Untargeted Defining Protein-Metabolites Interaction Based on Label-Free Kinetic Size Exclusion Chromatography-Mass Spectrometry.

Abstract

The specific interactions between protein and metabolites (PMIs) are closely related to many cellular processes and play a vital role in signal transduction and regulating material and energy metabolism. However, most of the available analytical strategies for PMIs involve chemical modification of metabolites or immobilization of protein, which has restricted current PMIs study mainly to lipid-protein and hydrophobic metabolites. In this work, a label-free online kinetic size exclusion chromatography-mass spectrometry (KSEC-MS) method combined with untargeted metabolomics was developed to define PMIs in a complex system. The metabolite mixture and target protein were injected into the SEC column sequentially without preincubation, and the separation results of KSEC were monitored by global metabolite profiling with mass spectrometry. The potential ligands in the metabolite mixture can be discovered if their migration patterns were affected by the target protein and the variation was positively correlated with the concentration of target protein. To verify this approach, carbonic anhydrase was first selected as a test protein, and acetazolamide as its known inhibitor was successfully defined. Furthermore, human serum albumin (HSA) as the common transport carrier of metabolites was selected as a target protein to demonstrate the usefulness of this approach. Multiple endogenous ligands of HSA were simultaneously defined from the extracted metabolites of human serum; most of them are polar metabolites rather than nonpolar lipids. This approach can provide a novel way for mapping and identifying unknown PMIs in a complex system, especially for polar metabolites-protein interactions.