We describe an affinity
purification–mass spectrometry (AP–MS)
method for probing the interactome of a special targeting protein.
The AP was implemented with monolithic micro immobilized metal ion
affinity chromatography columns (m-IMAC) which were prepared by photoinitiated
polymerization in the tip of a pipet (spin-tip columns). The recombinant
His6-tagged protein (bait protein) was reversibly immobilized
on the affinity column through the chelating group nitrilotriacetic
acid (NTA)–Ni2+. The bait protein and its interacting
partners can be easily eluted from the affinity matrix. The pulled-down
cellular proteins were then analyzed with label-free quantitative
proteomics. We used this method for probing the interactome concerning
the GOLD (Golgi dynamics) domain of the autophagy-associated adaptor
protein FYCO1. Totally, 96 proteins including seven literature-reported
FYCO1-associating proteins were identified. Among them CCZ1 and MON1A
were further biochemically validated, and the direct interaction between
the FYCO1 GOLD domain with CCZ1 was confirmed by co-immunoprecipitation
experiments.
of main observation and conclusion We developed a method for comprehensively profiling drug-protein interactions using micro-column affinity purification (AP) combined with label-free quantitative (LFQ) proteomics as well as the statistical and bioinformatics analysis. FK506 was used as the experimental model for proof of concept. The true interacting proteins were distinguished from the background proteins by their fold changes of FLQ intensities combined with p-values. Totally 116 FK506 interacting proteins including 5 known target proteins were identified. The method was validated by using the LFQ intensity of the endogenous known drug targets together with statistical analysis. We demonstrated that the micro-column-based affinity purification in combination with LFQ proteomics provides a highly reproducible and robust approach for profiling drug-protein interactions.
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