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.

Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Enables Time-Resolved Analysis of Protein Synthesis in Native Plant Tissue

“…Moreover, traditional shotgun proteomics strategies provide reduced coverage in samples where a few highly abundant proteins predominate. For example, Rubisco comprises 30% to 60% of the leaf proteome and obstructs detection of less abundant proteins in leaf samples (Kim et al, 2013). To counter this problem specifically, Kim et al (2013) developed a protamine sulfate precipitation method to selectively deplete Rubisco and thus enrich for less abundant proteins.…”

“…For example, Rubisco comprises 30% to 60% of the leaf proteome and obstructs detection of less abundant proteins in leaf samples (Kim et al, 2013). To counter this problem specifically, Kim et al (2013) developed a protamine sulfate precipitation method to selectively deplete Rubisco and thus enrich for less abundant proteins. Pulsed stable isotope labeling by amino acids in cell culture was introduced to monitor de novo protein synthesis, but poor label incorporation has prevented it from being widely adopted in plant systems.…”

Proteomic profile of Piper tuberculatum (Piperaceae)

“…RuBisCO, identified in the leaves, is an enzyme involved in the Calvin cycle which catalyzes the first major atmospheric carbon fixation steps. This multimeric enzyme is also the most abundant protein in the leaves and the most abundant protein on earth (Kim et al, 2013). Given its importance in the biosphere, there are currently efforts to genetically engineer crop plants with more efficient RuBisCO.…”

High Abundance Proteins: Proteomer’s Thorns in the Flesh?

“…These depletion based methods utilize different chemicals [12,13], solvents [14] or antibodies against the target proteins [15] to efficiently remove the high-abundance proteins from the total protein samples. Previous reports have shown that depletion of albumin from plasma [15], RuBisCO from leaf [5,12,13] and storage proteins from seed [16,17] and tuber [14] samples lead to the significant increase in the proteome coverage and identification of low-abundance proteins. However, although the precipitation based methods are rapid and costeffective, there is always a risk of loss of some non-target proteins that might precipitate along with the depleted target proteins.…”