Fig. 3.

Comparative proteomic analysis of the RBC membrane proteome during parasite egress. A, iRBC membranes (I) and postrupture vesicles derived from parasite egress (R) were isolated and compared with uninfected RBC membranes (U). Samples for the proteomic analysis were resolved on a 4–12% SDS-PAGE gel and stained with colloidal Coomassie. 34 slices were excised from each sample lane, digested with trypsin, extracted, and analyzed by LC/MS/MS mass spectrometry-based sequencing. The overall migration pattern for the post-rupture vesicle membrane proteome was different, as the characteristic high molecular weight spectrin doublet appeared to be degraded and with the appearance of more small molecular weight bands. B, Peptides from each gel slice were sequenced by mass spectrometry and the percentage distribution of identified proteins through the SDS-PAGE lanes for all samples (U, I, and R) were expressed in a heat map format. Red color indicates high percentage of peptides per protein (10%) whereas yellow indicates minimum (0%). This representation indicates that proteins were well-distributed throughout the SDS-PAGE gel. C, Spectral counting was used to compare the relative abundance of each protein from the three proteomes. The total number of peptides was summed for each identified protein in the iRBC and post-rupture vesicle proteome samples and was individually compared with the uninfected preparation to determine fold changes in relative protein abundance among the three proteomes. The abundance of most proteins remained similar (within a threefold difference) among the three proteomes, whereas a small proportion (∼10%) showed markedly lower abundance in the postrupture vesicle proteome.