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Multistage Tandem Mass Spectrometry Strategies for the Targeted Analysis of Oxidative Protein Modifications

Jennifer M. Froelich

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Description:The origin and control of ex vivo sample handling related oxidative modifications of methionine-, S-alkyl cysteine- and tryptophan-containing peptides obtained from typical in-solution or in-gel proteolytic digestion strategies, have been examined by capillary HPLC and MS/MS. The origin of increased oxidation levels was found to be predominately associated with the extensive ex vivo sample handling steps required for gel electrophoresis and/or in-gel proteolytic digestion of proteins prior to analysis by MS. Conditions for deliberately controlling the oxidation state (both oxidation and reduction) of these peptides, as well as for those containing cysteine, have also been evaluated using a series of model synthetic peptides and standard tryptic protein digests. Optimal conditions for oxidation and reduction were achieved via reaction with 30% hydrogen peroxide/5% acetic acid and reaction with 1 M dimethylsulfide/10 M hydrochloric acid, respectively.;The mechanisms for the gas-phase fragmentation reactions of singly and multiply protonated precursor ions of a series of model S-alkyl cysteine sulfoxide-containing peptides prepared by reaction with iodomethane, iodoacetamide, iodoacetic acid, acrylamide or 4-vinylpyridine, followed by oxidation with hydrogen peroxide have also been examined using multistage tandem mass spectrometry (MS/MS, MS3 and MS4), hydrogen/deuterium exchange and molecular orbital calculations (at the B3LYP/6-31 + G(d,p) level of theory). Dissociation of uniformly deuterated precursor ions of these model peptides confirmed that the non-sequence neutral loss of alkyl sulfenic acid (XSOH) in each case occurred via a charge-remote five-centered cis-1,2 elimination reaction. Similarly, the charge state dependence to the mechanisms and product ion structures for the losses of CO2, CO2 + H2O and CO2 + CH2O from S-carboxymethyl cysteine sulfoxide-containing peptides, and for the losses of CH2CHCONH 2 and CH2CHC5H4N, respectively from S-amidoethyl and S-pyridylethyl cysteine sulfoxide-containing peptide ions have also been determined.;A strategy involving the fixed-charge sulfonium ion derivatization, stable isotope labeling, capillary HPLC and automated neutral loss MS/MS and data dependent "pseudo MS3" scans in a triple quadrupole mass spectrometer has also been developed for the targeted gas-phase identification, characterization and quantitative analysis of low abundance methionine-containing peptides present within complex protein digests. In contrast to MS-based quantitative analysis strategies, the neutral loss scan mode MS/MS method was able to achieve accurate quantification for individual peptides at levels as low as 100 fmol and at abundance ratios ranging from 0.1 to 10, present within a complex protein digest. Using a similar fixed-charge sulfonium ion derivatization and tandem mass spectrometry-based analysis strategy, methionine oxidation was successfully quantified following hydrogen peroxide treatment of the Ca2+/calmodulin dependent serine/threonine phosphatase calcineurin.We have made it easy for you to find a PDF Ebooks without any digging. And by having access to our ebooks online or by storing it on your computer, you have convenient answers with Multistage Tandem Mass Spectrometry Strategies for the Targeted Analysis of Oxidative Protein Modifications. To get started finding Multistage Tandem Mass Spectrometry Strategies for the Targeted Analysis of Oxidative Protein Modifications, you are right to find our website which has a comprehensive collection of manuals listed.
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ISBN: 1109036094

Description: The origin and control of ex vivo sample handling related oxidative modifications of methionine-, S-alkyl cysteine- and tryptophan-containing peptides obtained from typical in-solution or in-gel proteolytic digestion strategies, have been examined by capillary HPLC and MS/MS. The origin of increased oxidation levels was found to be predominately associated with the extensive ex vivo sample handling steps required for gel electrophoresis and/or in-gel proteolytic digestion of proteins prior to analysis by MS. Conditions for deliberately controlling the oxidation state (both oxidation and reduction) of these peptides, as well as for those containing cysteine, have also been evaluated using a series of model synthetic peptides and standard tryptic protein digests. Optimal conditions for oxidation and reduction were achieved via reaction with 30% hydrogen peroxide/5% acetic acid and reaction with 1 M dimethylsulfide/10 M hydrochloric acid, respectively.;The mechanisms for the gas-phase fragmentation reactions of singly and multiply protonated precursor ions of a series of model S-alkyl cysteine sulfoxide-containing peptides prepared by reaction with iodomethane, iodoacetamide, iodoacetic acid, acrylamide or 4-vinylpyridine, followed by oxidation with hydrogen peroxide have also been examined using multistage tandem mass spectrometry (MS/MS, MS3 and MS4), hydrogen/deuterium exchange and molecular orbital calculations (at the B3LYP/6-31 + G(d,p) level of theory). Dissociation of uniformly deuterated precursor ions of these model peptides confirmed that the non-sequence neutral loss of alkyl sulfenic acid (XSOH) in each case occurred via a charge-remote five-centered cis-1,2 elimination reaction. Similarly, the charge state dependence to the mechanisms and product ion structures for the losses of CO2, CO2 + H2O and CO2 + CH2O from S-carboxymethyl cysteine sulfoxide-containing peptides, and for the losses of CH2CHCONH 2 and CH2CHC5H4N, respectively from S-amidoethyl and S-pyridylethyl cysteine sulfoxide-containing peptide ions have also been determined.;A strategy involving the fixed-charge sulfonium ion derivatization, stable isotope labeling, capillary HPLC and automated neutral loss MS/MS and data dependent "pseudo MS3" scans in a triple quadrupole mass spectrometer has also been developed for the targeted gas-phase identification, characterization and quantitative analysis of low abundance methionine-containing peptides present within complex protein digests. In contrast to MS-based quantitative analysis strategies, the neutral loss scan mode MS/MS method was able to achieve accurate quantification for individual peptides at levels as low as 100 fmol and at abundance ratios ranging from 0.1 to 10, present within a complex protein digest. Using a similar fixed-charge sulfonium ion derivatization and tandem mass spectrometry-based analysis strategy, methionine oxidation was successfully quantified following hydrogen peroxide treatment of the Ca2+/calmodulin dependent serine/threonine phosphatase calcineurin.We have made it easy for you to find a PDF Ebooks without any digging. And by having access to our ebooks online or by storing it on your computer, you have convenient answers with Multistage Tandem Mass Spectrometry Strategies for the Targeted Analysis of Oxidative Protein Modifications. To get started finding Multistage Tandem Mass Spectrometry Strategies for the Targeted Analysis of Oxidative Protein Modifications, you are right to find our website which has a comprehensive collection of manuals listed.
Our library is the biggest of these that have literally hundreds of thousands of different products represented.

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Multistage Tandem Mass Spectrometry Strategies for the Targeted Analysis of Oxidative Protein Modifications

Multistage Tandem Mass Spectrometry Strategies for the Targeted Analysis of Oxidative Protein Modifications

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