Protein Complex Identification and Quantitative Complexome by CN-PAGE

Overview

Journal Sci Rep

Specialty Science

Date 2019 Aug 10

PMID 31395906

Citations 16

Authors

Michal Gorka

Corne Swart

Beata Siemiatkowska

Silvia Martinez-Jaime

Aleksandra Skirycz

Sebastian Streb

Alexander Graf

Affiliations

Soon will be listed here.

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.

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