Figure 1

Experimental design and TMT labelling workflow for the experiment.

(A) Summary of the experiment conditions of the control, host soluble factor and co-incubation treatments. (B) Explanation of the TMT-labelling strategy utilised in the experiments. Peptides from the triplicates of the 3 conditions and a pooled control were labeled with one each of the TMT 10plex reagents. These reagents are observed as a monoisotopic complex in the first round of MS analysis on a high resolution mass spectrometer. During MS/MS and HCD based fragmentation, the TMT labels are fragmented to produce 10 reporter ions with distinguishable masses in the low m/z range, which allow relative protein quantitation (C) Overarching experimental design and workflow. Biological triplicates of BRIS/95/HEPU/2041 were grown to confluence in parasite culture in TYI-S-33 medium, before replicates were split into a replicate of each control for cell culture conditions (Con), co-incubation with IEC monolayers (CI IEC) and incubation in host soluble factors generated by IECs (HSF). Proteins were extracted from the 9 replicates and a pooled control was generated from equal aliquots of protein from the control triplicates. After proteolytic digestion, samples were labelled in a 10 plex TMT reaction and then pooled. The combined sample was fractionated by SCX chromatography and desalted using a C18 ZipTip prior to LC-MS/MS on a Q-Exactive Orbitrap.