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Isolation, Purity Assessment, and Proteomic Analysis of Endoplasmic Reticulum

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Plant Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2139))

Abstract

Subcellular proteomics include, in its experimental workflow, steps aimed at purifying organelles. The purity of the subcellular fraction should be assessed before mass spectrometry analysis, in order to confidently conclude the presence of associated specific proteoforms, deepening the knowledge of its biological function. In this chapter, a protocol for isolating endoplasmic reticulum (ER) and purity assessment is reported, and it precedes the proteomic analysis through a gel-free/label-free proteomic approach. Dysfunction of quality-control mechanisms of protein metabolism in ER leads to ER stress. Additionally, ER, which is a calcium-storage organelle, is responsible for signaling and homeostatic function, and calcium homeostasis is required for plant tolerance. With such predominant cell functions, effective protocols to fractionate highly purified ER are needed. Here, isolation methods and purity assessments of ER are described. In addition, a gel-free/label-free proteomic approach of ER is presented.

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Acknowledgments

This work was supported by JSPS KAKENHI Grant Number 15H04445.

Author information

Authors and Affiliations

  1. College of Agronomy and Biotechnology, China Agricultural University, Beijing, China

    Xin Wang

  2. Faculty of Environmental and Information Sciences, Fukui University of Technology, Fukui, Japan

    Setsuko Komatsu

Authors
  1. Xin Wang
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  2. Setsuko Komatsu
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Corresponding author

Correspondence to Setsuko Komatsu .

Editor information

Editors and Affiliations

  1. Agroforestry and Plant Biochemistry, Proteomics and Systems Biology, Department of Biochemistry and Molecular Biology, University of Cordoba, Cordoba, Spain

    Jesus V. Jorrin-Novo

  2. Department of Organisms and Systems Biology, Institute of Biotechnology of Asturias, University of Oviedo, Oviedo, Asturias, Spain

    Luis Valledor

  3. Agroforestry and Plant Biochemistry, Proteomics and Systems Biology, Department of Biochemistry and Molecular Biology, University of Cordoba UCO-CeiA3, Cordoba, Cordoba, Spain

    Mari Angeles Castillejo

  4. Agroforestry and Plant Biochemistry, Proteomics and Systems Biology, Department of Biochemistry and Molecular Biology, University of Cordoba, Cordoba, Spain

    Maria-Dolores Rey

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Wang, X., Komatsu, S. (2020). Isolation, Purity Assessment, and Proteomic Analysis of Endoplasmic Reticulum. In: Jorrin-Novo, J., Valledor, L., Castillejo, M., Rey, MD. (eds) Plant Proteomics. Methods in Molecular Biology, vol 2139. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0528-8_9

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  • DOI: https://doi.org/10.1007/978-1-0716-0528-8_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0527-1

  • Online ISBN: 978-1-0716-0528-8

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