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Analysis of Rice Proteins Using SDS-PAGE Shotgun Proteomics

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Book cover Plant Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1072))

Abstract

In this chapter we describe the workflow used in our laboratory to analyze rice leaf samples using label-free shotgun proteomics based on SDS-PAGE fractionation of proteins. Rice proteomics has benefitted substantially from successful execution of shotgun proteomics techniques. We describe steps on how to proceed starting from rice protein extraction, SDS-PAGE, in-gel protein digestion with trypsin, nanoLC-MS/MS, and database searching using the GPM. Data from these experiments can be used for spectral counting, where simultaneous quantitation of several thousand proteins can be obtained.

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Abbreviations

2-DE:

Two-dimensional electrophoresis

ACN:

Acetonitrile

BCA:

Bicinchoninic acid

BSA:

Bovine serum albumin

DTT:

Dithiothreitol

FDR:

False discovery rate

GO:

Gene ontology

GPM:

Global proteome machine

IAA:

Iodoacetamide

MS:

Mass spectrometry

MS/MS:

Tandem mass spectrometry

MudPIT:

Multidimensional protein identification technology

NSAF:

Normalized spectral abundance factor

RP:

Reversed phase

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TCA:

Trichloroacetic acid

WEGO:

Web gene ontology annotation plot

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Acknowledgements

KAN, ISG, and SM acknowledge support in the form of iMQRES awards. SJE acknowledges support in the form of an APA scholarship. PAH acknowledges funding support from the Australian Research Council and wishes to thank Gayani Gammulla for providing images.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW, Australia

    Karlie A. Neilson, Sridevi Muralidharan & Mehdi Mirzaei

  2. Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, Australia

    Iniga S. George & Samantha J. Emery

  3. Department of Chemistry and Biomolecular Sciences, Macquarie University, Macquarie Park, Australia

    Paul A. Haynes

Authors
  1. Karlie A. Neilson
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  2. Iniga S. George
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  3. Samantha J. Emery
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  4. Sridevi Muralidharan
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  5. Mehdi Mirzaei
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  6. Paul A. Haynes
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Editor information

Editors and Affiliations

  1. Dept of Biochem & Molecular Bio, University of Cordoba, Cordoba, Spain

    Jesus V. Jorrin-Novo

  2. University of Tsukuba National Institute of Crop Science, Tsukuba, Japan

    Setsuko Komatsu

  3. Molecular Systems Biology, University of Vienna, Vienna, Austria

    Wolfram Weckwerth

  4. Dept. Molecular Systems Biology, University of Vienna, Vienna, Austria

    Stefanie Wienkoop

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Neilson, K.A., George, I.S., Emery, S.J., Muralidharan, S., Mirzaei, M., Haynes, P.A. (2014). Analysis of Rice Proteins Using SDS-PAGE Shotgun Proteomics. In: Jorrin-Novo, J., Komatsu, S., Weckwerth, W., Wienkoop, S. (eds) Plant Proteomics. Methods in Molecular Biology, vol 1072. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-631-3_21

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  • DOI: https://doi.org/10.1007/978-1-62703-631-3_21

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-630-6

  • Online ISBN: 978-1-62703-631-3

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