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Fast protein analysis enabled by high-temperature hydrolysis
While the bottom-up protein analysis serves as a mainstream method for biological studies, its efficiency is limited by the time-consuming process for enzymatic digestion or hydrolysis as well as the post-digestion treatment prior to mass spectrometry analysis. In this work, we developed an enzyme-f...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Royal Society of Chemistry
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8162451/ https://www.ncbi.nlm.nih.gov/pubmed/34094309 http://dx.doi.org/10.1039/d0sc03237a |
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author | Wang, Yuchen Zhang, Wenpeng Ouyang, Zheng |
author_facet | Wang, Yuchen Zhang, Wenpeng Ouyang, Zheng |
author_sort | Wang, Yuchen |
collection | PubMed |
description | While the bottom-up protein analysis serves as a mainstream method for biological studies, its efficiency is limited by the time-consuming process for enzymatic digestion or hydrolysis as well as the post-digestion treatment prior to mass spectrometry analysis. In this work, we developed an enzyme-free microreaction system for fast and selective hydrolysis of proteins, and a direct analysis of the protein digests was achieved by nanoESI (electrospray ionization) mass spectrometry. Using the microreactor, proteins in aqueous solution could be selectively hydrolyzed at the aspartyl sites within 2 min at high temperatures (∼150 °C). Being free of salts, the protein digest solution could be directly analyzed using a mass spectrometer with nanoESI without further purification or post-digestion treatment. This method has been validated for the analysis of a variety of proteins with molecular weights ranging from 8.5 to 67 kDa. With introduction of a reducing agent into the protein solutions, fast cleavage of disulfide bonds was also achieved along with high-temperature hydrolysis, allowing for fast analysis of large proteins such as bovine serum albumin. The high-temperature microreaction system was also used with a miniature mass spectrometer for the determination of highly specific peptides from Mycobacterium tuberculosis antigens, showing its potential for point-of-care analysis of protein biomarkers. |
format | Online Article Text |
id | pubmed-8162451 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | The Royal Society of Chemistry |
record_format | MEDLINE/PubMed |
spelling | pubmed-81624512021-06-04 Fast protein analysis enabled by high-temperature hydrolysis Wang, Yuchen Zhang, Wenpeng Ouyang, Zheng Chem Sci Chemistry While the bottom-up protein analysis serves as a mainstream method for biological studies, its efficiency is limited by the time-consuming process for enzymatic digestion or hydrolysis as well as the post-digestion treatment prior to mass spectrometry analysis. In this work, we developed an enzyme-free microreaction system for fast and selective hydrolysis of proteins, and a direct analysis of the protein digests was achieved by nanoESI (electrospray ionization) mass spectrometry. Using the microreactor, proteins in aqueous solution could be selectively hydrolyzed at the aspartyl sites within 2 min at high temperatures (∼150 °C). Being free of salts, the protein digest solution could be directly analyzed using a mass spectrometer with nanoESI without further purification or post-digestion treatment. This method has been validated for the analysis of a variety of proteins with molecular weights ranging from 8.5 to 67 kDa. With introduction of a reducing agent into the protein solutions, fast cleavage of disulfide bonds was also achieved along with high-temperature hydrolysis, allowing for fast analysis of large proteins such as bovine serum albumin. The high-temperature microreaction system was also used with a miniature mass spectrometer for the determination of highly specific peptides from Mycobacterium tuberculosis antigens, showing its potential for point-of-care analysis of protein biomarkers. The Royal Society of Chemistry 2020-09-10 /pmc/articles/PMC8162451/ /pubmed/34094309 http://dx.doi.org/10.1039/d0sc03237a Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by/3.0/ |
spellingShingle | Chemistry Wang, Yuchen Zhang, Wenpeng Ouyang, Zheng Fast protein analysis enabled by high-temperature hydrolysis |
title | Fast protein analysis enabled by high-temperature hydrolysis |
title_full | Fast protein analysis enabled by high-temperature hydrolysis |
title_fullStr | Fast protein analysis enabled by high-temperature hydrolysis |
title_full_unstemmed | Fast protein analysis enabled by high-temperature hydrolysis |
title_short | Fast protein analysis enabled by high-temperature hydrolysis |
title_sort | fast protein analysis enabled by high-temperature hydrolysis |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8162451/ https://www.ncbi.nlm.nih.gov/pubmed/34094309 http://dx.doi.org/10.1039/d0sc03237a |
work_keys_str_mv | AT wangyuchen fastproteinanalysisenabledbyhightemperaturehydrolysis AT zhangwenpeng fastproteinanalysisenabledbyhightemperaturehydrolysis AT ouyangzheng fastproteinanalysisenabledbyhightemperaturehydrolysis |