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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

The protein-centric OFFGEL™ strategy. Proteins in a freeze-thaw lysate were separated by OFFGEL™ electrophoresis in the first dimension followed by SDS-PAGE for the second dimension. Bands in the gel area between the masses of 50 to 70 kDa and lanes 5 to 9 were analysed by mass spectrometry. Between ten and twelve 1 mm thick bands were analysed for each lane between these masses.
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Figure 2: The protein-centric OFFGEL™ strategy. Proteins in a freeze-thaw lysate were separated by OFFGEL™ electrophoresis in the first dimension followed by SDS-PAGE for the second dimension. Bands in the gel area between the masses of 50 to 70 kDa and lanes 5 to 9 were analysed by mass spectrometry. Between ten and twelve 1 mm thick bands were analysed for each lane between these masses.

Mentions: The preliminary observations described above suggested to us that whilst OFFGEL™ separation at the protein level had significant potential, this could not be properly delivered from extracts containing high levels of HDP. Further investigation therefore sought to identify a parasite lysis procedure that could release cytosolic parasite proteins free from contamination with HDP. This was ultimately achieved using five cycles of freezing and thawing in deionized water. After acetone precipitation, resuspension in OFFGEL™ sample buffer and overnight OFFGEL™ fractionation, SDS-PAGE analysis of the resulting fractions (Figure 2) revealed little evidence of HDP and, presumably as a consequence of this, excellent focussing of plasmodial proteins across the entire range of pI values.


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)

The protein-centric OFFGEL™ strategy. Proteins in a freeze-thaw lysate were separated by OFFGEL™ electrophoresis in the first dimension followed by SDS-PAGE for the second dimension. Bands in the gel area between the masses of 50 to 70 kDa and lanes 5 to 9 were analysed by mass spectrometry. Between ten and twelve 1 mm thick bands were analysed for each lane between these masses.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The protein-centric OFFGEL™ strategy. Proteins in a freeze-thaw lysate were separated by OFFGEL™ electrophoresis in the first dimension followed by SDS-PAGE for the second dimension. Bands in the gel area between the masses of 50 to 70 kDa and lanes 5 to 9 were analysed by mass spectrometry. Between ten and twelve 1 mm thick bands were analysed for each lane between these masses.
Mentions: The preliminary observations described above suggested to us that whilst OFFGEL™ separation at the protein level had significant potential, this could not be properly delivered from extracts containing high levels of HDP. Further investigation therefore sought to identify a parasite lysis procedure that could release cytosolic parasite proteins free from contamination with HDP. This was ultimately achieved using five cycles of freezing and thawing in deionized water. After acetone precipitation, resuspension in OFFGEL™ sample buffer and overnight OFFGEL™ fractionation, SDS-PAGE analysis of the resulting fractions (Figure 2) revealed little evidence of HDP and, presumably as a consequence of this, excellent focussing of plasmodial proteins across the entire range of pI values.

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