Polyethylene Glycol (PEG) Fractionation Of Proteome And Its Application In Proteomic Analysis Of Cold Stress Responses In Arabidopsis (Arabidopsis Thaliana)

Poor detection of low-abundant proteins is a common problem in two-dimensional electrophoresis (2-DE) for separation of proteins in a proteome analysis. This is attributed partially, at least, to the existence of high-abundant proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants. They engage a large proportion of the whole-cell proteins and thus prevent low-abundant proteins from being up-taken by immobilized pH gradient (IPG) strip, consequently making the latter poorly detectable by 2-DE. In this work, we report a straightforward protocol for preparation of whole-cell proteins through differential polyethylene glycol (PEG) precipitation aiming at elimination of Rubisco from plant protein samples. In comparison with 2-DE analysis of protein samples prepared using a conventional TCA/acetone method, a relatively high reproducibility of proteins was achieved using a PEG fractionation protocol in terms of protein yield and protein species. As expected, the large subunit of Rubisco was precipitated predominantly in the 16% PEG fraction. This allowed proteins of the Rubisco-containing fraction to be analyzed separately from those of other PEG fractions. After taking into account the overlapping protein spots among 2-DE gels of all fractions through image and statistical analyses, we detected with this protocol a total 5077 protein spots, among which about 80% are proteins undetectable with the TCA/acetone method, while the rest of proteins exhibited a significant increase in their abundance. This protocol was developed using Arabidopsis as a source of protein and thus may also be applicable to protein preparations of other plants. A proteomic analysis was carried out aiming at understanding of the molecular adaptation mechanisms of cold stress in Arabidiopsis (Arabidopsis thanliana, ecotype Col-0). The seedlings were exposed to 4℃for 8 and 24 h respectively, together with the controls which were grown at normal temperature for 8 and 24 h. In PEG fractionation, the whole-cell proteins were extracted from both control and stressed seedlings. Sixty-seven protein spots were found to exhibit different dynamic patterns on 2-D gels in responding to the low temperature stress and fifty-seven of them were identified using MALDI-TOF MS. These cold responsive proteins, besides eight proteins of unknown function, are involved in regulation of metabolism, energy, cell defense, biogenesis of cellular component, etc. by MIPS function category, TAIR database and related references. Proteins (43.3%) were predicted to be located in the chloroplasts, implying that chloroplast proteins are likely involved in the cold stress adaptation in Arabidopsis. The physiological implications, which were derived from the experimental data, are discussed, suggesting that there is a complex metabolic network in plants responsive to cold stress.