Human malaria caused by Plasmodium falciparum is a major global health burden, killing around 1 million people every year . 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 . With the sequencing of the genome , 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 . 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  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 identification of proteins of low abundance, such as those of the folate and many other metabolic pathways, due to the complexity of the proteome. It is estimated that even in the most comprehensive proteomic studies of the P. falciparum life-cycle to date, only ~46% of the predicted gene products were detected [6, 7].
As well as identifying proteins of interest by fractionation followed by mass spectrometry, quantification of these proteins is also highly valuable in understanding the dynamics of biological systems. It has been shown a number of times that the expression of a particular gene as measured by mRNA quantification does not necessarily correlate with the level of protein within the cell [[8–11]]. Moreover, it is known that the P. falciparum proteome contains many unstructured proteins that can experience rapid degradation at both mRNA and protein levels [12, 13]. In addition, the nature and degree of post-translational modifications, which can determine a number of protein properties including function, localization and activity, can only be analysed at the protein level.
Quantitative proteomics of the P. falciparum parasite is a field still in its infancy. In 2004, relative quantification was performed in this laboratory using a SILAC (stable-isotope labelling of amino acids in cell culture) -based method . While a significant step forward for plasmodial proteomics, this study necessarily focused on proteins of high abundance to establish proof of principle. Since then, other significant studies have been undertaken to assess plasmodial proteins in a quantitative manner [14, 15], but again, these studies determined only relative quantification.
In order to truly understand a biological system, we must aspire to absolute quantification of proteins across their entire dynamic range. By doing this, data from different studies and different laboratories can be more easily compared and proteomic, transcriptomic and metabolomic data can be more meaningfully correlated. One potential method for absolute protein quantification is the use of a heterologously expressed so-called QconCAT protein .
In this method, proteotypic tryptic peptides from proteins of interest, 'Q-peptides', are chosen. A number of these peptides are then expressed together from an artificial gene to produce a concatenated protein containing many different Q-peptides. Thus, when the QconCAT protein is digested using trypsin, peptides representing a number of proteins of interest are produced in equal molar amounts. If such QconCAT proteins are expressed, from Escherichia coli, in a labelled form using stable-isotope labelling, they can then be introduced, in a known amount, into biological samples prior to tryptic digestion and mass spectrometry. The resulting 'heavy' peaks on mass spectra, derived from the Q-peptides can be compared with the corresponding 'light' peaks, obtained concurrently from the native peptides, allowing accurate, absolute quantification. Producing a labelled QconCAT, rather than a labelled form of an entire native protein, allows several proteins to be efficiently quantified in the same experiment.
Here a QconCAT-based method, combined with SRM mass spectrometry, is described for the absolute quantification of proteins of low abundance in erythrocytic-stage P. falciparum.