Jun Song Gordon Braun Eric Bevis Kristen Doncaster Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, Kentville, Canada Received October 18, 2005 Revised January 16, 2006 Accepted January 21, 2006 Research Article A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Three phenol-free protein extraction procedures for 2-DE were compared and evaluated on apple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone pre- cipitation was found to be the most effective protocol. The results from SDS-PAGE and 2-DE analysis showed high quality proteins. More than 500 apple polypeptides were separated on a small scale 2-DE gel. The successful protocol was further tested on banana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues, respectively. To demonstrate the quality of the extracted proteins, several protein spots from apple and banana peels were cut from 2-DE gels, analyzed by MS and have been tentatively identified. The protocol described in this study is a simple procedure which could be routinely used in proteomic studies of many types of recalcitrant fruit tissues. Keywords: Apple / Banana / 2-DE / Protein extraction / SDS DOI 10.1002/elps.200500921 1 Introduction Proteomics is a global term for the study of protein populations in tissue, cell or subcellular compartments related to changes in protein structure and abundance in response to development, treatment and environmental stress [1]. In recent years, research on Arabidopsis tha- liana (L.) Heynh. and other plant tissues has demon- strated that proteomics is a very powerful tool in the study of molecular mechanisms in plants [2–5]. One of the pri- mary advantages of proteomics research based on 2-DE is the ability to simultaneously investigate hundreds or thousands of proteins with high accuracy. The ability to precisely determine molecular weight by MS and the development of genomic sequence databases for pep- tide mass matches make it possible to achieve high throughput of plant protein identification [1, 6]. 2-DE is especially useful for comparative studies between pairs of samples. Comparative proteomics has the poten- tial in postharvest research to help understand the physio- logical and biochemical changes related to fruit ripening and senescence as well as the effects of handling and storage treatments on stored fruit quality. Although many protocols have been reported, optimum conditions for the preparation of protein extracts from plant tissue that are suitable for 2-DE have primarily been developed for young vegetative tissues that have high protein content and low amounts of contaminants [7–10]. Fruit are considered recalcitrant plant tissues for proteomic analysis, from which it has been difficult to obtain high quality protein suitable for 2-DE analysis due to a low protein content and the presence of interfering substances such as pigments, carbohydrates, polyphenols, polysaccharides and starch. Establishing a reliable and effective protein extraction pro- cedure is an essential first step in conducting proteomic research. Apples (Malus6domestica Borkh.) are among the most popular fruits consumed in the world, yet to our knowledge there has been no successful protocol reported for the 2-DE separation of apple fruit proteins, to date. The objective of this study was to compare two previously published protein extraction protocols, widely applied on plant tissue, with a protocol we developed to extract and purify protein from apple, a recalcitrant fruit tissue. Protein yield, quality, the number of well resolved polypeptides after 2-DE, and their suitability for MS analysis were evaluated. Our protocol was tested further on banana fruit, known for the difficulty of protein extraction from both peel and flesh tissue. Correspondence: Dr. Jun Song, Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, 32 Main St., Kent- ville, N.S., B4N 1J5, Canada E-mail: songj@agr.gc.ca Fax: 11-902-679-2311 Abbreviations: SB 3-10, N-decyl-N, N-dimethyl-3-ammonio-1-pro- pane sulfonate; TCEP , tris(2-carboxyethyl) phosphine hydrochloride 3144 Electrophoresis 2006, 27, 3144–3151  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com