Limits...
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.

Show MeSH

Related in: MedlinePlus

Western blot analysis. A. SDS-PAGE gel of OFFGEL™ fractions probed with anti-DHFS-FPGS. B. SDS-PAGE gel of OFFGEL™ fractions probed with anti-GTPCI. Anti-PTPS was used as a positive control after blotting and probing for DHFS-FPGS. The isoelectric point and molecular mass of both DHFS-FPGS and GTPCI differ significantly from their theoretical values. DHFS-FPGS would be expected to be identified from lane 6 at molecular mass 60 kDa, yet western blotting analysis places it in lane 3. GTPCI has a theoretical mass of 46 kDa and would be expected to be identified from lane 12, yet this analysis identifies it as being found in lanes 9 and 10 and at mass 60 kDa
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2967559&req=5

Figure 5: Western blot analysis. A. SDS-PAGE gel of OFFGEL™ fractions probed with anti-DHFS-FPGS. B. SDS-PAGE gel of OFFGEL™ fractions probed with anti-GTPCI. Anti-PTPS was used as a positive control after blotting and probing for DHFS-FPGS. The isoelectric point and molecular mass of both DHFS-FPGS and GTPCI differ significantly from their theoretical values. DHFS-FPGS would be expected to be identified from lane 6 at molecular mass 60 kDa, yet western blotting analysis places it in lane 3. GTPCI has a theoretical mass of 46 kDa and would be expected to be identified from lane 12, yet this analysis identifies it as being found in lanes 9 and 10 and at mass 60 kDa

Mentions: The full complement of folate pathway enzymes was not identified by mass spectrometry in this study. Of the two proteins that were not identified, DHFS-FPGS has a theoretical isoelectric point of 6.25 based on its sequence, while that for GTPCI is estimated to be 9.4. On the SDS-PAGE gel, this would place DHFS-FPGS in lane 6 (pH range 5.9 to 6.5) and GTPCI in lane 12 (pH range 9.2 and upwards) with molecular masses of 60 and 46 kDa respectively. However, these enzymes were not identified at those positions. Their presence in the gel was however confirmed by western blotting (Figures 5a and 5b). As a positive control, an antibody to the protein previously identified by mass spectrometry as PTPS (pI 6.0, MW 20 kDa) was also used to probe a filter prepared by transfer of proteins from an SDS-PAGE analysis of an OFFGEL™ separation. As revealed in this experiment, its location confirmed the mass spectrometry results, with its position on the SDS-PAGE gel corresponding to its calculated molecular mass and isoelectric point. However, western blotting also localized DHFS-FPGS to lane 3 (pH range of 4.2 to 4.7) on the same filter, and GTPCI was determined to be in both lanes 9 and 10 (pH range 7.6 to 8.8) of a second filter. The observed mass for DHFS-FPGS was estimated to be 60 kDa, similar to its theoretical mass, while GTPCI was estimated to have a mass also of approximately 60 kDa, which differs significantly from its theoretical mass of 46 kDa. Although it was thus apparent that the two unidentified proteins were not running as expected on OFFGEL™ and/or SDS-PAGE, further excision of bands in areas of the gel highlighted by western blotting followed by subsequent mass spectrometry still failed to identity these two enzymes. It is also noted that the western analysis reported here reveals the possible existence of at least two forms of GTPCI differing slightly in their mobility on SDS-PAGE,


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)

Western blot analysis. A. SDS-PAGE gel of OFFGEL™ fractions probed with anti-DHFS-FPGS. B. SDS-PAGE gel of OFFGEL™ fractions probed with anti-GTPCI. Anti-PTPS was used as a positive control after blotting and probing for DHFS-FPGS. The isoelectric point and molecular mass of both DHFS-FPGS and GTPCI differ significantly from their theoretical values. DHFS-FPGS would be expected to be identified from lane 6 at molecular mass 60 kDa, yet western blotting analysis places it in lane 3. GTPCI has a theoretical mass of 46 kDa and would be expected to be identified from lane 12, yet this analysis identifies it as being found in lanes 9 and 10 and at mass 60 kDa
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Western blot analysis. A. SDS-PAGE gel of OFFGEL™ fractions probed with anti-DHFS-FPGS. B. SDS-PAGE gel of OFFGEL™ fractions probed with anti-GTPCI. Anti-PTPS was used as a positive control after blotting and probing for DHFS-FPGS. The isoelectric point and molecular mass of both DHFS-FPGS and GTPCI differ significantly from their theoretical values. DHFS-FPGS would be expected to be identified from lane 6 at molecular mass 60 kDa, yet western blotting analysis places it in lane 3. GTPCI has a theoretical mass of 46 kDa and would be expected to be identified from lane 12, yet this analysis identifies it as being found in lanes 9 and 10 and at mass 60 kDa
Mentions: The full complement of folate pathway enzymes was not identified by mass spectrometry in this study. Of the two proteins that were not identified, DHFS-FPGS has a theoretical isoelectric point of 6.25 based on its sequence, while that for GTPCI is estimated to be 9.4. On the SDS-PAGE gel, this would place DHFS-FPGS in lane 6 (pH range 5.9 to 6.5) and GTPCI in lane 12 (pH range 9.2 and upwards) with molecular masses of 60 and 46 kDa respectively. However, these enzymes were not identified at those positions. Their presence in the gel was however confirmed by western blotting (Figures 5a and 5b). As a positive control, an antibody to the protein previously identified by mass spectrometry as PTPS (pI 6.0, MW 20 kDa) was also used to probe a filter prepared by transfer of proteins from an SDS-PAGE analysis of an OFFGEL™ separation. As revealed in this experiment, its location confirmed the mass spectrometry results, with its position on the SDS-PAGE gel corresponding to its calculated molecular mass and isoelectric point. However, western blotting also localized DHFS-FPGS to lane 3 (pH range of 4.2 to 4.7) on the same filter, and GTPCI was determined to be in both lanes 9 and 10 (pH range 7.6 to 8.8) of a second filter. The observed mass for DHFS-FPGS was estimated to be 60 kDa, similar to its theoretical mass, while GTPCI was estimated to have a mass also of approximately 60 kDa, which differs significantly from its theoretical mass of 46 kDa. Although it was thus apparent that the two unidentified proteins were not running as expected on OFFGEL™ and/or SDS-PAGE, further excision of bands in areas of the gel highlighted by western blotting followed by subsequent mass spectrometry still failed to identity these two enzymes. It is also noted that the western analysis reported here reveals the possible existence of at least two forms of GTPCI differing slightly in their mobility on SDS-PAGE,

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