Chapter title |
Peptide Separation Methodologies for In-depth Proteomics.
|
---|---|
Chapter number | 17 |
Book title |
Plant Cell Expansion
|
Published in |
Methods in molecular biology, October 2014
|
DOI | 10.1007/978-1-4939-1902-4_17 |
Pubmed ID | |
Book ISBNs |
978-1-4939-1901-7, 978-1-4939-1902-4
|
Authors |
Sajad Majeed Zargar, Rie Kurata, Randeep Rakwal, Yoichiro Fukao |
Editors |
José M. Estevez |
Abstract |
The integration of proteomics to other omics technologies and generation of proteome maps of a particular cell/tissue requires the identification and quantification of a maximum number of proteins. Traditional 2-D gel-based approach though provides a clear proteome map has its limitations, such as time consuming, requiring high skill, and most importantly, inability to identify low-abundance proteins. The most common drawback of 2-D gel electrophoresis is the masking of low amount proteins by the highly expressed (high abundance) proteins. Therefore, the elucidation of complete regulatory networks of a cell/tissue demands identification of low-abundance proteins. Low-abundance protein identification requires the use of usually gel-free mass spectrometry (MS)-based approaches. Using Arabidopsis thaliana as a model system, in this chapter, we describe all the steps followed for the extraction of microsomal proteins to MS analysis of separated peptides with a major focus on three different methods, namely, OFFGEL fractionation, 2D-LC, and long-column method for the identification of low-abundance proteins. Separation of such peptides will lead to in-depth proteomics-based investigations to answer biological questions. |
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