Mapping the Interactome of c-Myc/Max Heterodimer by Micro-affinity Purification Coupling Mass Spectrometry

We developed a micro-immobilized metal affinity chromatography (m-IMAC) protein purification method coupled with label-free quantification (LFQ) to enable mapping of protein-protein interactions (PPIs). We applied this method to reveal the interactome of c-Myc/Max, a master transcriptional regulator that controls the expression of thousands of genes and plays a key role in tumorigenesis. The m-IMAC columns were fabricated from 2-hydroxyethyl methacrylate phosphate (HEMAP) and the cross-linker pentaerythritol triacrylate via photoinitiated polymerization inside pipet tips. The phosphate groups of the column bed were activated by chelating Ni²⁺ ions, enabling immobilization of recombinant bait proteins through complexation with their His₆ tag, thereby allowing efficient enrichment of c-Myc/Max-interacting proteins from cell lysate. Using data-independent acquisition (DIA)-based LFQ, we identified 574 c-Myc/Max-interacting proteins, the majority of which have not been reported previously. GO analysis of the c-Myc/Max interactome revealed significant enrichment in RNA processing, chromatin organization and chromatin remodeling, protein-DNA assembly, and ribosome biogenesis. Our work provided the most comprehensive and up-to-date view of PPIs involved in c-Myc/Max-mediated transcriptional regulation. The straightforward workflow, ease of operation, and robust performance of the m-IMAC purification make it a versatile and efficient strategy for high-throughput interactome analysis.