title: Peptide separation methodologies for in-depth proteomics.
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.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0192, Japan.
authors.forename: Sajad Majeed
authors.initials: SM
authors.lastname: Zargar
authors.forename: Rie
authors.initials: R
authors.lastname: Kurata
authors.forename: Randeep
authors.initials: R
authors.lastname: Rakwal
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Fukao
mesh_terms: D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism*; D008954:Models, Biological; D010455:Peptides / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods
chemical_list: D029681:Arabidopsis Proteins; D010455:Peptides