Depletion of abundant plant RuBisCO protein using the protamine sulfate precipitation method

Authors


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Correspondence: Dr. 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

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.

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