A bioanalytical method for the proteome wide display and analysis of protein complexes from whole plant cell lysates

Abstract

While protein interaction studies and protein network modeling come to the forefront, the isolation and identification of protein complexes in a cellular context remains a major challenge for plant science. To this end, a nondenaturing extraction procedure was optimized for plant whole cell matrices and the combined use of gel filtration and BN-PAGE for the separation of protein complexes was studied. Hyphenation to denaturing electrophoresis and mass spectrometric analysis allows for the simultaneous identification of multiple (previously unidentified) protein interactions in single samples. The reliability and efficacy of the technique was confirmed (i) by the identification of well-studied plant protein complexes, (ii) by the presence of nonplant interologs for several of the novel complexes (iii) by presenting physical evidence of previously hypothetical plant protein interactions and (iv) by the confirmation of found interactions using co-IP. Furthermore practical issues concerning the use of this 2-D BN/SDS-PAGE display method for the analysis of protein-protein interactions are discussed.

Fourteen years of plant proteomics reflected in Proteomics: Moving from model species and 2DE‐based approaches to orphan species and gel‐free platforms

“…Within proteomics, PTMs in plant systems including phosphorylation , thiol oxidation , nitrosylation , and glycosylation have been reported, and in the interactomics area, protein partners for thioredoxins , phytochrome , 14–3–3 proteins , mannose , and cupper have been described. Because of the reduced number of papers published, these areas are mostly skipped, taking the reader to those general reviews published in Proteomics .…”

Proteomics insights into plant signaling and development

“…Alternatively, protein fusions to green fluorescent protein (GFP) are being used, which allow the direct visualization of the protein expression and subcellular localization in planta 78. Combination of affinity purification and separation by size exclusion and/or blue native‐PAGE potentially enables the detection of distinct complexes formed by one protein 79. Recently, the first systematic proteomics efforts to unravel ‘interactomes’ of specific signaling processes have been accomplished.…”

Isolation and characterization of plant protein complexes by mass spectrometry

“…Obtaining a description of multi‐protein complexes corresponding to their actual in vivo state requires isolating them in close‐to‐physiological conditions before further MS analysis. This can be performed by diverse methods such as native fractionation on sucrose gradients 12, chromatography 13–15, Blue Native (BN)‐PAGE 16–18 or multidimensional electrophoresis 19. Nonetheless, affinity purification has been favored by the majority of studies aiming to characterize protein complexes.…”