Analyzing In Vivo Metabolite‐Protein Interactions by Large‐Scale Systematic Analyses

Abstract

Metabolites interact with proteins in vivo in various ways other than enzymatic reactions.Profiling of such interactions may help disclose unknown molecular mechanisms that regulate protein functions, and provide potential targets for disease treatment. Here we describe a procedure for systematic analyses of metabolite-protein interactions in vivo. This procedure couples protein affinity purification and mass spectrometry to identify metabolite-protein interactions. The primary effort can be completed within one day and scaled to process hundreds of samples in a batch. Originally developed in yeast, the same principle and protocol can be adapted to other organisms.

Optimized small‐molecule pull‐downs define MLBP1 as an acyl‐lipid‐binding protein

“…Nevertheless, the endogenous small molecular ligands of a large number of proteins with predicted ligand-binding domains have still not been identified (Anantharaman and Aravind, 2001;Schrick et al, 2004;Shinohara et al, 2012;Das and Rahman, 2014;Grotewold et al, 2017;Hohmann et al, 2017). While several methods allowing identification of endogenous protein ligands have been demonstrated in yeast, bacteria and animal systems (Saghatelian et al, 2004;Li and Snyder, 2012;Piazza et al, 2018), only a handful have been applied in planta. These include profiling metabolomes in unique genetic backgrounds and the application of affinity co-purification of a target protein in complex with its ligand (Casanal et al, 2013;Luzarowski et al, 2017).…”

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

“…Methods include affinity chromatography 6 , drug affinity responsive target-stability assay 7 , chemo-proteomics 8 , and thermal proteome profiling 9 . The second group consists of a single method that starts with a known protein in order to identify small-molecule ligands 10 11 .…”

“…Previously, we could demonstrate that, similarly to lipids, polar and semi-polar compounds also remain bound to protein complexes isolated from the cellular lysates 13 . Based on these findings, we decided to optimize the AP method published previously 10 11 for plant cells and hydrophilic compounds 14 . For this purpose, we used TAP vectors described by Van Leene et al 2010, successfully used in plant PPI studies 15 .…”

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

“…The first group comprises techniques that start with a known-metabolite bait in order to trap novel protein partners. Methods include affinity chromatography The second group consists of a single method that starts with a known protein in order to identify small-molecule ligands 10,11 .…”