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Title: Plant iTRAQ-based proteomics

Abstract

We present a simple one-­pot extraction protocol, which rapidly isolates hydrophyllic metabolites, lipids, and proteins from the same pulverized plant sample. Also detailed is a global plant proteomics sample preparation method utilizing iTRAQ multiplexing reagents that enables deep proteome coverage due to the use of HPLC fractionation of the peptides prior to mass spectrometric analysis. We have successfully used this protocol on several different plant tissues (e.g., roots, stems, leaves) from different plants (e.g., sorghum, poplar, Arabidopsis, soybean), and have been able to successfully detect and quantify thousands of proteins. Multiplexing strategies such as iTRAQ and the bioinformatics strategy outlined here, ultimately provide insight into which proteins are significantly changed in abundance between two or more groups (e.g., control, perturbation). Our bioinformatics strategy yields z-­score values, which normalize the expression data into a format that can easily be cross-­compared with other expression data (i.e., metabolomics, transcriptomics) obtained from different analytical methods and instrumentation.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1406693
Report Number(s):
PNNL-SA-123355
Journal ID: ISBN 9781119077367; 49706; 49250; KP1601020
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Current Protocols in Plant Biology
Additional Journal Information:
Journal Volume: 2; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; quantitative proteomics; plant; iTRAQ; Sorghum; Environmental Molecular Sciences Laboratory

Citation Formats

Handakumbura, Pubudu, Hixson, Kim K., Purvine, Samuel O., Jansson, Georg C., and Pasa Tolic, Ljiljana. Plant iTRAQ-based proteomics. United States: N. p., 2017. Web. doi:10.1002/cppb.20052.
Handakumbura, Pubudu, Hixson, Kim K., Purvine, Samuel O., Jansson, Georg C., & Pasa Tolic, Ljiljana. Plant iTRAQ-based proteomics. United States. doi:10.1002/cppb.20052.
Handakumbura, Pubudu, Hixson, Kim K., Purvine, Samuel O., Jansson, Georg C., and Pasa Tolic, Ljiljana. Wed . "Plant iTRAQ-based proteomics". United States. doi:10.1002/cppb.20052.
@article{osti_1406693,
title = {Plant iTRAQ-based proteomics},
author = {Handakumbura, Pubudu and Hixson, Kim K. and Purvine, Samuel O. and Jansson, Georg C. and Pasa Tolic, Ljiljana},
abstractNote = {We present a simple one-­pot extraction protocol, which rapidly isolates hydrophyllic metabolites, lipids, and proteins from the same pulverized plant sample. Also detailed is a global plant proteomics sample preparation method utilizing iTRAQ multiplexing reagents that enables deep proteome coverage due to the use of HPLC fractionation of the peptides prior to mass spectrometric analysis. We have successfully used this protocol on several different plant tissues (e.g., roots, stems, leaves) from different plants (e.g., sorghum, poplar, Arabidopsis, soybean), and have been able to successfully detect and quantify thousands of proteins. Multiplexing strategies such as iTRAQ and the bioinformatics strategy outlined here, ultimately provide insight into which proteins are significantly changed in abundance between two or more groups (e.g., control, perturbation). Our bioinformatics strategy yields z-­score values, which normalize the expression data into a format that can easily be cross-­compared with other expression data (i.e., metabolomics, transcriptomics) obtained from different analytical methods and instrumentation.},
doi = {10.1002/cppb.20052},
journal = {Current Protocols in Plant Biology},
number = 2,
volume = 2,
place = {United States},
year = {2017},
month = {6}
}

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