Protein Complex Identification and quantitative complexome by CN-PAGE

Abstract

The majority of cellular processes are carried out by protein complexes. Various size fractionation methods have previously been combined with mass spectrometry to identify protein complexes. However, most of these approaches lack the quantitative information which is required to understand how changes of protein complex abundance and composition affect metabolic fluxes. In this paper we present a proof of concept approach to quantitatively study the complexome in the model plant Arabidopsis thaliana at the end of the day (ED) and the end of the night (EN). We show that size-fractionation of native protein complexes by Clear-Native-PAGE (CN-PAGE), coupled with mass spectrometry can be used to establish abundance profiles along the molecular weight gradient. Furthermore, by deconvoluting complex protein abundance profiles, we were able to drastically improve the clustering of protein profiles. To identify putative interaction partners, and ultimately protein complexes, our approach calculates the Euclidian distance between protein profile pairs. Acceptable threshold values are based on a cut-off that is optimized by a receiver-operator characteristic (ROC) curve analysis. Our approach shows low technical variation and can easily be adapted to study in the complexome in any biological system.

Global mapping of protein–metabolite interactions in Saccharomyces cerevisiae reveals that Ser-Leu dipeptide regulates phosphoglycerate kinase activity

“…Given that the majority of the proteins and metabolites had complex elution patterns characterised by more than one elution maximum, we split the data profiles into single peaks; this is referred to as deconvolution 26 . By doing so, we obtained 1320 and 125 peaks for unknown and annotated metabolites, respectively, and 5834 protein peaks.…”

Combination of network and molecule structure accurately predicts competitive inhibitory interactions

“…Analysis of the metabolite and protein elution profiles was performed as describe before Sokolowska et al [25] , Gorka M [27] , and included data filtering, normalization and deconvolution. Obtained data were integrated together using Pearson correlation assuming that what correlates, and hence co-eluates, together is potentially in the complex.…”

Metabolons, enzyme–enzyme assemblies that mediate substrate channeling, and their roles in plant metabolism

“…Additional technologies, such AP–MS combined with crosslinking, can preserve the crosslinked proteins during the washing steps in affinity purification ( Wu and Minteer, 2015 ; Leitner et al., 2016 ). In addition, size fractionation of native protein complexes by clear-native PAGE and blue-native PAGE can also be used to establish an information-rich protein interaction network by separating the protein complex on a molecular weight gradient, followed by MS ( Senkler et al., 2017 ; Gorka et al., 2019 ). Both methods have been successfully used to detect specific interactions between large protein complexes, such as the glycolytic complex of the mitochondrial outer membrane ( Graham et al., 2007 ).…”