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.
Highlights
Protein function and activity are highly regulated within living cells
Great care should be taken to select the appropriate methods that are used for protein complex extraction, fractionation and sample preparation for mass spectrometry, any or all of which could serve as the source of technical variation
With this in mind we established a workflow to allow processing of native protein extracts for the comparative analysis of the plant complexome at different diurnal time points. In this approach the isolated protein extracts are separated by native polyacrylamide gel electrophoresis (PAGE) fractionation, followed by a modified in gel digestion procedure and analysis by tandem mass spectrometry (Fig. 1)
Summary
Protein function and activity are highly regulated within living cells. To become functional, proteins commonly depend on their interactions with other molecules. AP-MS strategies often require modifications of the biological system, which alters the native structure of the protein by the introduction of a tag, and the quantity as a result of overexpression This method is labour intensive and limited to studying a small number of proteins in parallel[8,9]. In an attempt to overcome the challenges of the aforementioned methods, researchers combined classical methods of protein fractionation, such as polyacrylamide gel electrophoresis (PAGE) or column based chromatography with quantitative mass spectrometry. One such method, 2-D PAGE remains an accessible method for www.nature.com/scientificreports/. The importance of minimization of technical variation implementation has been recently highlighted in complexome studies[13]
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