Affinity columns as a key to unravel the potential of biochemical pathways

The vast majority of natural metabolic pathways remain uncharacterized, limiting the ability to harness biological systems for industrial biotechnology and environmental bioremediation. Classical functional genomics and activity-guided purification protocols frequently fail to map multi-step metabolic cascades due to transient protein-substrate interactions, rapid metabolic flux, and the inherent complexity of complex cellular proteomes. Here, I present the comprehensive protocol for Sequential Affinity-Based Pathway Mapping (SAMP) an iterative, multi-phase chromatographic framework designed to systematically trace metabolic cascades across different chemical states of matter (solid, liquid, and gas). By utilizing the native or immobilized state of a metabolic substrate as an affinity bait for cellular proteomes, SAMP enables the layer-by-layer isolation, elution, and structural characterization of sequential enzymes within an uncharacterized cascade. I highlight the utility of this method using bacterial plastic degradation as an archetypal model for solid-phase systems, detail the physical encapsulation frameworks required for unstable or liquid intermediates, and propose a pressurized micro-perfusion strategy to handle volatile gaseous substrates.