Proteomics 2015, 15, 1765–1769 1765 DOI 10.1002/pmic.201400526 TECHNICAL BRIEF Abundant storage protein depletion from tuber proteins using ethanol precipitation method: Suitability to proteomics study Hye Min Lee 1∗ , Ravi Gupta 1∗ , Sun Hyung Kim 2 , Yiming Wang 3 , Randeep Rakwal 4,5,6,7 , Ganesh Kumar Agrawal 6,7 and Sun Tae Kim 1 1 Department of Plant Bioscience, Life and Industry Convergence Research Institute, Pusan National University, Miryang, South Korea 2 Department of Environmental Horticulture, University of Seoul, Seoul, South Korea 3 Department of Plant Microbe Interaction, Max Planck Institute for Plant Breeding Research, K¨ oln, Germany 4 Organization for Educational Initiatives, University of Tsukuba, Tsukuba, Ibaraki, Japan 5 Department of Anatomy I, Showa University School of Medicine, Shinagawa, Tokyo, Japan 6 Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal 7 GRADE Academy Private Limited, Birgunj, Nepal Received: November 7, 2014 Revised: January 21, 2015 Accepted: February 11, 2015 High-abundance proteins (HAPs) hamper in-depth proteome study necessitating development of a HAPs depletion method. Here, we report a novel ethanol precipitation method (EPM) for HAPs depletion from total tuber proteins. Ethanol showed a dose-dependent effect on depletion of sporamin from sweet potato and patatin from potato tubers, respectively. The 50% ethanol was an optimal concentration. 2DE analysis of EPM-prepared sweet potato proteins also revealed enrichment of storage proteins (SPs) in ethanol supernatant (ES) resulting in detection of new low-abundance proteins in ethanol pellet (EP), compared to total fraction. The ES fraction showed even higher trypsin inhibitor activity than total proteins, further showing the efficacy of EPM in enrichment of sporamin in ES fraction. Application of this method was demonstrated for comparative proteomics of two sweet potato cultivars (Hwang-geum and Ho-bac) and purification of SP (sporamin) in its native form, as examples. Comparative proteomics identified many cultivar specific protein spots and selected spots were confidently assigned for their protein identity using MALDI-TOF-TOF analysis. Overall, the EPM is simple, reproducible, and economical for depletion of SPs and is suitable for downstream proteomics study. This study opens a door for its potential application to other tuber crops or fruits rich in carbohydrates. Keywords: Ethanol precipitation method / Plant proteomics / Sporamin / Storage proteins / Sweet potato tuber / Trypsin inhibitor  Additional supporting information may be found in the online version of this article at the publisher’s web-site Correspondence: Professor Sun Tae Kim, Department of Plant Bioscience, Pusan National University, Miryang, 627–706, South Korea E-mail: stkim71@pusan.ac.kr Fax: +82-55-350-5509 Abbreviations: EPM, ethanol precipitation method; EP, ethanol pellet; ES, ethanol supernatant; HAP, high-abundance protein; SP, storage protein Tubers are plant storage organs. They accumulate high amount of storage proteins (SPs) to serve as carbon and ni- trogen sources during tuber germination. Patatin in potato, dioscorin in yams, and sporamin in sweet potatoes are some of the major SP complexes that hamper an in-depth study ∗ These authors contributed equally to this work. Colour Online: See the article online to view Figs. 1 and 2 in colour. C  2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com