Giardia duodenalis is a gastrointestinal parasite causing ~200 million symptomatic infections annually, disproportionately in lower socioeconomic tiers and children, leading to malnutrition, stunting in physical and cognitive growth. Chemotherapeutic interventions are limited to nitroheterocyclic antibiotics such as metronidazole. However, high doses are toxic and treatment failure related to drug-resistance occurs in up to 20% of cases, highlighting the urgency of novel and safer chemotherapeutics.
We previously identified a potent, drug-like series of kinase inhibitor compounds (IC50=114nM); its target is the BRAF kinase in humans, but there is no known BRAF ortholog in Giardia. To identify the kinase target(s) in Giardia, we selected a representative compound and immobilise through “Click” chemistry, to azide-agarose (ClickChemistryTools) and azide-magnetic beads (ClickChemistryTools) as a bait to “pulldown” the high affinity target(s). Pilot mass spectrometry (MS) studies of this approach showed different enrichment and non-specific binding profiles for either sets of beads, and manufacturers reported different compound support affinities. To optimise this pulldown approach, we have investigated washing buffers and workflows to reduce non-specific binding, and different protein elution/digest strategies. We use “blank” azide-beads as negative controls and “baited” azide-beads for the samples. For azide-agarose bead specifically, we implemented a secondary control by binding the baited beads in the presence of excess inhibitor; for azide-magnetic beads, we use excess inhibitor as an elution strategy, hence we implemented the use of a vehicle-elution as a secondary control.
Data-independent acquisition (DIA), library-free searches of these samples identified 2065 and 931 proteins from these respective pulldowns, out of the 4900 proteins in the annotated G. duodenalis proteome. Relative to “blank” agarose and magnetic beads, we pulled down eleven and one significant proteins respectively. When further cross-referenced to the secondary controls, we identify nine significantly enriched proteins from the baited agarose only. Two of these nine proteins were annotated as mitogen-activated and cell-cycle-dependent kinases. Overall, despite both beads being appropriate for Click chemistry approaches, we had to use different workflows for both reagents. This was largely due to non-specific binding between reagents, and sample-processing had to be adapted for magnetic or agarose beads. Our magnetic bead workflow showed less significantly enriched proteins; this highlights that the choice of beads selected in a pulldown may dramatically alter the protein targets identified, and may delay downstream drug-discovery experiments.