A mass spectrometric strategy for absolute quantification of Plasmodium falciparum proteins of low abundance

Human malaria caused by Plasmodium falciparum is a major global health burden, killing around 1 million people every year [1]. Africa bears the greatest proportion of this burden, with over three-quarters of deaths occurring in African children, accounting for nearly a fifth of all child deaths in sub-Saharan Africa [2]. With the sequencing of the genome [3], as well as great advances in mass spectrometry (MS), characterization of the P. falciparum proteome is now technically possible. Information obtained using these techniques should be valuable in informing our future understanding of parasite/human interactions, disease progression and the selection of novel drug targets.One important recent advance in MS is the development of so-called selected reaction monitoring (SRM). This is an analysis method used in triple quadrupole mass spectrometers. The first quadrupole acts as a mass filter, allowing through only ions of selected mass/charge ratios before they are fragmented, using the second quadrupole as a collision cell. The final quadrupole then acts as a mass filter of resulting fragment ions in a similar way to the first quadrupole, allowing through only fragment ions of a particular, selected mass [4]. Since different peptides of the same mass would be expected to show different fragmentation patterns, this method provides an extra degree of certainty of protein identification compared to MS of the peptides alone.Further to these advances, recent work from this laboratory [5] has demonstrated that a form of 'soft' extraction of erythrocytic-stage P. falciparum parasites can resolve the issue of haemoglobin-derived products, which previously caused great hindrance in downstream methods of protein analysis. In addition, this work demonstrated the utility of the OFFGEL？ (Agilent, UK) isoelectric fractionation system in separating whole P. falciparum proteins, rather than tryptic peptides, according to their pI. This fractionation is vitally important in the ident