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A proteomic approach employing a two dimensional electrophoresis (2-DE) technique with SYPRO Ruby, a fluorescent stain with improved sensitivity and quantitative accuracy, was performed to separate the total proteins from apple fruit at different stages of ripening and senescence. After imaging and statistical analyses were performed on 2340 spots, a total of 316 spots, or approximately 13.5% of the total protein population, was found to be significantly changed in this study. Of the 316 proteins, 219 spots were only present at a specific ripening stage, while 97 spots were significantly different (p<0.05) throughout fruit ripening and in response to ethylene treatment. From 316 candidate spots, 221 proteins were further identified by liquid chromatography and mass spectrometry analysis with protein sequence and express sequence tag (EST) data searching. Analysis and identification of proteins revealed that apple fruit ripening is associated with increase of abundance of many proteins with functions such as ethylene production, antioxidation and redox, carbohydrate metabolism, oxidative stress, energy, and defense response. Ethylene treatment increased a group of unique proteins that were not present during normal fruit ripening and have not been previously reported. It also reduced some proteins involved in primary metabolism, including those of the last few steps of the glycolytic pathway. This study demonstrated the complexity and dynamic changes of protein profiles of apple fruit during ripening and in response to exogenous ethylene treatment. Identifying and tracking protein changes may allow us to better understand the mechanism of ripening in climacteric fruit., Biological Significance: Postharvest physiology and biochemistry has been conducted on apple fruit for many years. Ethylene plays an important role in ripening and senescence in many climacteric fruit. However, little information is available at the proteome level to investigate fruit ripening and effect of ethylene treatment. The significance of this paper is that it is the first study employing 2-DE and fluorescent dye in the investigation of the apple fruit ripening and influence of ethylene treatment. It reveals some significant biological changes in association with these events and demonstrates significant changed proteins under these conditions. Therefore, our study links the biological events with proteomic information and provides detailed peptide information on all identified proteins. Through the function analysis, those significantly changed proteins are also analyzed. These findings from this paper provide not only proteome information on fruit ripening, but also pave the ground for further quantitative studies using SMR to investigate certain proteins and pathways under the hypothesis involved in fruit ripening. This article is part of a Special Issue entitled: Translational Plant Proteomics., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)