title: Abundant storage protein depletion from tuber proteins using ethanol precipitation method: Suitability to proteomics study.
abstract: 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.
authors.affiliation: Department of Plant Bioscience, Life and Industry Convergence Research Institute, Pusan National University, Miryang, South Korea.
authors.forename: Hye Min
authors.initials: HM
authors.lastname: Lee
authors.affiliation: Department of Plant Bioscience, Life and Industry Convergence Research Institute, Pusan National University, Miryang, South Korea.
authors.forename: Ravi
authors.initials: R
authors.lastname: Gupta
authors.affiliation: Department of Environmental Horticulture, University of Seoul, Seoul, South Korea.
authors.forename: Sun Hyung
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Department of Plant Microbe Interaction, Max Planck Institute for Plant Breeding Research, Köln, Germany.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Organization for Educational Initiatives, University of Tsukuba, Tsukuba, Ibaraki, Japan.|Department of Anatomy I, Showa University School of Medicine, Shinagawa, Tokyo, Japan.|Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal.|GRADE Academy Private Limited, Birgunj, Nepal.
authors.forename: Randeep
authors.initials: R
authors.lastname: Rakwal
authors.affiliation: Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal.|GRADE Academy Private Limited, Birgunj, Nepal.
authors.forename: Ganesh Kumar
authors.initials: GK
authors.lastname: Agrawal
authors.affiliation: Department of Plant Bioscience, Life and Industry Convergence Research Institute, Pusan National University, Miryang, South Korea.
authors.forename: Sun Tae
authors.initials: ST
authors.lastname: Kim
mesh_terms: D011232:Chemical Precipitation; D015180:Electrophoresis, Gel, Two-Dimensional; D000431:Ethanol / Q000737:chemistry*; D035281:Plant Tubers / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D055314:Seed Storage Proteins / Q000378:metabolism*; D011198:Solanum tuberosum / Q000378:metabolism*
chemical_list: D020543:Proteome; D055314:Seed Storage Proteins; D000431:Ethanol
keywords: Ethanol precipitation method; Plant proteomics; Sporamin; Storage proteins; Sweet potato tuber; Trypsin inhibitor