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Two dimensional nanoflow liquid chromatography - tandem mass spectrometry of proteins extracted from rice leaves and roots

Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.14/41933

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Title: Two dimensional nanoflow liquid chromatography - tandem mass spectrometry of proteins extracted from rice leaves and roots
Related: Thiellement, Hervé; Zivy, Michel; Damerval, Catherine and Méchin, Valérie. Plant proteomics : methods and protocols, p.249-266
DOI: 10.1385/1-59745-227-0:249
Related: Methods in molecular biology 355
Publisher: Totowa, NJ : Humana Press
Date: 2006
FoR/RFCD Code(s): 030108 Separation Science 030199 Analytical Chemistry not elsewhere classified 060109 Proteomics and Intermolecular Interactions (excl. Medical Proteomics)
Author/Creator: Breci, Linda
Author/Creator: Haynes, Paul, A
Description: In this chapter we present a detailed protocol for the large-scale identification of proteins present in rice leaf and root tissue samples using 2D liquid chromatography-tandem mass spectrometry of protein extracts. This is performed using biphasic (strong cation exchange/reversed phase) columns with integral electrospray emitters operating at nanoliter flow rates, a technique known by the acronym Mudpit (for multidimensional protein identification technique). The protocol involves harvesting of leaves and roots from rice plants, preparing protein extracts from the harvested tissues, preparing proteolytic digests of the extracted proteins, making a biphasic capillary column with an integral electrospray emitter, performing two-dimensional chromatographic separation of peptides with data-dependent tandem mass spectrometry, and the use of database searching of the acquired tandem mass spectra to identify peptides and proteins. This protocol is adaptable for use with a wide variety of plant materials and can be used to identify large numbers of proteins present in a specific tissue, organ, organelle, or other subcellular fraction. In addition to the detailed protocol, we also present the results of a representative experiment showing the identification of more than 1000 distinct proteins from rice leaf and root samples in two Mupdit experiments.
Subject Keyword: 030108 Separation Science
Subject Keyword: 030199 Analytical Chemistry not elsewhere classified
Subject Keyword: 060109 Proteomics and Intermolecular Interactions (excl. Medical Proteomics)
Subject Keyword: Nanoflow liquid chromatography-tandem mass spectrometry
Subject Keyword: two-dimensional nanoflow chromatography
Subject Keyword: multidimensional protein identification technique (Mudpit)
Subject Keyword: plant protein extraction
Subject Keyword: rice leaf
Subject Keyword: rice root
Resource Type: book chapter
Organisation: Macquarie University. Dept. of Chemistry and Biomolecular Sciences

Identifier: http://hdl.handle.net/1959.14/41933
Identifier: ISBN:9781588296351
Identifier: mq-rm-2007003082
Language: eng

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