Ymes of the folate pathway were obtained using UniProt [19]. These Menthone data
Ymes of the folate pathway were obtained using UniProt [19]. These data were used to determine their expected positions within the SDS-PAGE gel following OFFGELTM fractionation. To increase the probability of identifying the enzymes of interest, a mass position of plus and minus 10 of a particular enzyme’s theoretical mass was used. Similarly, for isoelectric point, the lanes adjacent to that predicted to contain the protein (using the observed PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/11011031 pH ranges) were also excised and subjected to analysis by mass spectrometry. By applying a significance threshold rate of p < 0.001 against a decoy database, the false discovery rate for the identified folate proteins was estimated to be less than 1 . Of the eight experimentally validated enzymes of the malarial folate pathway, six were detected in this study (Table 2). HPPK-DHPS and DHFR-TS are bifunctional so that a statistically significant identification to either protein domain meant that, by definition, the bifunctional enzyme had been identified.Identification of GTPCI and DHFS-FPGS by western blottingThe 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-FPGSin 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 OFFGELTM 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 DHFSFPGS 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 OFFGELTM 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,Table 2 Proteins of the folate pathway identified in this studyName PTPS DHFR-TS HPPK-DHPS SHMT PlasmoDB identifier PFF1360w PFD0830w PF08_0095 PFL1720w Average sequence coverage ( ) 31 5 25 28 Mass (kDa) 20280 72660 84062 49749 pI 5.97 6.86 6.73 8.19 False discovery rate ( ) 0.58 0.3 0.24For each protein the observed protein molecular mass and isoelectric point corresponds to the theoretical values as obtained from UNIPROT. The percentage false discovery rate was calculated using a decoy database and the pepti.
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