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A protein-centric approach for the identification of folate enzymes from the malarial parasite, Plasmodium falciparum, using OFFGEL™ solution-based isoelectric focussing and mass spectrometry

BACKGROUND: Plasmodium species are difficult to study using proteomic technology because they contain large amounts of haemoglobin-derived products (HDP), generated by parasite breakdown of host haemoglobin. HDP are known to interfere with isoelectric focussing, a cornerstone of fractionation strate...

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Autores principales: O'Cualain, Ronan DM, Hyde, John E, Sims, Paul FG
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2967559/
https://www.ncbi.nlm.nih.gov/pubmed/20955557
http://dx.doi.org/10.1186/1475-2875-9-286
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author O'Cualain, Ronan DM
Hyde, John E
Sims, Paul FG
author_facet O'Cualain, Ronan DM
Hyde, John E
Sims, Paul FG
author_sort O'Cualain, Ronan DM
collection PubMed
description BACKGROUND: Plasmodium species are difficult to study using proteomic technology because they contain large amounts of haemoglobin-derived products (HDP), generated by parasite breakdown of host haemoglobin. HDP are known to interfere with isoelectric focussing, a cornerstone of fractionation strategies for the identification of proteins by mass spectrometry. In addition to the challenge presented by this material, as in most proteomes, there exists in this parasite a considerable dynamic range between proteins of high and low abundance. The enzymes of the folate pathway, a proven and widely used drug target, are included in the latter class. METHODS: This report describes a work-flow utilizing a parasite-specific extraction protocol that minimizes release of HDP into the lysate, followed by in-solution based OFFGEL™ electrophoresis at the protein level, trypsin digestion and mass spectrometric analysis. RESULTS: It is demonstrated that, by removing HDP from parasite lysates, OFFGEL™-mediated protein separation is able to deliver reduced complexity protein fractions. Importantly, proteins with similar and predictable physical properties are sharply focussed within such fractions. CONCLUSIONS: By following this novel workflow, data have been obtained which allow the unequivocal experimental identification by mass spectrometry of four of the six proteins involved in folate biosynthesis and recycling.
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spelling pubmed-29675592010-11-02 A protein-centric approach for the identification of folate enzymes from the malarial parasite, Plasmodium falciparum, using OFFGEL™ solution-based isoelectric focussing and mass spectrometry O'Cualain, Ronan DM Hyde, John E Sims, Paul FG Malar J Methodology BACKGROUND: Plasmodium species are difficult to study using proteomic technology because they contain large amounts of haemoglobin-derived products (HDP), generated by parasite breakdown of host haemoglobin. HDP are known to interfere with isoelectric focussing, a cornerstone of fractionation strategies for the identification of proteins by mass spectrometry. In addition to the challenge presented by this material, as in most proteomes, there exists in this parasite a considerable dynamic range between proteins of high and low abundance. The enzymes of the folate pathway, a proven and widely used drug target, are included in the latter class. METHODS: This report describes a work-flow utilizing a parasite-specific extraction protocol that minimizes release of HDP into the lysate, followed by in-solution based OFFGEL™ electrophoresis at the protein level, trypsin digestion and mass spectrometric analysis. RESULTS: It is demonstrated that, by removing HDP from parasite lysates, OFFGEL™-mediated protein separation is able to deliver reduced complexity protein fractions. Importantly, proteins with similar and predictable physical properties are sharply focussed within such fractions. CONCLUSIONS: By following this novel workflow, data have been obtained which allow the unequivocal experimental identification by mass spectrometry of four of the six proteins involved in folate biosynthesis and recycling. BioMed Central 2010-10-18 /pmc/articles/PMC2967559/ /pubmed/20955557 http://dx.doi.org/10.1186/1475-2875-9-286 Text en Copyright ©2010 O'Cualain et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology
O'Cualain, Ronan DM
Hyde, John E
Sims, Paul FG
A protein-centric approach for the identification of folate enzymes from the malarial parasite, Plasmodium falciparum, using OFFGEL™ solution-based isoelectric focussing and mass spectrometry
title A protein-centric approach for the identification of folate enzymes from the malarial parasite, Plasmodium falciparum, using OFFGEL™ solution-based isoelectric focussing and mass spectrometry
title_full A protein-centric approach for the identification of folate enzymes from the malarial parasite, Plasmodium falciparum, using OFFGEL™ solution-based isoelectric focussing and mass spectrometry
title_fullStr A protein-centric approach for the identification of folate enzymes from the malarial parasite, Plasmodium falciparum, using OFFGEL™ solution-based isoelectric focussing and mass spectrometry
title_full_unstemmed A protein-centric approach for the identification of folate enzymes from the malarial parasite, Plasmodium falciparum, using OFFGEL™ solution-based isoelectric focussing and mass spectrometry
title_short A protein-centric approach for the identification of folate enzymes from the malarial parasite, Plasmodium falciparum, using OFFGEL™ solution-based isoelectric focussing and mass spectrometry
title_sort protein-centric approach for the identification of folate enzymes from the malarial parasite, plasmodium falciparum, using offgel™ solution-based isoelectric focussing and mass spectrometry
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2967559/
https://www.ncbi.nlm.nih.gov/pubmed/20955557
http://dx.doi.org/10.1186/1475-2875-9-286
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