Fig. 2

Workflow for protein identification and characterization. Identification and characterization of Eimeria proteins are carried out by several biochemical, genetic and in silico approaches. Exogenous stimuli can propel parasites to secrete proteins in vitro and subjection of parasite stages to sonication/organellar fractionation can produce parasite lysates. The crude protein components of parasite lysate can be resolved by chromatography (LC/GC) coupled with gel-based techniques (e.g. SDS-PAGE, 2D-PAGE, 2D-DIGE) and then subjected to MS or MS/MS. Commonly used ionization methods in conjunction with MS include MALDI, SELDI and ESI followed by curation of peptide sequences in the database. Besides, parasite lysate can be subjected to quantitative proteomics techniques (e.g. iTRAQ, ICAT, TMT, SILAC) to identify the relative quantity of each characterised protein curated from web-based library screening. Alternatively, specific protein coding genes could be identified, cloned and expressed in bacterial vectors. The recombinant protein is then used to raise antibody in animals with which protein size (from western blotting) and sub-cellular location (by immunolocalisation/immunofluorescence) of protein in parasite stages are determined. Overall, quantitative proteomics techniques give precise, differential expression of proteins and can predict the underlying functional mechanism that may resolve various overlapping functions of several eimerian proteins. It is however notable that very few studies have used quantitative proteomics methods to characterise Eimeria proteins. SDS-PAGE: sodium dodecyl sulphate polyacrylamide gel electrophoresis; 2-DE: two-dimensional gel electrophoresis; 2D-DIGE: two-dimensional differential gel electrophoresis; LC: liquid chromatography; GC: gas chromatography; MS/MS: tandem mass spectrometry; SELDI: surface-enhanced laser desorption/ionization; ESI: electrospray ionization; MALDI-TOF: matrix-assisted laser desorption ionisation time of flight; iTRAQ: isobaric tags for relative and absolute quantification; SILAC: stable isotope labelling by amino acids in cell culture; TMT: tandem mass tag; ICAT: isotope-coded affinity tags