Abundance of tegument surface proteins in the human blood fluke Schistosoma mansoni determined by QconCAT proteomics
Graphical absract
Research highlights
► Construction of an artificial protein concatamer comprising. ► 30 tryptic peptides from the tegument of S. mansoni. ► Its use revealed that aquaporin, three phosphohydrolases, dysferlin and tetraspanin Tsp2 are the principal membrane proteins. ► The demonstration that Sm200 surface protein is in a different tegument compartment, possibly the secreted membranocalyx.
Introduction
Schistosomes are platyhelminth parasites that can live for decades in the human portal vasculature, surrounded by humoral and cellular components of the immune system but apparently invulnerable to attack. Even more remarkable, these centimetre-long worms are covered by a naked syncytial layer of cytoplasm, called a tegument, which by rights should provide the perfect target. The outer surface of this layer, which forms the parasite-host interface, has an unusual multilaminate appearance [1] and its properties must be intimately linked with the worm's ability to evade attack. It is often referred to as a heptalaminate membrane [2], but the alternative interpretation, followed here, is that it represents a normal plasma membrane, overlain by a membrane-like secretion, termed a membranocalyx by analogy with the glycocalyx of conventional eukaryotic cells [3], [4]. The cell bodies, containing the machinery for protein synthesis and export, lie below the musculature of the body wall and are connected to the tegument by narrow tubes of cytoplasm [5]. Multilaminate vesicles originating there traffic to the syncytium to deliver their contents to the exterior at the base of tegument pits [1], [3], thus contributing to a slow turnover of the membranocalyx into the blood stream [6], [7]. The most plausible explanation for the properties and function of the membranocalyx is that it provides a relatively inert barrier to protect the underlying plasma membrane where conventional membrane enzymes and transporters reside. If this is correct, the membranocalyx must itself contain few macromolecules of parasite origin. However, its hydrophobic nature allows it to acquire lipophilic host molecules such as GPI-anchored membrane proteins [8] and erythrocyte glycolipids [9].
Methods have been developed to enrich the surface for biochemical analysis, with freeze/thaw/vortexing (F/T/V) being widely used to detach the membranes [10], [11]. The advent of proteomic techniques permitted the composition of the total material released by F/T/V, to be investigated [12] but relatively few membrane-spanning proteins were identified. The application of density gradient centrifugation to enrich the surface membranes [11], followed by differential extraction with chaotropic agents of increasing strength yielded a final insoluble pellet containing primarily membrane proteins [13]. Subsequently, proteins on the surface of live worms accessible to biotinylation with sulfo-NHS-biotin were identified [14]. More recently, the surface proteins of live worms accessible to the enzymes trypsin and phosphatidyl-inositol phospholipase C (PiPLC) have also been documented [8]. The cumulative result of these studies is that we have a growing list of proteins which comprise the principal macromolecular constituents of the parasite interface. However, there is still much work required to place them in their correct context within the multilayered surface. In addition, and important for the identification of possible vaccine targets, we have no idea of the relative abundance of the various constituents.
The proteomic approach, linked to suitable fractionation procedures, is ideally suited to the qualitative identification of proteins in a complex mixture, but obtaining quantitative information has proved less tractable. The fractionation of soluble schistosome proteins by 2-dimensional electrophoresis (2-DE) before quantitation by staining and image analysis [15], [16] provides a good indication of their relative abundance but the approach is not suitable for integral membrane proteins as ionic detergents are often required for their optimal extraction. The number of peptides identified per protein in tandem mass spectrometry has been used as a protein abundance index (PAI), normalised relative to the theoretical number of peptides [17]. The PAI was subsequently modified to an exponential form [18], and validated using synthetic peptides, giving a score roughly proportional to abundance. However, the response of a peptide in the mass spectrometer is a function of its amount, physiochemical properties and ability to ionise, which can all vary widely. The QconCAT approach has been developed to circumvent these limitations, allowing for the measurement of both relative and absolute abundance of multiple proteins within a mixture.
In the QconCAT approach, absolute quantification relies on the addition of isotopically labelled peptide internal standards (Q peptides) to the protein sample under investigation. The Q peptide has a different mass but otherwise identical physiochemical properties to the target peptide. These peptides therefore ionise similarly, meaning their relative abundances can be directly quantified from their responses in the mass spectrometer. Furthermore, since a known amount of Q peptide is added, the absolute amount of unmodified peptide can be calculated [19]. The key advance provided by the QconCAT technique is that, instead of chemically synthesising each Q peptide individually, it relies on the production of an artificial protein that is a concatamer of Q peptides (hence the term QconCAT) [20], [21], [22]. These are selected from proteins known to be present in the sample and facilitate the simultaneous quantitation of numerous target proteins. A gene encoding the requisite peptides is designed, and the concatamer is expressed heterologously in Escherichia coli. It can be labelled with stable isotopes by growth in the presence of e.g. [13C6]lysine and [13C6]arginine [22]. The labelled QconCAT protein is then purified, quantified and a known amount added to the complex protein sample under investigation. Tryptic digestion of the QconCAT-sample mix releases each of the QconCAT peptides in a strict 1:1 stoichiometry; subsequent MS analysis allows the quantification of each peptide present in the sample. We report here the design of a QconCAT gene encoding 33 signature peptides selected from different classes of protein identified in our previous proteomic studies of the schistosome tegument surface [13], [14]. The labelled QconCAT standard was added to two tegument surface preparations, and the abundance of the selected peptides, and thus the proteins from which they were released, in each sample determined by mass spectrometry. Our results provide insights into the relative importance of processes occurring at the tegument surface of adult worms resident in the host bloodstream.
Section snippets
Ethics statement
The procedures involving animals were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986, and authorised on personal and project licences issued by the UK Home Office. The study protocol was approved by the Biology Department Ethical Review Committee at the University of York.
Selection of peptides, preparation and purification of the SmQconCAT construct
A total of 31 proteins was selected for quantification based on their previous identification in proteomic studies of the S. mansoni tegument [13], [14]. Representative unique peptides were
Characterization and verification of the SmQconCAT
The sequences of the peptides selected for quantification of the 31 tegument proteins, the m/z values of their protonated molecules and the accession numbers of the parent proteins are presented in Table 1. To assess the reproducibility of the method, alkaline phosphatase and Sm200 were each represented by two peptides in the protein construct, (Fig. 1A, C). In order to assess the homogeneity of the recombinant SmQconCAT after purification by nickel-affinity chromatography, a 3 μg aliquot was
Discussion
The QconCAT technique was developed to quantitate known protein constituents in a biological sample, using signature peptides selected on the basis that they were found in previous MS/MS analyses. This means that they should have a good chance of generating a mass spectrometric response and being detected. However, there are constraints that do limit choice. Because the quantification was performed by MALDI-TOF MS, individual peptides in the concatamer must differ sufficiently in molecular
Acknowledgements
This work received support from the UK Biotechnology and Biological Sciences Research Council via a grant to RAW and JTO. QconCAT design, construction and expression were supported by BBSRC Grant BB/C007433 to RJB. The authors are also grateful to Ann Bamford for her excellent snail husbandry.
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