Peptide Separation Methodologies for In-depth Proteomics
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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.
Key wordsPeptide fractionation Low-abundance proteins C-18 resin Peptide desalting MS
This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (No. 23119512 to Y.F.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; a Grant-in-Aid for Scientific Research from Nara Institute of Science and Technology supported by The Ministry of Education, Culture, Sports, Science and Technology, Japan. This research was supported by Japan Advanced Plant Science Network. S.M.Z. acknowledges the DBT, New Delhi, India for award of CREST, Overseas postdoc fellowship. R.R. acknowledges the great support of Professors Yoshihiro Shiraiwa (Provost, Faculty of Life and Environmental Sciences, University of Tsukuba) and Koji Nomura (Organization for Educational Initiatives, University of Tsukuba) in promoting interdisciplinary research and unselfish encouragement.
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