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

Malaria Journal

Methodology

Ronan DM O'Cualain¹  John E Hyde¹  Paul FG Sims¹ 

[1] Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK;

关键词: Isoelectric Point;    Theoretical Isoelectric Point;    Acetone Precipitation;    Ammonium Bicarbonate;    Serine Hydroxymethyltransferase;   

DOI  :  10.1186/1475-2875-9-286

 received in 2010-07-08, accepted in 2010-10-18,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundPlasmodium 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.MethodsThis 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.ResultsIt 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.ConclusionsBy 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.

【 授权许可】

Unknown   
© O'Cualain et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. 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.