Chloroplast isolation and affinity chromatography for enrichment of low-abundant proteins in complex proteomes

Roman G Bayer, Simon Stael, Markus Teige

Publications: Contribution to bookChapterPeer Reviewed

Abstract

Detailed knowledge of the proteome is crucial to advance the biological sciences. Low-abundant proteins are of particular interest to many biologists as they include, for example those proteins involved in signal transduction. Recent technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS). However, the dynamic range in protein abundance still forms a fundamental problem that limits the detection of low-abundant proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of other proteins at an abundance several-fold higher in order of magnitude. Therefore, specific enrichment strategies are required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundant proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles.

Original languageEnglish
Title of host publicationProteomic Profiling
Subtitle of host publicationMethods and Protocols
EditorsAnton Posch
Place of PublicationNew York, NY
PublisherHumana Press
Pages211-223
Number of pages13
Volume1295
ISBN (Electronic)978-1-4939-2550-6
ISBN (Print)978-1-4939-2549-0
DOIs
Publication statusPublished - 2015

Publication series

SeriesMethods in Molecular Biology
Volume1295
ISSN1064-3745

Austrian Fields of Science 2012

  • 106031 Plant physiology

Keywords

  • Cell Fractionation
  • Chloroplasts
  • Chromatography, Liquid
  • Proteins
  • Proteome
  • Proteomics
  • Journal Article
  • Research Support, Non-U.S. Gov't
  • Chloroplast isolation
  • Affinity chromatography
  • Calcium-binding protein
  • ATP-binding protein
  • Mass spectrometry
  • Organellar proteome

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