Time to dig deep into the plant proteome: a hunt for low-abundance proteins.

Abstract

Two-dimensional gel electrophoresis (2-DGE) has come a long way since its introduction around 40 years by the pioneering work of these three researchers (Klose, 1975; O'Farrell, 1975; Scheele, 1975). 2-DGE was one of the major breakthroughs in proteomics, enabling researchers to detect, analyze and identify the whole set of proteins of a cell or tissue, simultaneously. With the advancement in technology, some modifications to this technique like development of immobilized pH gradient (IPG) strips were introduced, which undoubtedly, made this technique more simple, rapid and autonomous (Bjellqvist et al., 1982). After its introduction to the present, 2-DGE has been the method of choice for analyzing the complex proteomes of plants. 2-DGE has been used extensively to investigate the effects of biotic and abiotic stress, role of hormones, and developmental changes of plants, among others (Agrawal and Rakwal, 2008). However, it was slowly realized that identification of the plant proteins led to the repeated detection of high-abundance proteins (HAPs) including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and other housekeeping proteins, which are present at 106-105 order of magnitude (Gygi et al., 2000; Patterson and Aebersold, 2003; Gorg et al., 2004). Signaling and other regulatory proteins are generally present 100 molecules per cells. Subsequently, these proteins are difficult to identify by either gel-based or gel-free proteomic approaches, even with access to the latest mass spectrometers. RuBisCO comprises of a large percentage in the total proteins and thus hinders the absorption of low-abundance proteins (LAPs) on the IPG strips, which subsequently results in the poor detection and identification of LAPs on 2D gels and by mass spectrometry (MS), respectively. Therefore, the time has come for all plant proteomers to realize the need to hunt for the LAPs, moving one step ahead from the present. As RuBisCO is the major HAP in plant leaves, here we recommend the incorporation of a RuBisCO depletion/removal method in every plant protein extraction step to look deeper into the plant proteome. RuBisCO depletion will definitely improve the proteome coverage and will lead to the detection of novel unidentified LAPs.