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Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nomin...

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Autores principales: Gunning, Yvonne, Watson, Andrew D., Rigby, Neil M., Philo, Mark, Peazer, Joshua K., Kemsley, E. Kate
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5092036/
https://www.ncbi.nlm.nih.gov/pubmed/27685654
http://dx.doi.org/10.3791/54420
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author Gunning, Yvonne
Watson, Andrew D.
Rigby, Neil M.
Philo, Mark
Peazer, Joshua K.
Kemsley, E. Kate
author_facet Gunning, Yvonne
Watson, Andrew D.
Rigby, Neil M.
Philo, Mark
Peazer, Joshua K.
Kemsley, E. Kate
author_sort Gunning, Yvonne
collection PubMed
description We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture.
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spelling pubmed-50920362016-11-15 Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication Gunning, Yvonne Watson, Andrew D. Rigby, Neil M. Philo, Mark Peazer, Joshua K. Kemsley, E. Kate J Vis Exp Biochemistry We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture. MyJove Corporation 2016-09-20 /pmc/articles/PMC5092036/ /pubmed/27685654 http://dx.doi.org/10.3791/54420 Text en Copyright © 2016, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
spellingShingle Biochemistry
Gunning, Yvonne
Watson, Andrew D.
Rigby, Neil M.
Philo, Mark
Peazer, Joshua K.
Kemsley, E. Kate
Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication
title Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication
title_full Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication
title_fullStr Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication
title_full_unstemmed Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication
title_short Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication
title_sort species determination and quantitation in mixtures using mrm mass spectrometry of peptides applied to meat authentication
topic Biochemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5092036/
https://www.ncbi.nlm.nih.gov/pubmed/27685654
http://dx.doi.org/10.3791/54420
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