In a publishing event they hoped would generate buzz, Science and Nature simultaneously released a group of papers last week on the genome and proteome of the malaria parasite Plasmodium falciparum (in Nature), as well as malaria’s vector, the mosquito Anopheles gambiae (in Science). In addition, Nature published the genome of the related rodent parasite Plasmodium yoelii yoelii.
Among this group, two papers offered a proteomic analysis of P. falciparum, providing evidence of just how much this new science has become inextricably intertwined with genomics. The results also demonstrate the synergistic potential of analyzing both genome and proteome.
For instance, while an analysis of the P. falciparum genome sequence did not reveal any genes similar to those that encode proteins key to metabolism and mitochondria in other species, the proteomic analyses did detect fragments of enzymes similar to those involved in mitochondrial processes in other species, indicating that the organism does in fact employ mitochondria at some stage of its life cycle.
In the first of these papers, “Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry,” scientists in Matthias Mann’s lab at the University of Southern Denmark and Hendrik Stunnenberg’s lab at the University of Nijmegen in the Netherlands used the human and P. falciparum genome databases to match spectra of peptides obtained to predicted proteins, and performed a direct genome search in areas where no exons had been previously predicted. Using these two different search methods, the group was able to identify 1,289 unique malaria proteins.
In this paper, the researchers extracted the parasite at different stages, sexual and asexual, from samples of red blood cells through freeze-thawing and centrifugation, deriving soluble and insoluble fractions. Next, they separated these mixtures into ten molecular mass fractions using 1D gels, then extracted hemoglobin and globin, and digested the mixtures using trypsin. Finally, the researchers separated out the peptides by combining reverse phase liquid chromatography and quadrupole time-of-flight mass spectrometry (using an Applied Biosystems/MDS Sciex QSTAR). Using iterative calibration algorithms, the group reported that it had achieved mass accuracy of over 20 ppm or a deviation of 0.03 Da per 1,300 Da peptide. They performed the stages of this analysis at least in duplicate.
Stunnenberg said the group decided to use online nano-LC-MS/MS because it “allows a very rapid analysis of highly complex protein mixtures in an extremely short time span,” once the procedure is established. “Secondly, one of the aims of our study was to identify membrane proteins of various developmental stages of the malaria parasite. Two-D gel technology is pretty useless, or very difficult and inefficient, at identifying membrane proteins. The nano-LC-MS/MS approach enabled us to do an exhaustive analysis.”
Further analysis resulted in a large number of orphan peptides and the identification of genes that had been missed in the annotation, Stunnenberg said.
In a broader paper, “A proteomic view of the Plasmodium falciparum life cycle,” a far-flung group led by Laurence Florens of the University of Washington Department of Genome Sciences more explicitly identified the different proteins expressed during four different phases of the parasite’s life cycle. They found marked differences between the merozoite of the P. falciparum, released after the parasite has infected a red blood cell, the trophozoite, which can digest the contents of the host cell, the gametocyte, which is taken up by the mosquito vector, and the sporozoite, which is injected into the human. The group used MudPIT (Multi-dimensional protein identification technology), a combination of high-resolution liquid chromatography and tandem mass spectrometry, to identify 2,415 proteins, or 45 percent of those predicted by the open reading frames of the genome.
This increased knowledge of the proteins behind the machinery of the malaria parasite, the authors said, could potentially aid researchers in designing drugs that “substantially and persistently interrupt the life cycle of this complex parasite.”
— MMJ