Chapter title |
Chloroplast Isolation and Affinity Chromatography for Enrichment of Low-Abundant Proteins in Complex Proteomes
|
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Chapter number | 16 |
Book title |
Proteomic Profiling
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2550-6_16 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2549-0, 978-1-4939-2550-6
|
Authors |
Roman G. Bayer, Simon Stael, Markus Teige |
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. |
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