title: Depletion of abundant plant RuBisCO protein using the protamine sulfate precipitation method.
abstract: Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant plant leaf protein, hampering deep analysis of the leaf proteome. Here, we describe a novel protamine sulfate precipitation (PSP) method for the depletion of RuBisCO. For this purpose, soybean leaf total proteins were extracted using Tris-Mg/NP-40 extraction buffer. Obtained clear supernatant was subjected to the PSP method, followed by 13% SDS-PAGE analysis of total, PS-supernatant and -precipitation derived protein samples. In a dose-dependent experiment, 0.1% w/v PS was found to be sufficient for precipitating RuBisCO large and small subunits (LSU and SSU). Western blot analysis confirmed no detection of RuBisCO LSU in the PS-supernatant proteins. Application of this method to Arabidopsis, rice, and maize leaf proteins revealed results similar to soybean. Furthermore, 2DE analyses of PS-treated soybean leaf displayed enriched protein profile for the protein sample derived from the PS-supernatant than total proteins. Some enriched 2D spots were subjected to MALDI-TOF-TOF analysis and were successfully assigned for their protein identity. Hence, the PSP method is: (i) simple, fast, economical, and reproducible for RuBisCO precipitation from the plant leaf sample; (ii) applicable to both dicot and monocot plants; and (iii) suitable for downstream proteomics analysis.
authors.affiliation: Department of Plant Bioscience, Pusan National University, Miryang, South Korea.
authors.forename: Yu Ji
authors.initials: YJ
authors.lastname: Kim
authors.forename: Hye Min
authors.initials: HM
authors.lastname: Lee
authors.forename: Yiming
authors.initials: Y
authors.lastname: Wang
authors.forename: Jingni
authors.initials: J
authors.lastname: Wu
authors.forename: Sang Gon
authors.initials: SG
authors.lastname: Kim
authors.forename: Kyu Young
authors.initials: KY
authors.lastname: Kang
authors.forename: Ki Hun
authors.initials: KH
authors.lastname: Park
authors.forename: Yong Chul
authors.initials: YC
authors.lastname: Kim
authors.forename: In Soo
authors.initials: IS
authors.lastname: Choi
authors.forename: Ganesh Kumar
authors.initials: GK
authors.lastname: Agrawal
authors.forename: Randeep
authors.initials: R
authors.lastname: Rakwal
authors.forename: Sun Tae
authors.initials: ST
authors.lastname: Kim
mesh_terms: D011232:Chemical Precipitation; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D018515:Plant Leaves / Q000737:chemistry / Q000201:enzymology; D011479:Protamines / Q000737:chemistry*; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000302:isolation & purification*; D013025:Glycine max / Q000737:chemistry / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011479:Protamines; D020543:Proteome; D012273:Ribulose-Bisphosphate Carboxylase
keywords: 2DE; Abundant protein; Depletion; Plant proteomics; Protamine sulfate; RuBisCO