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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.

O'Cualain RD, Hyde JE, Sims PF - Malar. J. (2010)

Bottom Line: The enzymes of the folate pathway, a proven and widely used drug target, are included in the latter class.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.Importantly, proteins with similar and predictable physical properties are sharply focussed within such fractions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

ABSTRACT

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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Related in: MedlinePlus

Theoretical and observed pH ranges of individual OFFGEL™ fractions. A. The theoretical pH range was calculated by dividing by 12 the 7 available pH units between pH 3 and 10 to give a value of approximately 0.6 pH units for the range covered by each fraction. The median value for each fraction was then plotted. The observed pH for each fraction was calculated by averaging the pH measured for equivalent fractions from three OFFGEL™ separations. B. Plots of the median theoretical and mean observed pH values for each OFFGEL™ fraction. The standard deviation for each individual OFFGEL™ fraction pH measurement is also displayed.
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Figure 4: Theoretical and observed pH ranges of individual OFFGEL™ fractions. A. The theoretical pH range was calculated by dividing by 12 the 7 available pH units between pH 3 and 10 to give a value of approximately 0.6 pH units for the range covered by each fraction. The median value for each fraction was then plotted. The observed pH for each fraction was calculated by averaging the pH measured for equivalent fractions from three OFFGEL™ separations. B. Plots of the median theoretical and mean observed pH values for each OFFGEL™ fraction. The standard deviation for each individual OFFGEL™ fraction pH measurement is also displayed.

Mentions: Commercially available IPG strips from GE Healthcare were used in validating the OFFGEL™ fractionation. These are strips of 13 cm in length containing an immobilized pH gradient ranging from pH 3 to 10. A frame consisting of 12 equal sized fractions was placed on top of the IPG strip. To assign pH ranges to the individual OFFGEL™ fractions, a "theoretical pH range" was constructed. The assumption was made that the distribution of immobilized ampholytes on the IPG strip comprised a linear gradient within the range from pH 3 to 10. Dividing the 7 available pH units by 12 gave a graduation of approximately 0.6 between each fraction. Therefore, fraction 1 had a theoretical pH range from 3 to 3.6, fraction 2 ranged from 3.6 to 4.2 and so on up to pH 10. This is displayed in Figure 4a. An "observed pH range" was also determined. For this, three OFFGEL™ fractionations were performed using only OFFGEL™ buffer as sample, and the pH of each fraction was directly measured and corresponding values averaged. This information is also displayed in Figure 4a. From graphical analysis of these data (Figure 4b), it is evident that the immobilized pH gradient measured after an OFFGEL™ separation is linear, but begins at approximately pH 4.2, and not pH 3 as described by the manufacturer. Thus, proteins with a pI 4.7 and lower are focussed to fraction 1, while proteins whose pI lies between 4.7 and 5.2 are focussed to fraction 2 and so on. It has been noted previously that the immobilized pH gradient in commercial IPG strips can vary both in length and position [18]. This may explain the distribution of the IPG gradient as determined by this study and is a phenomenon to be aware of when utilizing an isoelectric point-based identification approach. Table 1 further illustrates this point. Using the annotation information of the 91 proteins categorized in Figure 3, the mean and median isoelectric point of the proteins identified in each fraction was calculated. These show reasonable agreement with the theoretically expected values but agree rather less well with pH ranges observed in the earlier experiments. Importantly however, the sequence coverage of proteins separated and identified using OFFGEL™ was excellent, with an average sequence coverage of 25% (Table 1).


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 RD, Hyde JE, Sims PF - Malar. J. (2010)

Theoretical and observed pH ranges of individual OFFGEL™ fractions. A. The theoretical pH range was calculated by dividing by 12 the 7 available pH units between pH 3 and 10 to give a value of approximately 0.6 pH units for the range covered by each fraction. The median value for each fraction was then plotted. The observed pH for each fraction was calculated by averaging the pH measured for equivalent fractions from three OFFGEL™ separations. B. Plots of the median theoretical and mean observed pH values for each OFFGEL™ fraction. The standard deviation for each individual OFFGEL™ fraction pH measurement is also displayed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2967559&req=5

Figure 4: Theoretical and observed pH ranges of individual OFFGEL™ fractions. A. The theoretical pH range was calculated by dividing by 12 the 7 available pH units between pH 3 and 10 to give a value of approximately 0.6 pH units for the range covered by each fraction. The median value for each fraction was then plotted. The observed pH for each fraction was calculated by averaging the pH measured for equivalent fractions from three OFFGEL™ separations. B. Plots of the median theoretical and mean observed pH values for each OFFGEL™ fraction. The standard deviation for each individual OFFGEL™ fraction pH measurement is also displayed.
Mentions: Commercially available IPG strips from GE Healthcare were used in validating the OFFGEL™ fractionation. These are strips of 13 cm in length containing an immobilized pH gradient ranging from pH 3 to 10. A frame consisting of 12 equal sized fractions was placed on top of the IPG strip. To assign pH ranges to the individual OFFGEL™ fractions, a "theoretical pH range" was constructed. The assumption was made that the distribution of immobilized ampholytes on the IPG strip comprised a linear gradient within the range from pH 3 to 10. Dividing the 7 available pH units by 12 gave a graduation of approximately 0.6 between each fraction. Therefore, fraction 1 had a theoretical pH range from 3 to 3.6, fraction 2 ranged from 3.6 to 4.2 and so on up to pH 10. This is displayed in Figure 4a. An "observed pH range" was also determined. For this, three OFFGEL™ fractionations were performed using only OFFGEL™ buffer as sample, and the pH of each fraction was directly measured and corresponding values averaged. This information is also displayed in Figure 4a. From graphical analysis of these data (Figure 4b), it is evident that the immobilized pH gradient measured after an OFFGEL™ separation is linear, but begins at approximately pH 4.2, and not pH 3 as described by the manufacturer. Thus, proteins with a pI 4.7 and lower are focussed to fraction 1, while proteins whose pI lies between 4.7 and 5.2 are focussed to fraction 2 and so on. It has been noted previously that the immobilized pH gradient in commercial IPG strips can vary both in length and position [18]. This may explain the distribution of the IPG gradient as determined by this study and is a phenomenon to be aware of when utilizing an isoelectric point-based identification approach. Table 1 further illustrates this point. Using the annotation information of the 91 proteins categorized in Figure 3, the mean and median isoelectric point of the proteins identified in each fraction was calculated. These show reasonable agreement with the theoretically expected values but agree rather less well with pH ranges observed in the earlier experiments. Importantly however, the sequence coverage of proteins separated and identified using OFFGEL™ was excellent, with an average sequence coverage of 25% (Table 1).

Bottom Line: The enzymes of the folate pathway, a proven and widely used drug target, are included in the latter class.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.Importantly, proteins with similar and predictable physical properties are sharply focussed within such fractions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus