PROMIS: Global Analysis of PROtein‐Metabolite Interactions. Issue 4 (21st November 2019)

• Record Type: Journal Article
• Authors: Sokolowska, Ewelina M.; Schlossarek, Dennis; Luzarowski, Marcin; Skirycz, Aleksandra
• Editors: Birchler, James; Ecker, Joseph; Martin, Cathie R.; Stitt, Mark; Zhou, Jian-Min
• Other Names: Stacey Gary editorInChief.
• Is Part Of: Current protocols in plant biology. Volume 4:Issue 4(2019)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2019-11-21
• Subjects: complexes -- metabolomics -- protein–metabolite interactions -- proteomics -- size‐exclusion chromatography; Botany -- Laboratory manuals; Plants; Botany; Botany; Laboratory Manual; Periodical; Laboratory manuals; 580.28
• DOI: 10.1002/cppb.20101
• Languages: English
• ISSNs: 2379-8068

Abstract

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