RESEARCH ARTICLE The Arabidopsis thaliana 2-D gel mitochondrial proteome: Refining the value of reference maps for assessing protein abundance, contaminants and post-translational modifications Nicolas L. Taylor 1,2 , Joshua L. Heazlewood 1,3 and A. Harvey Millar 1,2 1 ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley WA, Australia 2 Centre for Comparative Analysis of Biomolecular Networks, The University of Western Australia, Crawley WA, Australia 3 Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Received: September 30, 2010 Revised: November 30, 2010 Accepted: December 5, 2010 Mitochondria undertake the process of oxidative phosphorylation yielding ATP for plant cell maintenance and growth. The principles of isolation and fractionation of plant mitochondrial proteins have been improved over decades, and surveys of the mitochondrial proteome in a number of plants species have been performed. Over time, many quantitative analyses of changes in the plant mitochondrial proteome have been performed by 2-D gel analyses revealing the induction, degradation and modification of mitochondrial proteins in responses to mutation, stress and development. Here, we present a saturating MS analysis of 2-D gel separable protein spots from a typical purification of Arabidopsis mitochondria identifying 264 proteins, alongside an LC-MS/MS survey by non-gel methods identifying 220 proteins. This allowed us to characterise the major mitochondrial proteins that are not observed on 2-D gels, the common contaminants and the abundance of the protein machinery of key mito- chondrial biochemical pathways, and consider the impact of N-terminal pre-sequence clea- vage and phosphorylation as explanations of multiple protein spots and the co-ordinates of proteins on 2-D gels. Keywords: Arabidopsis / Mass spectra / Mitochondrial proteome / Multidimensional gel experiments / Plant proteomics / Phosphorylation 1 Introduction Mitochondria undertake the process of oxidative phosphor- ylation yielding ATP for plant cell maintenance and growth. However, they are also involved in a wide array of additional catabolic and biosynthetic pathways in plants including vitamin synthesis, amino acid metabolism, photorespiration and C1 metabolism. The principles of isolation and frac- tionation of plant mitochondrial proteins have been improved over decades. Importantly, the introduction and refinement of sucrose and Percoll density gradients allowed the extraction of high-purity mitochondrial samples from a wide variety of plant tissues [1–3]. Recent improvements in the process including the use of protoplast formation to improve yield, successive density gradients and free flow electrophoresis have further aided the purity of these orga- nelle isolations [4–8]. Two-dimensional gel separation based on IEF and SDS- PAGE has been the most commonly used method for the separation and display of the protein composition of isolated plant mitochondrial samples. The mitochondrial proteome Abbreviations: FDR, false detection rate; emPAI, exponentially modified protein abundance index; TCA, tricarboxylic acid Correspondence: Dr. A. Harvey Millar, ARC Centre of Excellence in Plant Energy Biology, 4th Floor MCS Building M316, Univer- sity of Western Australia, 35 Stirling Highway, Crawley 6009 WA, Australia E-mail: harvey.millar@uwa.edu.au Fax: 161-8-6488-4401 & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 1720 Proteomics 2011, 11, 1720–1733 DOI 10.1002/pmic.201000620