title: Chloroplast isolation and affinity chromatography for enrichment of low-abundant proteins in complex proteomes.
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.
authors.affiliation: Department of Ecogenomics and Systems Biology, University of Vienna, Althanstr. 14, A-1090, Vienna, Austria.
authors.forename: Roman G
authors.initials: RG
authors.lastname: Bayer
authors.forename: Simon
authors.initials: S
authors.lastname: Stael
authors.forename: Markus
authors.initials: M
authors.lastname: Teige
mesh_terms: D002458:Cell Fractionation / Q000379:methods; D002736:Chloroplasts; D002853:Chromatography, Liquid / Q000379:methods; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D020543:Proteome; D040901:Proteomics / Q000379:methods
chemical_list: D011506:Proteins; D020543:Proteome