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Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins

Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) have been successfully applied for understanding the dynamics of protein complexes. Here, we developed a highly specific and reproducible method to quantify a...

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Autores principales: Kosaka, Yuishin, Aoki, Wataru, Mori, Megumi, Aburaya, Shunsuke, Ohtani, Yuta, Minakuchi, Hiroyoshi, Ueda, Mitsuyoshi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735604/
https://www.ncbi.nlm.nih.gov/pubmed/33315868
http://dx.doi.org/10.1371/journal.pone.0236850
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author Kosaka, Yuishin
Aoki, Wataru
Mori, Megumi
Aburaya, Shunsuke
Ohtani, Yuta
Minakuchi, Hiroyoshi
Ueda, Mitsuyoshi
author_facet Kosaka, Yuishin
Aoki, Wataru
Mori, Megumi
Aburaya, Shunsuke
Ohtani, Yuta
Minakuchi, Hiroyoshi
Ueda, Mitsuyoshi
author_sort Kosaka, Yuishin
collection PubMed
description Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) have been successfully applied for understanding the dynamics of protein complexes. Here, we developed a highly specific and reproducible method to quantify all ribosomal proteins (r-proteins) by combining selected reaction monitoring (SRM) and isotope labeling. We optimized the SRM methods using purified ribosomes and Escherichia coli lysates and verified this approach as detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a highly specific analytical tool in the research of E. coli ribosomes, and this methodology have potential to accelerate the understanding of ribosome biogenesis, function, and the development of engineered ribosomes with additional functions.
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spelling pubmed-77356042020-12-22 Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins Kosaka, Yuishin Aoki, Wataru Mori, Megumi Aburaya, Shunsuke Ohtani, Yuta Minakuchi, Hiroyoshi Ueda, Mitsuyoshi PLoS One Research Article Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) have been successfully applied for understanding the dynamics of protein complexes. Here, we developed a highly specific and reproducible method to quantify all ribosomal proteins (r-proteins) by combining selected reaction monitoring (SRM) and isotope labeling. We optimized the SRM methods using purified ribosomes and Escherichia coli lysates and verified this approach as detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a highly specific analytical tool in the research of E. coli ribosomes, and this methodology have potential to accelerate the understanding of ribosome biogenesis, function, and the development of engineered ribosomes with additional functions. Public Library of Science 2020-12-14 /pmc/articles/PMC7735604/ /pubmed/33315868 http://dx.doi.org/10.1371/journal.pone.0236850 Text en © 2020 Kosaka et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Kosaka, Yuishin
Aoki, Wataru
Mori, Megumi
Aburaya, Shunsuke
Ohtani, Yuta
Minakuchi, Hiroyoshi
Ueda, Mitsuyoshi
Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins
title Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins
title_full Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins
title_fullStr Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins
title_full_unstemmed Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins
title_short Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins
title_sort selected reaction monitoring for the quantification of escherichia coli ribosomal proteins
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735604/
https://www.ncbi.nlm.nih.gov/pubmed/33315868
http://dx.doi.org/10.1371/journal.pone.0236850
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