Experimental design of the N. caninum secretome study. Purified N. caninum tachyzoites were stimulated with 1% ethanol, and the ESA proteins were separated from the discharged tachyzoites. For the Secreted Fraction Approach, the ESA was concentrated, dried and after separation with 1D SDS-PAGE, the proteins were digested. The tryptic peptides were analysed with the following: an LTQ-Orbitrap-XL, equipped with a peptide fragmentation system by collision induced dissociation (CID), and an LTQ-Orbitrap-Velos, using a decision tree-guided peptide fragmentation, equipped with electron transfer dissociation (ETD) with LTQ mass analysis, ETD with Orbitrap readout (ETD-FT), and higher-energy collisional dissociation (HCD-FT) with Orbitrap readout. For the Quantitative Approach, unstimulated tachyzoites (control) and the tachyzoites recovered after ESA collection (discharged), separately, had their total protein extract (TE) produced and digested with Lys-C and trypsin. The peptides were dimethyl labelled with light (control) and medium (discharged) labelling reagents, mixed in a 1:1 ratio, fractionated by strong-cation exchange (SCX) and analysed on an LTQ-Orbitrap-XL using a decision tree for the fragmentation methods CID and ETD. For both approaches, combined MS data were analysed using Mascot software and in silico analyses were performed (details in Methods).