Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome

Jinghui Xi, Xu Wang, Shanyu Li, Xin Zhou, Lin Yue, Jia Fan, Dongyun Hao

Research output: Contribution to journalArticlepeer-review

65 Scopus citations

Abstract

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 ca. 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.

Original languageEnglish (US)
Pages (from-to)2341-2348
Number of pages8
JournalPhytochemistry
Volume67
Issue number21
DOIs
StatePublished - Nov 2006

Keywords

  • Arabidopsis thaliana
  • Cruciferae
  • Low-abundant proteins
  • Polyethylene glycol
  • Protein preparation
  • Rubisco
  • Two-dimensional electrophoresis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Plant Science
  • Horticulture

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