi-PGF2α in humans using liquid chromatography/tandem mass spectrometryNasser E. BastaniThomas E. GundersenRune Blomhoff2012-03-30T00:00:00-05:00doi:10.1002/rcm.6149John Wiley & Sons, Inc.10.1002/rcm.6149http://dx.doi.org/10.1002%2Frcm.6149Research Article645652

RATIONALE

F2-isoprostanes are a series of prostaglandin F2-like compounds that are formed by free-radical-catalyzed peroxidation of arachidonic acid (ARA). Several F2-isoprostanes, but in particular 8-epi-PGF2α, are widely used as oxidative stress biomarkers. In this study we have developed an analytical tool for finger-tip blood sampling and analysis of 8-epi-PGF2α from dried blood spots (DBS).

METHODS

We have applied solid-phase extraction (SPE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the extraction, separation and detection of 8-epi-PGF2α in DBS and have studied the stability of this marker using the DBS collection platform.

RESULTS

The mass limit of detection (mLOD) for 8-epi-PGF2α extracted from DBS samples was 1.5 pg while the concentration limit of detection (cLOD) and concentration limit of quantitation (cLOQ) were 6 pg/mL and 18 pg/mL, respectively. All values based on triplicate analysis. Sufficient stability of 8-epi-PGF2α in DBS was achieved by preconditioning DBS paper with vitamin E and BHT.

CONCLUSIONS

The developed method is sensitive, specific, robust, efficient, and can accurately measure endogenous concentrations of 8-epi-PGF2α in DBS. Thus, it offers an analytical approach to measure 8-epi-PGF2α by a novel sample collection technique that is less invasive and costly than conventional techniques. Copyright © 2012 John Wiley & Sons, Ltd.

]]>RATIONALEF2-isoprostanes are a series of prostaglandin F2-like compounds that are formed by free-radical-catalyzed peroxidation of arachidonic acid (ARA). Several F2-isoprostanes, but in particular 8-epi-PGF2α, are widely used as oxidative stress biomarkers. In this study we have developed an analytical tool for finger-tip blood sampling and analysis of 8-epi-PGF2α from dried blood spots (DBS).METHODSWe have applied solid-phase extraction (SPE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the extraction, separation and detection of 8-epi-PGF2α in DBS and have studied the stability of this marker using the DBS collection platform.RESULTSThe mass limit of detection (mLOD) for 8-epi-PGF2α extracted from DBS samples was 1.5 pg while the concentration limit of detection (cLOD) and concentration limit of quantitation (cLOQ) were 6 pg/mL and 18 pg/mL, respectively. All values based on triplicate analysis. Sufficient stability of 8-epi-PGF2α in DBS was achieved by preconditioning DBS paper with vitamin E and BHT.CONCLUSIONSThe developed method is sensitive, specific, robust, efficient, and can accurately measure endogenous concentrations of 8-epi-PGF2α in DBS. Thus, it offers an analytical approach to measure 8-epi-PGF2α by a novel sample collection technique that is less invasive and costly than conventional techniques. Copyright © 2012 John Wiley & Sons, Ltd.Optimizing the enrichment of cross-linked products for mass spectrometric protein analysishttp://dx.doi.org/10.1002%2Frcm.6150Optimizing the enrichment of cross-linked products for mass spectrometric protein analysisRomy FritzscheChristian H. IhlingMichael GötzeAndrea Sinz2012-03-30T00:00:00-05:00doi:10.1002/rcm.6150John Wiley & Sons, Inc.10.1002/rcm.6150http://dx.doi.org/10.1002%2Frcm.6150Research Article653658

RATIONALE

Chemical cross-linking in combination with a mass spectrometric analysis of the created cross-linked products is an area of growing interest for deriving low-resolution structural information of proteins and protein complexes. One of the greatest challenges is the complexity of the created cross-linking mixtures, which can be met by a charge-based enrichment of cross-linked peptides after proteolytic digestion using strong cation-exchange (SCX) chromatography.

METHODS

SCX chromatography was used for the enrichment of cross-linked peptides with the N-hydroxysuccinimide ester bis(sulfosuccinimidyl)succinate (BS3) prior to a mass spectrometric analysis by nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS. Bovine serum albumin (BSA) and glutathione S-transferase (GST) were employed as model proteins.

RESULTS

Conditions for SCX enrichment were optimized for obtaining as many interpeptide cross-linked peptides as possible in order to maximize the amount of structural information from a single experiment. With an SCX-based enrichment step of cross-linked products within BSA using the cross-linker BS3, 154 interpeptidal cross-linking products were identified during nano-HPLC/nano-ESI-MS/MS analyses, whereas analyses without a prior SCX enrichment allowed the identification of merely 20 cross-linked products. The application of the SCX enrichment strategy for the analysis of cross-linked products of GST with BS3 allowed the identification of 26 interpeptidal cross-linked products compared with 16 without SCX enrichment.

CONCLUSIONS

For both proteins investigated herein, BSA and GST, the introduction of an SCX-based enrichment step prior to nano-HPLC/nano-ESI-MS/MS of cross-linked products led to a considerable gain in structural information. Copyright © 2012 John Wiley & Sons, Ltd.

]]>RATIONALEChemical cross-linking in combination with a mass spectrometric analysis of the created cross-linked products is an area of growing interest for deriving low-resolution structural information of proteins and protein complexes. One of the greatest challenges is the complexity of the created cross-linking mixtures, which can be met by a charge-based enrichment of cross-linked peptides after proteolytic digestion using strong cation-exchange (SCX) chromatography.METHODSSCX chromatography was used for the enrichment of cross-linked peptides with the N-hydroxysuccinimide ester bis(sulfosuccinimidyl)succinate (BS3) prior to a mass spectrometric analysis by nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS. Bovine serum albumin (BSA) and glutathione S-transferase (GST) were employed as model proteins.RESULTSConditions for SCX enrichment were optimized for obtaining as many interpeptide cross-linked peptides as possible in order to maximize the amount of structural information from a single experiment. With an SCX-based enrichment step of cross-linked products within BSA using the cross-linker BS3, 154 interpeptidal cross-linking products were identified during nano-HPLC/nano-ESI-MS/MS analyses, whereas analyses without a prior SCX enrichment allowed the identification of merely 20 cross-linked products. The application of the SCX enrichment strategy for the analysis of cross-linked products of GST with BS3 allowed the identification of 26 interpeptidal cross-linked products compared with 16 without SCX enrichment.CONCLUSIONSFor both proteins investigated herein, BSA and GST, the introduction of an SCX-based enrichment step prior to nano-HPLC/nano-ESI-MS/MS of cross-linked products led to a considerable gain in structural information. Copyright © 2012 John Wiley & Sons, Ltd.High-throughput screening for glutathione conjugates using stable-isotope labeling and negative electrospray ionization precursor-ion mass spectrometryhttp://dx.doi.org/10.1002%2Frcm.6135High-throughput screening for glutathione conjugates using stable-isotope labeling and negative electrospray ionization precursor-ion mass spectrometryShengkai LiaoNigel P. EwingBrian BoucherOlivier MaterneChristopher L. Brummel2012-03-30T00:00:00-05:00doi:10.1002/rcm.6135John Wiley & Sons, Inc.10.1002/rcm.6135http://dx.doi.org/10.1002%2Frcm.6135Research Article659669

RATIONALE

It has been proposed that the increase in the instances of idiosyncratic adverse drug reactions (IADRs) and black box warnings may be attributed to the occurrence of reactive metabolites. Consequently, a high-throughput screen for reactive metabolites formed from liver microsome extracts with added glutathione (GSH) was developed for use in the early stages of drug discovery.

METHODS

To enhance sensitivity and specificity, as well as accelerate data processing, a mixture of a stable-isotope probe consisting of natural GSH (light GSH) and stable-isotope-labeled [15 N,13 C2] GSH (heavy GSH) at a ratio of 1:1 was used. Any metabolite that reacted with the GSH results in the formation of light and heavy GSH conjugates with a 3 Da difference. Employing a precursor-ion scan using negative ion electrospray ionization (ESI) corresponding to the expected fragments, signals with the appropriate ratio in the precursor ion scan are then further examined.

RESULTS

The new method greatly simplifies data collection by assuming molecules containing GSH will fragment to form specific ions. As such, this approach accelerates data processing (and collection) at the risk of missing compounds that do not fragment as expected. The assay was validated with 33 diverse drugs known to form GSH conjugates, 5 drugs known to not form GSH adducts and over 100 samples containing components of the normal in vitro matrix. In all cases data collected matched the expected result.

CONCLUSIONS

The observed sensitivity, specificity, and fast data processing make this assay an excellent fit for high-throughput screening of reactive metabolites in the early stages of drug discovery. This method is not intended to eliminate compounds or terminate their development. Instead, it is to bring forward molecules with one less liability and thus a greater probability of ultimate success. Copyright © 2012 John Wiley & Sons, Ltd.

]]>RATIONALEIt has been proposed that the increase in the instances of idiosyncratic adverse drug reactions (IADRs) and black box warnings may be attributed to the occurrence of reactive metabolites. Consequently, a high-throughput screen for reactive metabolites formed from liver microsome extracts with added glutathione (GSH) was developed for use in the early stages of drug discovery.METHODSTo enhance sensitivity and specificity, as well as accelerate data processing, a mixture of a stable-isotope probe consisting of natural GSH (light GSH) and stable-isotope-labeled [15 N,13 C2] GSH (heavy GSH) at a ratio of 1:1 was used. Any metabolite that reacted with the GSH results in the formation of light and heavy GSH conjugates with a 3 Da difference. Employing a precursor-ion scan using negative ion electrospray ionization (ESI) corresponding to the expected fragments, signals with the appropriate ratio in the precursor ion scan are then further examined.RESULTSThe new method greatly simplifies data collection by assuming molecules containing GSH will fragment to form specific ions. As such, this approach accelerates data processing (and collection) at the risk of missing compounds that do not fragment as expected. The assay was validated with 33 diverse drugs known to form GSH conjugates, 5 drugs known to not form GSH adducts and over 100 samples containing components of the normal in vitro matrix. In all cases data collected matched the expected result.CONCLUSIONSThe observed sensitivity, specificity, and fast data processing make this assay an excellent fit for high-throughput screening of reactive metabolites in the early stages of drug discovery. This method is not intended to eliminate compounds or terminate their development. Instead, it is to bring forward molecules with one less liability and thus a greater probability of ultimate success. Copyright © 2012 John Wiley & Sons, Ltd.Comparison of different tandem mass spectrometric techniques (ESI-IT, ESI- and IP-MALDI-QRTOF and vMALDI-TOF/RTOF) for the analysis of crocins and picrocrocin from the stigmas of Crocus sativus L.http://dx.doi.org/10.1002%2Frcm.6142Comparison of different tandem mass spectrometric techniques (ESI-IT, ESI- and IP-MALDI-QRTOF and vMALDI-TOF/RTOF) for the analysis of crocins and picrocrocin from the stigmas of Crocus sativus L.Nikolaos Stavros KoulakiotisErnst PittenauerMaria HalabalakiAnthony TsarbopoulosGünter Allmaier2012-03-30T00:00:00-05:00doi:10.1002/rcm.6142John Wiley & Sons, Inc.10.1002/rcm.6142http://dx.doi.org/10.1002%2Frcm.6142Research Article670678

RATIONALE

The expensive spice saffron originating from the stigmas of Crocus sativus L. and also applied in traditional Chinese medicine (TCM) constitutes a complex mixture of glycoconjugates varying not only in the aglycon structure, but also in glycosylation pattern. Therefore, various tandem mass spectrometric techniques were evaluated for their usefulness in structural elucidation.

METHODS

Three selected constituents of the stigmas of Crocus sativus L., trans- and cis-crocin-4 as well as picrocrocin, were isolated and purified by HPLC and finally analyzed by ESI-MS (ion trap, QqRTOF), IP-MALDI-MS (QqRTOF) and vMALDI-MS (TOF/RTOF) in combination with tandem mass spectrometry in collision energy regimes ranging from a few eV (LE) to 20 keV (HE) collisions for the first time. These data aid in structurally elucidating minor, unknown glycoconjugates originating from this plant-derived spice.

RESULTS

LE-CID of isomeric crocins on either an ion trap with ESI or a QqRTOF-instrument with ESI or IP-MALDI as desorption/ionization technique only yielded a limited number of structurally diagnostic sodiated product ions related to the carbohydrate moiety as well as to the intact aglycon in contrast to true HE-CID. The low MW constituent picrocrocin did not yield useful LE-CID spectra, but showed a high number of structurally diagnostic product ions by HE-CID utilizing a vMALDI TOF/RTOF-instrument.

CONCLUSIONS

The highest number of structurally diagnostic product ions allowing also determination of the carbohydrate linkage of the gentiobiose-moiety of isomeric crocins (0,4A2, 3,5A2 product ions indicating a 1→6 carbohydrate linkage) was only achievable by HE-CID. Fragmentation of the aglycon was not observed by any collision energy regime applied. Copyright © 2012 John Wiley & Sons, Ltd.

]]>RATIONALEThe expensive spice saffron originating from the stigmas of Crocus sativus L. and also applied in traditional Chinese medicine (TCM) constitutes a complex mixture of glycoconjugates varying not only in the aglycon structure, but also in glycosylation pattern. Therefore, various tandem mass spectrometric techniques were evaluated for their usefulness in structural elucidation.METHODSThree selected constituents of the stigmas of Crocus sativus L., trans- and cis-crocin-4 as well as picrocrocin, were isolated and purified by HPLC and finally analyzed by ESI-MS (ion trap, QqRTOF), IP-MALDI-MS (QqRTOF) and vMALDI-MS (TOF/RTOF) in combination with tandem mass spectrometry in collision energy regimes ranging from a few eV (LE) to 20 keV (HE) collisions for the first time. These data aid in structurally elucidating minor, unknown glycoconjugates originating from this plant-derived spice.RESULTSLE-CID of isomeric crocins on either an ion trap with ESI or a QqRTOF-instrument with ESI or IP-MALDI as desorption/ionization technique only yielded a limited number of structurally diagnostic sodiated product ions related to the carbohydrate moiety as well as to the intact aglycon in contrast to true HE-CID. The low MW constituent picrocrocin did not yield useful LE-CID spectra, but showed a high number of structurally diagnostic product ions by HE-CID utilizing a vMALDI TOF/RTOF-instrument.CONCLUSIONSThe highest number of structurally diagnostic product ions allowing also determination of the carbohydrate linkage of the gentiobiose-moiety of isomeric crocins (0,4A2, 3,5A2 product ions indicating a 1→6 carbohydrate linkage) was only achievable by HE-CID. Fragmentation of the aglycon was not observed by any collision energy regime applied. Copyright © 2012 John Wiley & Sons, Ltd.Potential biomarkers for identification of mycobacterial cultures by proton transfer reaction mass spectrometry analysishttp://dx.doi.org/10.1002%2Frcm.6139Potential biomarkers for identification of mycobacterial cultures by proton transfer reaction mass spectrometry analysisE. CrespoH. RondeS. KuijperA. PolA. H. J. KolkS. M. CristescuR. M. AnthonyF. J. M. Harren2012-03-30T00:00:00-05:00doi:10.1002/rcm.6139John Wiley & Sons, Inc.10.1002/rcm.6139http://dx.doi.org/10.1002%2Frcm.6139Research Article679685

RATIONALE

Several mycobacterial species can produce serious infections in humans, and the treatment required depends on the infecting species. Fast identification, ideally with minimal manipulation of the infecting species, is therefore critical; here, we propose a method potentially allowing cultures to be identified by headspace analysis and use it to screen for differences between mycobacterial species based on the volatiles released during growth.

METHODS

Short-chain volatile organic compound emissions from two non-tuberculosis slow growing mycobacterial species, Mycobacterium avium and Mycobacterium kansasii, and a non-pathogenic fast growing species, Mycobacterium smegmatis, in Middlebrook M7H9 culturing media were followed online with a proton transfer reaction quadrupole mass spectrometer.

RESULTS

Measurable differences between the headspace of the two slow growing mycobacteria M. kansasii and M. avium were found, as well as differences with respect to the faster growing mycobacteria M. smegmatis. Three compounds, attributed to sulfur-containing volatiles – dimethyl sulfide, propanethiol and dimethyl disulfide – were found to be specific to M. avium.

CONCLUSIONS

Clear differences were detected in the low molecular weight volatile emissions compounds of the mycobacterial species under study, without the need for sample manipulation. Further studies with other mycobacterial species will reveal if the differences observed are specific to the species studied here. Furthermore, the use of an ion trap as a mass analyzer with the same ionization technique, allowing molecular detection over a wider molecular range, could allow the detection of additional biomarkers thus capturing a wider molecular range. Copyright © 2012 John Wiley & Sons, Ltd.

]]>RATIONALESeveral mycobacterial species can produce serious infections in humans, and the treatment required depends on the infecting species. Fast identification, ideally with minimal manipulation of the infecting species, is therefore critical; here, we propose a method potentially allowing cultures to be identified by headspace analysis and use it to screen for differences between mycobacterial species based on the volatiles released during growth.METHODSShort-chain volatile organic compound emissions from two non-tuberculosis slow growing mycobacterial species, Mycobacterium avium and Mycobacterium kansasii, and a non-pathogenic fast growing species, Mycobacterium smegmatis, in Middlebrook M7H9 culturing media were followed online with a proton transfer reaction quadrupole mass spectrometer.RESULTSMeasurable differences between the headspace of the two slow growing mycobacteria M. kansasii and M. avium were found, as well as differences with respect to the faster growing mycobacteria M. smegmatis. Three compounds, attributed to sulfur-containing volatiles – dimethyl sulfide, propanethiol and dimethyl disulfide – were found to be specific to M. avium.CONCLUSIONSClear differences were detected in the low molecular weight volatile emissions compounds of the mycobacterial species under study, without the need for sample manipulation. Further studies with other mycobacterial species will reveal if the differences observed are specific to the species studied here. Furthermore, the use of an ion trap as a mass analyzer with the same ionization technique, allowing molecular detection over a wider molecular range, could allow the detection of additional biomarkers thus capturing a wider molecular range. Copyright © 2012 John Wiley & Sons, Ltd.Characterization and identification of a C-terminal amidated mechano growth factor (MGF) analogue in black market productshttp://dx.doi.org/10.1002%2Frcm.6144Characterization and identification of a C-terminal amidated mechano growth factor (MGF) analogue in black market productsSimone EspositoKoen DeventerPeter Van Eenoo2012-03-30T00:00:00-05:00doi:10.1002/rcm.6144John Wiley & Sons, Inc.10.1002/rcm.6144http://dx.doi.org/10.1002%2Frcm.6144Research Article686692

RATIONALE

Mechano growth factor (MGF) is a splice variant of insulin-like growth factor that possesses anabolic properties and has not yet been approved for therapeutic use. Nevertheless, it is readily available on the black market. Although the World Anti-Doping Agency (WADA) has banned the use of MGF in sports, no routinely performed methods have been reported for its detection. In this work, two preparations from the black market containing an unknown MGF analogue were characterized.

METHODS

Mass spectrometry characterizations of unknown preparations and a reference human MGF were performed on an Orbitrap and a triple quadrupole mass spectrometers after separation by liquid chromatography. High accuracy measurements allowed protein identification from full scan MS data, and low-resolution full scan MS/MS provided further information on fragmentation.

RESULTS

HCD scans of the analytes showed the presence of common b series product ions in the black market preparations and the human MGF reference standard, but all the y series ions starting from (y1)+ exhibited a difference of 1 m/z unit in nominal mass. This difference was demonstrated to be due to a C-terminal amidation of MGF. High-resolution data demonstrated that the black market products were both C-terminal amidated analogues of human MGF. In addition, low-resolution MS/MS characterization revealed a potentially diagnostic transition (m/z 717.8 → 431.1) for the discrimination of C-amidated MGF from the endogenous form.

CONCLUSIONS

Qualitative identification of a MGF C-terminal amidated analogue in two black market products was successfully achieved. This report demonstrates that illegal MGF preparations are commercially available for use as doping agent in sports. Copyright © 2012 John Wiley & Sons, Ltd.

]]>RATIONALEMechano growth factor (MGF) is a splice variant of insulin-like growth factor that possesses anabolic properties and has not yet been approved for therapeutic use. Nevertheless, it is readily available on the black market. Although the World Anti-Doping Agency (WADA) has banned the use of MGF in sports, no routinely performed methods have been reported for its detection. In this work, two preparations from the black market containing an unknown MGF analogue were characterized.METHODSMass spectrometry characterizations of unknown preparations and a reference human MGF were performed on an Orbitrap and a triple quadrupole mass spectrometers after separation by liquid chromatography. High accuracy measurements allowed protein identification from full scan MS data, and low-resolution full scan MS/MS provided further information on fragmentation.RESULTSHCD scans of the analytes showed the presence of common b series product ions in the black market preparations and the human MGF reference standard, but all the y series ions starting from (y1)+ exhibited a difference of 1 m/z unit in nominal mass. This difference was demonstrated to be due to a C-terminal amidation of MGF. High-resolution data demonstrated that the black market products were both C-terminal amidated analogues of human MGF. In addition, low-resolution MS/MS characterization revealed a potentially diagnostic transition (m/z 717.8 → 431.1) for the discrimination of C-amidated MGF from the endogenous form.CONCLUSIONSQualitative identification of a MGF C-terminal amidated analogue in two black market products was successfully achieved. This report demonstrates that illegal MGF preparations are commercially available for use as doping agent in sports. Copyright © 2012 John Wiley & Sons, Ltd.On-target separation of analyte with 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix for matrix-assisted laser desorption/ionization mass spectrometryhttp://dx.doi.org/10.1002%2Frcm.6148On-target separation of analyte with 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix for matrix-assisted laser desorption/ionization mass spectrometrySadanori SekiyaKenichi TaniguchiKoichi Tanaka2012-03-30T00:00:00-05:00doi:10.1002/rcm.6148John Wiley & Sons, Inc.10.1002/rcm.6148http://dx.doi.org/10.1002%2Frcm.6148Research Article693700

3-Aminoquinoline/α-cyano-4-hydroxycinnamic acid (3AQ/CHCA) is a liquid matrix (LM), which was reported by Kumar et al. in 1996 for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It is a viscous liquid and has some advantages of durability of ion generation by a self-healing surface and quantitative performance. In this study, we found a novel aspect of 3AQ/CHCA as a MALDI matrix, which converges hydrophilic material into the center of the droplet of analyte-3AQ/CHCA mixture on a MALDI sample target well during the process of evaporation of water derived from analyte solvent. This feature made it possible to separate not only the buffer components, but also the peptides and oligosaccharides from one another within 3AQ/CHCA. The MALDI imaging analyses of the analyte-3AQ/CHCA droplet indicated that the oligosaccharides and the peptides were distributed in the center and in the whole area around the center of 3AQ/CHCA, respectively. This 'on-target separation' effect was also applicable to glycoprotein digests such as ribonuclease B. These features of 3AQ/CHCA liquid matrix eliminate the requirement for pretreatment, and reduce sample handling losses thus resulting in the improvement of throughput and sensitivity. Copyright © 2012 John Wiley & Sons, Ltd.

]]>3-Aminoquinoline/α-cyano-4-hydroxycinnamic acid (3AQ/CHCA) is a liquid matrix (LM), which was reported by Kumar et al. in 1996 for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It is a viscous liquid and has some advantages of durability of ion generation by a self-healing surface and quantitative performance. In this study, we found a novel aspect of 3AQ/CHCA as a MALDI matrix, which converges hydrophilic material into the center of the droplet of analyte-3AQ/CHCA mixture on a MALDI sample target well during the process of evaporation of water derived from analyte solvent. This feature made it possible to separate not only the buffer components, but also the peptides and oligosaccharides from one another within 3AQ/CHCA. The MALDI imaging analyses of the analyte-3AQ/CHCA droplet indicated that the oligosaccharides and the peptides were distributed in the center and in the whole area around the center of 3AQ/CHCA, respectively. This 'on-target separation' effect was also applicable to glycoprotein digests such as ribonuclease B. These features of 3AQ/CHCA liquid matrix eliminate the requirement for pretreatment, and reduce sample handling losses thus resulting in the improvement of throughput and sensitivity. Copyright © 2012 John Wiley & Sons, Ltd.An exclusion list based label-free proteome quantification approach using an LTQ Orbitraphttp://dx.doi.org/10.1002%2Frcm.6147An exclusion list based label-free proteome quantification approach using an LTQ OrbitrapJan MuntelMichael HeckerDörte Becher2012-03-30T00:00:00-05:00doi:10.1002/rcm.6147John Wiley & Sons, Inc.10.1002/rcm.6147http://dx.doi.org/10.1002%2Frcm.6147Research Article701709

RATIONALE

Label-based mass spectrometry is a powerful tool for large-scale protein identification and quantification. However, it requires the chemical or metabolic incorporation of the labeled compound(s) which can be difficult to attain, e.g. for non-cultivable organisms or scarce sample, such as biopsies. Therefore, we set out to develop and validate an efficient label-free liquid chromatography/tandem mass spectrometry (LC/MS/MS) workflow based on optimized instrument settings and incremental exclusion lists.

METHODS

To increase the number of quantified peptides an incremental exclusion list was incorporated along with optimized instrument settings for the used LTQ Orbitrap. As a proof of concept, label-free quantification data from this optimized approach were compared to the results of control measurements without exclusion lists and of an in vivo metabolic labeling GeLC/MS/MS experiment. The data were drawn from Staphylococcus aureus whole cell lysates of non-stressed and nitric oxide (NO)-stressed cells.

RESULTS

Compared to MS analysis without exclusion lists the new approach resulted in an increased number of identified peptides, enabling label-free quantification of more than 990 S. aureus proteins. With respect to the number of quantified proteins and differences in protein levels between the control and NO-treated samples the results of the new method were consistent with those of the GeLC/MS/MS experiment.

CONCLUSIONS

The application of exclusion lists and optimized instrument settings in LC/MS/MS analysis significantly enhances the sensitivity and resolution of label-free protein identification and quantification. Therefore, the new workflow is a powerful alternative to label-based quantification methods. Copyright © 2012 John Wiley & Sons, Ltd.

]]>RATIONALELabel-based mass spectrometry is a powerful tool for large-scale protein identification and quantification. However, it requires the chemical or metabolic incorporation of the labeled compound(s) which can be difficult to attain, e.g. for non-cultivable organisms or scarce sample, such as biopsies. Therefore, we set out to develop and validate an efficient label-free liquid chromatography/tandem mass spectrometry (LC/MS/MS) workflow based on optimized instrument settings and incremental exclusion lists.METHODSTo increase the number of quantified peptides an incremental exclusion list was incorporated along with optimized instrument settings for the used LTQ Orbitrap. As a proof of concept, label-free quantification data from this optimized approach were compared to the results of control measurements without exclusion lists and of an in vivo metabolic labeling GeLC/MS/MS experiment. The data were drawn from Staphylococcus aureus whole cell lysates of non-stressed and nitric oxide (NO)-stressed cells.RESULTSCompared to MS analysis without exclusion lists the new approach resulted in an increased number of identified peptides, enabling label-free quantification of more than 990 S. aureus proteins. With respect to the number of quantified proteins and differences in protein levels between the control and NO-treated samples the results of the new method were consistent with those of the GeLC/MS/MS experiment.CONCLUSIONSThe application of exclusion lists and optimized instrument settings in LC/MS/MS analysis significantly enhances the sensitivity and resolution of label-free protein identification and quantification. Therefore, the new workflow is a powerful alternative to label-based quantification methods. Copyright © 2012 John Wiley & Sons, Ltd.Comparing inactivation protocols of Yersinia organisms for identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometryhttp://dx.doi.org/10.1002%2Frcm.6152Comparing inactivation protocols of Yersinia organisms for identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometryCarine CoudercClaude NappezMichel Drancourt2012-03-30T00:00:00-05:00doi:10.1002/rcm.6152John Wiley & Sons, Inc.10.1002/rcm.6152http://dx.doi.org/10.1002%2Frcm.6152Protocol710714

RATIONALE

It is recommended that harmful Biosafety Level 3 (BSL-3) bacteria be inactivated prior to identification by mass spectrometry, yet optimal effects of inactivation protocol have not been defined.

METHODS

Here, we compare trifluoroacetic acid inactivation (protocol A) with ethanol inactivation (protocol B) of Yersinia organisms prior to identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

RESULTS

The total number of peaks detected was 10.5 ± 1.7 for protocol A and 15.7 ± 4.2 for protocol B (ρ <0.001, ANOVA test). The signal-to-noise ratio for the m/z 6049 peak present in all of the tested Yersinia isolates was 9.7 ± 3.1 for protocol A and 18.1 ± 4.6 for protocol B (ρ < 0.001). Compared with spectra in our local database containing 48 Yersinia spp., including 20 strains of Y. pestis, the identification score was 1.79 ± 0.2 for protocol A and 1.97 ± 0.19 for protocol B (ρ = 0.0024).

CONCLUSIONS

Our observations indicate that for the identification of Yersinia organisms, ethanol inactivation yielded MALDI-TOF-MS spectra of significantly higher quality than spectra derived from trifluoroacetic acid inactivation. Combined with previously published data, our results permit the updating of protocols for inactivating BSL-3 bacteria. Copyright © 2012 John Wiley & Sons, Ltd.

]]>RATIONALEIt is recommended that harmful Biosafety Level 3 (BSL-3) bacteria be inactivated prior to identification by mass spectrometry, yet optimal effects of inactivation protocol have not been defined.METHODSHere, we compare trifluoroacetic acid inactivation (protocol A) with ethanol inactivation (protocol B) of Yersinia organisms prior to identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).RESULTSThe total number of peaks detected was 10.5 ± 1.7 for protocol A and 15.7 ± 4.2 for protocol B (ρ <0.001, ANOVA test). The signal-to-noise ratio for the m/z 6049 peak present in all of the tested Yersinia isolates was 9.7 ± 3.1 for protocol A and 18.1 ± 4.6 for protocol B (ρ < 0.001). Compared with spectra in our local database containing 48 Yersinia spp., including 20 strains of Y. pestis, the identification score was 1.79 ± 0.2 for protocol A and 1.97 ± 0.19 for protocol B (ρ = 0.0024).CONCLUSIONSOur observations indicate that for the identification of Yersinia organisms, ethanol inactivation yielded MALDI-TOF-MS spectra of significantly higher quality than spectra derived from trifluoroacetic acid inactivation. Combined with previously published data, our results permit the updating of protocols for inactivating BSL-3 bacteria. Copyright © 2012 John Wiley & Sons, Ltd.Generation of tungstate ion clusters by Keggin-type silicopolyoxotungstate anions under matrix-assisted laser desorption/ionization conditions in the gas phasehttp://dx.doi.org/10.1002%2Frcm.6153Generation of tungstate ion clusters by Keggin-type silicopolyoxotungstate anions under matrix-assisted laser desorption/ionization conditions in the gas phaseYun-Peng BaiShu LiuFeng-Rui SongZhi-Qiang LiuShu-Ying Liu2012-03-30T00:00:00-05:00doi:10.1002/rcm.6153John Wiley & Sons, Inc.10.1002/rcm.6153http://dx.doi.org/10.1002%2Frcm.6153Letter to the Editor715718