PROMIS: Global Analysis of PROtein-Metabolite Interactions

Abstract

Small molecules are not only intermediates of metabolism, but also play important roles in signaling and in controlling cellular metabolism, growth, and development. Although a few systematic studies have been conducted, the true extent of protein–small molecule interactions in biological systems remains unknown. PROtein–metabolite interactions using size separation (PROMIS) is a method for studying protein–small molecule interactions in a non-targeted, proteome- and metabolome-wide manner. This approach uses size-exclusion chromatography followed by proteomics and metabolomics liquid chromatography–mass spectrometry analysis of the collected fractions. Assuming that small molecules bound to proteins would co-fractionate together, we found numerous small molecules co-eluting with proteins, strongly suggesting the formation of stable complexes. Using PROMIS, we identified known small molecule–protein complexes, such as between enzymes and cofactors, and also found novel interactions. © 2019 The Authors.

Basic Protocol 1: Preparation of native cell lysate from plant material

Support Protocol: Bradford assay to determine protein concentration

Basic Protocol 2: Separation of molecular complexes using size-exclusion chromatography

Basic Protocol 3: Simultaneous extraction of proteins and metabolites using single-step extraction protocol

Basic Protocol 4: Metabolomics analysis

Basic Protocol 5: Proteomics analysis