The Experts below are selected from a list of 108 Experts worldwide ranked by ideXlab platform
Jorg Bettmer - One of the best experts on this subject based on the ideXlab platform.
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absolute and relative protein quantification with the use of isotopically labeled p Hydroxymercuribenzoic Acid and complementary maldi ms and icpms detection
Analytical Chemistry, 2009Co-Authors: Daniel Kutscher, Jorg BettmerAbstract:Chemical labeling with subsequent mass spectrometric detection represents a common approach for protein quantification. Whereas most methods make use of stable isotope labels from natural elements such as 2D, 13C, 15N, or 18O, artificially introduced metals have gained interest as alternative markers. In this work we present the application of p-Hydroxymercuribenzoic Acid (pHMB) as a labeling reagent for cysteine-containing proteins. As a proof of concept, insulin was chosen as the model protein, and two different workflows were developed to its absolute and relative quantification with the use of complementary MALDI-MS and ICPMS. On the basis of the synthesis of isotopically labeled [199Hg]pHMB, and thus on the basis of the label-specific isotope dilution concept, a differential labeling procedure can be applied either to the comparative study of two different samples (relative quantification) or to the absolute quantification of insulin. In both cases, final detection by MALDI-MS followed by isotope pat...
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protein labelling with mercury tags fundamental studies on ovalbumin derivatised with p Hydroxymercuribenzoic Acid phmb
Journal of Analytical Atomic Spectrometry, 2008Co-Authors: Daniel Kutscher, Estela M Del Castillo Busto, Nico Zinn, Alfredo Sanzmedel, Jorg BettmerAbstract:Protein labelling in combination with mass spectrometry is appointed as a modern approach for quantifying biopolymers, especially proteins. With respect to elemental mass spectrometry, specifically inductively coupled plasma-mass spectrometry (ICP-MS), protein labelling approaches are still scarce, although they offer many advantages, e.g. in terms of detection sensitivity. In this fundamental work, we present results on the labelling of ovalbumin with p-Hydroxymercuribenzoic Acid (pHMB). After optimising the derivatisation procedure, the characterisation of the labelled species is necessary, and thus, the use of molecular MS techniques like MALDI-, and ESI-MS is required. Finally, the detection capabilities of ICP-MS are evaluated on the labelled species. Important factors to consider are the reaction yield, the selectivity, and the stoichiometry of the bioconjugate. For instance, the stoichiometry of the bioconjugate is determined by comparative measurements using MALDI-, and ESI-MS. It can be demonstrated that the label/protein ratio is determined to be ∼3 : 1 by MALDI-MS, which is lower than the number of expected binding sites (ovalbumin has four free sulfhydryl groups from cysteines). In contrast to these findings, the use of ESI-Q-ToF-MS with its superior mass resolution indicates a stoichiometry of 4 : 1. However, the overall strategy given here on the example of ovalbumin labelling with pHMB might be a promising approach for protein quantification as it provides a significant improvement in terms of detection limits (1 fmol for ovalbumin) in comparison to the use of sulfur as naturally occurring elemental tag.
Celia A. Sigua - One of the best experts on this subject based on the ideXlab platform.
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β-Alanine Betaine Synthesis in the Plumbaginaceae. Purification and Characterization of a Trifunctional, S -Adenosyl-l-Methionine-Dependent N -Methyltransferase from Limonium latifolium Leaves
Plant physiology, 2001Co-Authors: Bala Rathinasabapathi, Walid M. Fouad, Celia A. SiguaAbstract:beta-Alanine (beta-Ala) betaine is an osmoprotective compound accumulated by most members of the highly stress-tolerant family Plumbaginaceae. Its potential role in plant tolerance to salinity and hypoxia makes its synthetic pathway an interesting target for metabolic engineering. In the Plumbaginaceae, beta-Ala betaine is synthesized by S-adenosyl-L-methionine-dependent N-methylation of beta-Ala via N-methyl beta-Ala and N,N-dimethyl beta-Ala. It was not known how many N-methyltransferases (NMTases) participate in the three N-methylations of beta-Ala. An NMTase was purified about 1,890-fold, from Limonium latifolium leaves, using a protocol consisting of polyethylene glycol precipitation, heat treatment, anion-exchange chromatography, gel filtration, native polyacrylamide gel electrophoresis, and two substrate affinity chromatography steps. The purified NMTase was trifunctional, methylating beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that the native NMTase is a dimer of 43-kD subunits. The NMTase had an apparent K(m) of 45 microM S-adenosyl-l-methionine and substrate inhibition was observed above 200 microM. The apparent K(m) values for the methyl acceptor substrates were 5.3, 5.7, and 5.9 mM for beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala, respectively. The NMTase had an isoelectric point of 5.15 and was reversibly inhibited by the thiol reagent p-Hydroxymercuribenzoic Acid.
Daniel Kutscher - One of the best experts on this subject based on the ideXlab platform.
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absolute and relative protein quantification with the use of isotopically labeled p Hydroxymercuribenzoic Acid and complementary maldi ms and icpms detection
Analytical Chemistry, 2009Co-Authors: Daniel Kutscher, Jorg BettmerAbstract:Chemical labeling with subsequent mass spectrometric detection represents a common approach for protein quantification. Whereas most methods make use of stable isotope labels from natural elements such as 2D, 13C, 15N, or 18O, artificially introduced metals have gained interest as alternative markers. In this work we present the application of p-Hydroxymercuribenzoic Acid (pHMB) as a labeling reagent for cysteine-containing proteins. As a proof of concept, insulin was chosen as the model protein, and two different workflows were developed to its absolute and relative quantification with the use of complementary MALDI-MS and ICPMS. On the basis of the synthesis of isotopically labeled [199Hg]pHMB, and thus on the basis of the label-specific isotope dilution concept, a differential labeling procedure can be applied either to the comparative study of two different samples (relative quantification) or to the absolute quantification of insulin. In both cases, final detection by MALDI-MS followed by isotope pat...
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protein labelling with mercury tags fundamental studies on ovalbumin derivatised with p Hydroxymercuribenzoic Acid phmb
Journal of Analytical Atomic Spectrometry, 2008Co-Authors: Daniel Kutscher, Estela M Del Castillo Busto, Nico Zinn, Alfredo Sanzmedel, Jorg BettmerAbstract:Protein labelling in combination with mass spectrometry is appointed as a modern approach for quantifying biopolymers, especially proteins. With respect to elemental mass spectrometry, specifically inductively coupled plasma-mass spectrometry (ICP-MS), protein labelling approaches are still scarce, although they offer many advantages, e.g. in terms of detection sensitivity. In this fundamental work, we present results on the labelling of ovalbumin with p-Hydroxymercuribenzoic Acid (pHMB). After optimising the derivatisation procedure, the characterisation of the labelled species is necessary, and thus, the use of molecular MS techniques like MALDI-, and ESI-MS is required. Finally, the detection capabilities of ICP-MS are evaluated on the labelled species. Important factors to consider are the reaction yield, the selectivity, and the stoichiometry of the bioconjugate. For instance, the stoichiometry of the bioconjugate is determined by comparative measurements using MALDI-, and ESI-MS. It can be demonstrated that the label/protein ratio is determined to be ∼3 : 1 by MALDI-MS, which is lower than the number of expected binding sites (ovalbumin has four free sulfhydryl groups from cysteines). In contrast to these findings, the use of ESI-Q-ToF-MS with its superior mass resolution indicates a stoichiometry of 4 : 1. However, the overall strategy given here on the example of ovalbumin labelling with pHMB might be a promising approach for protein quantification as it provides a significant improvement in terms of detection limits (1 fmol for ovalbumin) in comparison to the use of sulfur as naturally occurring elemental tag.
Bala Rathinasabapathi - One of the best experts on this subject based on the ideXlab platform.
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β-Alanine Betaine Synthesis in the Plumbaginaceae. Purification and Characterization of a Trifunctional, S -Adenosyl-l-Methionine-Dependent N -Methyltransferase from Limonium latifolium Leaves
Plant physiology, 2001Co-Authors: Bala Rathinasabapathi, Walid M. Fouad, Celia A. SiguaAbstract:beta-Alanine (beta-Ala) betaine is an osmoprotective compound accumulated by most members of the highly stress-tolerant family Plumbaginaceae. Its potential role in plant tolerance to salinity and hypoxia makes its synthetic pathway an interesting target for metabolic engineering. In the Plumbaginaceae, beta-Ala betaine is synthesized by S-adenosyl-L-methionine-dependent N-methylation of beta-Ala via N-methyl beta-Ala and N,N-dimethyl beta-Ala. It was not known how many N-methyltransferases (NMTases) participate in the three N-methylations of beta-Ala. An NMTase was purified about 1,890-fold, from Limonium latifolium leaves, using a protocol consisting of polyethylene glycol precipitation, heat treatment, anion-exchange chromatography, gel filtration, native polyacrylamide gel electrophoresis, and two substrate affinity chromatography steps. The purified NMTase was trifunctional, methylating beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that the native NMTase is a dimer of 43-kD subunits. The NMTase had an apparent K(m) of 45 microM S-adenosyl-l-methionine and substrate inhibition was observed above 200 microM. The apparent K(m) values for the methyl acceptor substrates were 5.3, 5.7, and 5.9 mM for beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala, respectively. The NMTase had an isoelectric point of 5.15 and was reversibly inhibited by the thiol reagent p-Hydroxymercuribenzoic Acid.
Collin Sonia - One of the best experts on this subject based on the ideXlab platform.
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Occurrence of Ehrlich-Derived and Varietal Polyfunctional Thiols in Belgian White Wines Made from Chardonnay and Solaris Grapes
'American Chemical Society (ACS)', 2019Co-Authors: Chenot, Cécile Aline Andrée, Briffoz Laura, Lomartire Antonin, Collin SoniaAbstract:Although almost disappeared at the end of the 20th century, Belgian wine production reached a million liters in 2017. The aim of the present work was to identify aroma markers for two white cultivars widespread in Belgium: Chardonnay (the most commercially grown cultivar worldwide) and Solaris (an interspecific fungus-resistant variety). Specific p-Hydroxymercuribenzoic Acid extraction followed by gas chromatography with a pulsed flame photometric detector was applied to single-varietal wine samples from 2015 to 2018 harvests. Among the Ehrlich-derived thiols, all found below their sensory thresholds, 3-sulfanylpropyl acetate usually outranged 3-sulfanylpropan-1-ol, while 2-sulfanylethan-1-ol concentrations always exceeded 2-sulfanylethyl acetate levels. The data confirm the occurrence, in both Chardonnay and Solaris wines, of 3-sulfanylhexan-1-ol and phenylmethanethiol (grapefruit and gunflint aroma, respectively), at concentrations far above their thresholds. This work also revealed, for the first time, the presence of 3-sulfanylheptan-1-ol, the branched alcohols 2-methyl-3-sulfanyl propan-1-ol, 2-methyl-3-sulfanylbutan-1-ol, and 3-sulfanyl-3-methylbutan-1-ol, and the carbonyls 3-sulfanylbutan-2-one (not in sparkling wines) and 3-sulfanylpentanal
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Why aging is so different in dry-hopped beers: Fate of their bitterness, flavors, and polyphenols after two years of storage
2019Co-Authors: Silva Guimarães Ferreira, Carlos Antonio, Collin Sonia, 2019 Asbc MeetingAbstract:Dry-hopped (DH) beers have been rising in popularity all over the world in the last decade, partially due to the emergence of a large panel of interesting new hop varieties. Discriminant polyfunctional thiols and terpenols, found well above their sensorial threshold, give a unique profile to these beers, directly linked to the way the dry hopping was applied (hop cultivar, temperature, time, yeast, specific process…). Humulinones are known to impart (up to 28 % for Belgian DH beers) a particular bitterness to them while hop polyphenols reduce their colloidal stability. The aim of the present work is to assess why these beers behave so differently through ageing. 21 DH Belgian beers, including the famous DH trappist already on the market since 1931, have been followed during two years of storage. Besides basic parameters (extracts, alcohol content, color, pH…), our study focused on bitterness (official methods and newly developed HPLC assays more adapted for DH beers), flavors (Solvent Assisted Flavor Evaporation – SAFE and p-Hydroxymercuribenzoic Acid – pHMB extractions) and polyphenols (colloidal stability measurements and newly developed NP and RP-HPLC-ESI(-)-MS/MS methods). As expected, spectrophotometric bitterness and isohumulones concentrations strongly decreased (up to 16.4 ºBU and 18.2 mg/L isohumulones). trans-Isohumulones revealed much more impacted (up to 92 %) than its cis isomer. A very interesting data was the complete degradation, after two years of storage, of humulones and humulinones, by opposition to hulupones and tetrahydroisohumulones. The sensorial properties of the derived products were investigated. Aroma instability of DH beers is obviously the biggest hurdle for all brewers producing DH beers. Most of the so-pleasant polyfunctional thiols, including 3-sulfanylhexanol and 3-sulfanyl-4-methylpentanol, revealed completely degraded after ageing while half of the fruity ethyl hexanoate and isoamyl acetate disappeared in the same time. Linalool, usually found at very high levels in fresh beers (up to 498 μg/L for our samples), was the only terpenoid which remained above its sensorial threshold after ageing. Its loss correlated to that of geraniol and methyl geranate. Total polyphenols, determined by global methods, remained stable after two years, although drastic chemical transformations occurred among flavanoids. (+)-Catechin, (-)-epicatechin, procyanidins B3, B2, A2, C2 and C1 strongly dropped through ageing, with formation of new bridged compounds responsible of color and colloidal instability. This first review of DH beer ageing will be concluded by original proposals allowing to improve hop aroma, bitterness and colloidal stability of these beers. Their impact will be discussed in relation to other usual stale flavors, including trans-2-nonenal (cardboard), 3-sulfanyl-3-methylbutyl formate (ribes) and sotolon (madeira)
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Polyfunctional Thiols in Fresh and Aged Belgian Special Beers: Fate of Hop S-Cysteine Conjugates
'Scientific Societies', 2015Co-Authors: Tran, Thi Thu Hang, Kankolongo Cibaka Marie-lucie, Collin SoniaAbstract:Odorant polyfunctional thiols such as 4-sulfanyl-4-methylpentan-2-one, 3-sulfanylhexan-1-ol (3SHol), and 3-sulfanyl-3-methylbutan-1ol (3S3MBol) were shown to be strongly degraded in Belgian special beers after 1 year of aging. Yet, during the first 3 months, they also appeared to be synthesized in the bottle, even in the absence of yeast. Fresh filtered lager beer was spiked with nonvolatile S-cysteine conjugates of 3SHol, 3S3MBol, and 3-methylbut-2-en-1-thiol (MBT) before aging (1 to 3 months at 20°C or 5 days at 40 or 60°C). Thiol-specific para-Hydroxymercuribenzoic Acid extracts were analyzed by a GC-pulsed flame photometric detector, and HPLC-electrospray ionization/MS/MS was used to quantify the undegraded cysteinylated precursors. S-Cysteine conjugates were chemically degraded in the beer, releasing their corresponding thiols. The conversions were low (
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Occurrence of polyfunctional thiols in sorghum beer 'ikigage' made with Vernonia amygdalina 'umubirizi'
'Wiley', 2012Co-Authors: Lyumugabe François, Gros Jacques, Thonart Philippe, Collin SoniaAbstract:Several polyfunctional thiols have been previously identified in beers made from barley and hops. These compounds have not been investigated in beers brewed with 'non-Western' raw materials. Here we have performed a thiol-specific extraction with p-Hydroxymercuribenzoic Acid on a traditional ikigage sorghum beer from Rwandese peasants (use of Vernonia amygdalina just for yeast propagation), and on two pilot beers with addition (or not) of V. amygdalina in the boiling kettle, instead of hops. Gas chromatography-olfactometry, gas chromatography-mass spectrometry and gas chromatography with pulsed flame photometric detection analyses of the extracts enabled us to identify 14 polyfunctional thiols. The well-known hop constituent 3-methyl-2-buten-1-thiol emerged as a key flavour in the unhopped beers containing V. amygdalina (flavour dilution>262 144). The addition of V. amygdalina during boiling also resulted in the presence of 1-butanethiol, but the production of 2-sulfanylethanol and 2-sulfanylethyl acetate was inhibited. Complementary data are required to understand how V. amygdalina leaves are able to impact upon the Ehrlich pathway leading to cysteine and homocysteine-derived thiols. © 2012 John Wiley & Sons, Ltd
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Occurrence of polyfunctional thiols in fresh lager beers.
'American Chemical Society (ACS)', 2006Co-Authors: Vermeulen Catherine, Lejeune Ines, Tran T T H, Collin SoniaAbstract:Polyfunctional thiols are known to have a strong impact on the overall aroma of many fermented foods. Surprisingly, very little data is available on their occurrence in beer. A specific extraction with p-Hydroxymercuribenzoic Acid was performed on four different fresh light-protected lager beers. gas chromatography-olfactometry, gas chromatography-mass spectrometry, and gas chromatography-pulsed-flame photometer detector analyses of the extracts revealed the presence of more than 10 polyfunctional thiols. All of them were absent from wort, suggesting a key role of the H(2)S excreted by yeasts. 3-Methyl-2-buten-1-thiol, 2-mercapto-3-methylbutanol, 3-mercapto-3-methylbutanol seem to be created from hop allylic alcohols via four different mechanisms: nucleophilic substitution, addition-elimination, and radical anti-Markovnikov or electrophilic Markovnikov additions. 1,4 Addition of hydrogen sulfide to wort alpha,beta-unsaturated aldehydes or ketones may explain the synthesis of 1-mercapto-3-pentanol, 3-mercaptohexanol, and 4-mercapto-4-methyl-2-pentanone through fermentation. Finally, 2-mercaptoethanol, 3-mercaptopropanol, and their corresponding acetates may derive from Ehrlich degradation of sulfur amino Acids, while 2-methyl-3-furanthiol should be logically issued from Maillard reactions
