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An Integrated Approach for the Efficient Extraction and Solubilization of Rice Microsomal Membrane Proteins for High-Throughput Proteomics

The preparation of microsomal membrane proteins (MPs) is critically important to microsomal proteomics. To date most research studies have utilized an ultracentrifugation-based approach for the isolation and solubilization of plant MPs. However, these approaches are labor-intensive, time-consuming,...

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Autores principales: Nguyen, Truong Van, Gupta, Ravi, Annas, Dicky, Yoon, Jinmi, Kim, Yu-Jin, Lee, Gi Hyun, Jang, Jeong Woo, Park, Kang Hyun, Rakwal, Randeep, Jung, Ki-Hong, Min, Cheol Woo, Kim, Sun Tae
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8460067/
https://www.ncbi.nlm.nih.gov/pubmed/34567038
http://dx.doi.org/10.3389/fpls.2021.723369
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author Nguyen, Truong Van
Gupta, Ravi
Annas, Dicky
Yoon, Jinmi
Kim, Yu-Jin
Lee, Gi Hyun
Jang, Jeong Woo
Park, Kang Hyun
Rakwal, Randeep
Jung, Ki-Hong
Min, Cheol Woo
Kim, Sun Tae
author_facet Nguyen, Truong Van
Gupta, Ravi
Annas, Dicky
Yoon, Jinmi
Kim, Yu-Jin
Lee, Gi Hyun
Jang, Jeong Woo
Park, Kang Hyun
Rakwal, Randeep
Jung, Ki-Hong
Min, Cheol Woo
Kim, Sun Tae
author_sort Nguyen, Truong Van
collection PubMed
description The preparation of microsomal membrane proteins (MPs) is critically important to microsomal proteomics. To date most research studies have utilized an ultracentrifugation-based approach for the isolation and solubilization of plant MPs. However, these approaches are labor-intensive, time-consuming, and unaffordable in certain cases. Furthermore, the use of sodium dodecyl sulfate (SDS) and its removal prior to a mass spectrometry (MS) analysis through multiple washing steps result in the loss of proteins. To address these limitations, this study introduced a simple micro-centrifugation-based MP extraction (MME) method from rice leaves, with the efficacy of this approach being compared with a commercially available plasma membrane extraction kit (PME). Moreover, this study assessed the subsequent solubilization of isolated MPs in an MS-compatible surfactant, namely, 4-hexylphenylazosulfonate (Azo) and SDS using a label-free proteomic approach. The results validated the effectiveness of the MME method, specifically in the enrichment of plasma membrane proteins as compared with the PME method. Furthermore, the findings showed that Azo demonstrated several advantages over SDS in solubilizing the MPs, which was reflected through a label-free quantitative proteome analysis. Altogether, this study provided a relatively simple and rapid workflow for the efficient extraction of MPs with an Azo-integrated MME approach for bottom-up proteomics.
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spelling pubmed-84600672021-09-24 An Integrated Approach for the Efficient Extraction and Solubilization of Rice Microsomal Membrane Proteins for High-Throughput Proteomics Nguyen, Truong Van Gupta, Ravi Annas, Dicky Yoon, Jinmi Kim, Yu-Jin Lee, Gi Hyun Jang, Jeong Woo Park, Kang Hyun Rakwal, Randeep Jung, Ki-Hong Min, Cheol Woo Kim, Sun Tae Front Plant Sci Plant Science The preparation of microsomal membrane proteins (MPs) is critically important to microsomal proteomics. To date most research studies have utilized an ultracentrifugation-based approach for the isolation and solubilization of plant MPs. However, these approaches are labor-intensive, time-consuming, and unaffordable in certain cases. Furthermore, the use of sodium dodecyl sulfate (SDS) and its removal prior to a mass spectrometry (MS) analysis through multiple washing steps result in the loss of proteins. To address these limitations, this study introduced a simple micro-centrifugation-based MP extraction (MME) method from rice leaves, with the efficacy of this approach being compared with a commercially available plasma membrane extraction kit (PME). Moreover, this study assessed the subsequent solubilization of isolated MPs in an MS-compatible surfactant, namely, 4-hexylphenylazosulfonate (Azo) and SDS using a label-free proteomic approach. The results validated the effectiveness of the MME method, specifically in the enrichment of plasma membrane proteins as compared with the PME method. Furthermore, the findings showed that Azo demonstrated several advantages over SDS in solubilizing the MPs, which was reflected through a label-free quantitative proteome analysis. Altogether, this study provided a relatively simple and rapid workflow for the efficient extraction of MPs with an Azo-integrated MME approach for bottom-up proteomics. Frontiers Media S.A. 2021-09-09 /pmc/articles/PMC8460067/ /pubmed/34567038 http://dx.doi.org/10.3389/fpls.2021.723369 Text en Copyright © 2021 Nguyen, Gupta, Annas, Yoon, Kim, Lee, Jang, Park, Rakwal, Jung, Min and Kim. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Nguyen, Truong Van
Gupta, Ravi
Annas, Dicky
Yoon, Jinmi
Kim, Yu-Jin
Lee, Gi Hyun
Jang, Jeong Woo
Park, Kang Hyun
Rakwal, Randeep
Jung, Ki-Hong
Min, Cheol Woo
Kim, Sun Tae
An Integrated Approach for the Efficient Extraction and Solubilization of Rice Microsomal Membrane Proteins for High-Throughput Proteomics
title An Integrated Approach for the Efficient Extraction and Solubilization of Rice Microsomal Membrane Proteins for High-Throughput Proteomics
title_full An Integrated Approach for the Efficient Extraction and Solubilization of Rice Microsomal Membrane Proteins for High-Throughput Proteomics
title_fullStr An Integrated Approach for the Efficient Extraction and Solubilization of Rice Microsomal Membrane Proteins for High-Throughput Proteomics
title_full_unstemmed An Integrated Approach for the Efficient Extraction and Solubilization of Rice Microsomal Membrane Proteins for High-Throughput Proteomics
title_short An Integrated Approach for the Efficient Extraction and Solubilization of Rice Microsomal Membrane Proteins for High-Throughput Proteomics
title_sort integrated approach for the efficient extraction and solubilization of rice microsomal membrane proteins for high-throughput proteomics
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8460067/
https://www.ncbi.nlm.nih.gov/pubmed/34567038
http://dx.doi.org/10.3389/fpls.2021.723369
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