In Vivo Analysis of Proteomes and Interactomes Using Parallel Affinity Capture (iPAC) Coupled to Mass Spectrometry

Johanna S Rees,Irina M Armean,Helen Spriggs,Nick Lowe,Edward Ryder,John Roote,Kathryn S Lilley,Steven Russell,Daniel St Johnston,Glynnis Johnson,Emma Drummond

doi:10.1074/mcp.m110.002386

Abstract

Affinity purification coupled to mass spectrometry provides a reliable method for identifying proteins and their binding partners. In this study we have used Drosophila melanogaster proteins triple tagged with Flag, Strep II, and Yellow fluorescent protein in vivo within affinity pull-down experiments and isolated these proteins in their native complexes from embryos. We describe a pipeline for determining interactomes by Parallel Affinity Capture (iPAC) and show its use by identifying partners of several protein baits with a range of sizes and subcellular locations. This purification protocol employs the different tags in parallel and involves detailed comparison of resulting mass spectrometry data sets, ensuring the interaction lists achieved are of high confidence. We show that this approach identifies known interactors of bait proteins as well as novel interaction partners by comparing data achieved with published interaction data sets. The high confidence in vivo protein data sets presented here add new data to the currently incomplete D. melanogaster interactome. Additionally we report contaminant proteins that are persistent with affinity purifications irrespective of the tagged bait.

Highlights

From the ‡Cambridge Centre for Proteomics, §The Gurdon Institute, ¶Department of Genetics, ʈCambridge Systems Biology Centre, University of Cambridge, Cambridge, UK
The tandem affinity purification approach has been very successful in unicellular organisms such as yeast [9], the serial protein purifications employed by this method result in reduced final protein yields that can be a problem when dealing with limited material from metazoans
Our method of parallel affinity purification of individually tagged endogenous proteins from their native environment requires the incorporation of multiple affinity tags into the bait protein, and we assessed several alternative tags for use in Drosophila

Summary

Introduction

From the ‡Cambridge Centre for Proteomics, §The Gurdon Institute, ¶Department of Genetics, ʈCambridge Systems Biology Centre, University of Cambridge, Cambridge, UK. Homologous recombination is an efficient way to introduce affinity tags into endogenous proteins in yeast, but it is too laborious to use this approach on a large scale in multicellular organisms where the efficiency of homologous recombination is much lower To circumvent this difficulty, we generated endogenously tagged proteins by mobilizing a transposable element containing an exon encoding a series of affinity tags flanked by splice donor and acceptor sites [14]. Several protein trap screens have been performed using a transposon carrying a GFP reporter to generate lines useful for the study of protein expression patterns and subcellular localization (14 –16) We have adapted this approach by including affinity tags in the GFP exon so that the resulting lines can be used for interactome mapping

Methods

Results

Conclusion