Background
Methods
Culture of P. falciparum and parasite sampling
Sample preparation
Solution-based isoelectric focussing using the Agilent 3100 OFFGEL™ fractionator
Electrophoretic separation
Western blot identification of GTPCI and DHFS-FPGS
In-gel trypsin digestion
Mass spectrometric analyses
Protein identification criteria
Results
Influence of HDP on the quality of protein focussing
Freeze-thaw lysis avoids HDP extraction
Analysis of isoelectric focussing of proteins using OFFGEL™ electrophoresis
Validation of OFFGEL™ isoelectric protein focussing
Lane 5 (n = 24) | Lane 6 (n = 31) | Lane 7 (n = 34) | Lane 8 (n = 42) | Lane 9 (n = 38) | |
---|---|---|---|---|---|
Mean pI
| 6.2 ± 1.1 | 6.4 ± 0.9 | 6.7 ± 0.9 | 6.7 ± 1.0 | 7.3 ± 0.8 |
Median pI
| 5.7 | 6.3 | 6.5 | 6.7 | 7.2 |
Theoretical pH range
| 5.3 to 5.9 | 5.9 to 6.5 | 6.5 to 7.1 | 7.1 to 7.6 | 7.6 to 8.2 |
Observed pH range
| 6.1 to 6.5 | 6.5 to 7.0 | 7.0 to 7.5 | 7.5 to 7.9 | 7.9 to 8.4 |
Mean peptide coverage (%)
| 24.5 ± 15.3 | 23.1 ± 12.6 | 21.3 ± 9.8 | 19.6 ± 12.0 | 35.8 ± 14.8 |
Median peptide coverage (%)
| 23 | 24 | 21 | 17 | 34 |
Identification of folate enzymes using OFFGEL™ fractionation and mass spectrometry
Name | PlasmoDB identifier | Average sequence coverage (%) | Mass (kDa) | pI | False discovery rate (%) |
---|---|---|---|---|---|
PTPS
| PFF1360w | 31 | 20280 | 5.97 | 0.58 |
DHFR-TS
| PFD0830w | 5 | 72660 | 6.86 | 0.3 |
HPPK-DHPS
| PF08_0095 | 25 | 84062 | 6.73 | 0.24 |
SHMT
| PFL1720w | 28 | 49749 | 8.19 | 0 |