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An Ultrafast N-Glycoproteome Analysis Method Using Thermoresponsive Magnetic Fluid-Immobilized Enzymes
N-Glycosylation is one of the most common and important post-translational modification methods, and it plays a vital role in controlling many biological processes. Increasing discovery of abnormal alterations in N-linked glycans associated with many diseases leads to greater demands for rapid and e...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2021
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107388/ https://www.ncbi.nlm.nih.gov/pubmed/33981677 http://dx.doi.org/10.3389/fchem.2021.676100 |
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author | Fan, Zhiya Liu, Tong Zheng, Fei Qin, Weijie Qian, Xiaohong |
author_facet | Fan, Zhiya Liu, Tong Zheng, Fei Qin, Weijie Qian, Xiaohong |
author_sort | Fan, Zhiya |
collection | PubMed |
description | N-Glycosylation is one of the most common and important post-translational modification methods, and it plays a vital role in controlling many biological processes. Increasing discovery of abnormal alterations in N-linked glycans associated with many diseases leads to greater demands for rapid and efficient N-glycosylation profiling in large-scale clinical samples. In the workflow of global N-glycosylation analysis, enzymatic digestion is the main rate-limiting step, and it includes both protease digestion and peptide-N4–(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase) F deglycosylation. Prolonged incubation time is generally required because of the limited digestion efficiency of the conventional in-solution digestion method. Here, we propose novel thermoresponsive magnetic fluid (TMF)-immobilized enzymes (trypsin or PNGase F) for ultrafast and highly efficient proteome digestion and deglycosylation. Unlike other magnetic material-immobilized enzymes, TMF-immobilized enzymes display a unique temperature-triggered magnetic response behavior. At room temperature, a TMF-immobilized enzyme completely dissolves in an aqueous solution and forms a homogeneous system with a protein/peptide sample for efficient digestion but cannot be separated by magnetic force because of its excellent water dispersity. Above its lower critical solution temperature (LCST), thermoflocculation of a TMF-immobilized enzyme allows it to be easily recovered by increasing the temperature and magnetic force. Taking advantage of the unique homogeneous reaction of a TMF-immobilized enzyme, both protein digestion and glycopeptide deglycosylation can be finished within 3 min, and the whole sample processing time can be reduced by more than 20 times. The application of a TMF-immobilized enzyme in large-scale profiling of protein N-glycosylation in urine samples led to the successful identification of 2,197 N-glycopeptides and further demonstrated the potential of this strategy for fast and high-throughput analysis of N-glycoproteome in clinical samples. |
format | Online Article Text |
id | pubmed-8107388 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-81073882021-05-11 An Ultrafast N-Glycoproteome Analysis Method Using Thermoresponsive Magnetic Fluid-Immobilized Enzymes Fan, Zhiya Liu, Tong Zheng, Fei Qin, Weijie Qian, Xiaohong Front Chem Chemistry N-Glycosylation is one of the most common and important post-translational modification methods, and it plays a vital role in controlling many biological processes. Increasing discovery of abnormal alterations in N-linked glycans associated with many diseases leads to greater demands for rapid and efficient N-glycosylation profiling in large-scale clinical samples. In the workflow of global N-glycosylation analysis, enzymatic digestion is the main rate-limiting step, and it includes both protease digestion and peptide-N4–(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase) F deglycosylation. Prolonged incubation time is generally required because of the limited digestion efficiency of the conventional in-solution digestion method. Here, we propose novel thermoresponsive magnetic fluid (TMF)-immobilized enzymes (trypsin or PNGase F) for ultrafast and highly efficient proteome digestion and deglycosylation. Unlike other magnetic material-immobilized enzymes, TMF-immobilized enzymes display a unique temperature-triggered magnetic response behavior. At room temperature, a TMF-immobilized enzyme completely dissolves in an aqueous solution and forms a homogeneous system with a protein/peptide sample for efficient digestion but cannot be separated by magnetic force because of its excellent water dispersity. Above its lower critical solution temperature (LCST), thermoflocculation of a TMF-immobilized enzyme allows it to be easily recovered by increasing the temperature and magnetic force. Taking advantage of the unique homogeneous reaction of a TMF-immobilized enzyme, both protein digestion and glycopeptide deglycosylation can be finished within 3 min, and the whole sample processing time can be reduced by more than 20 times. The application of a TMF-immobilized enzyme in large-scale profiling of protein N-glycosylation in urine samples led to the successful identification of 2,197 N-glycopeptides and further demonstrated the potential of this strategy for fast and high-throughput analysis of N-glycoproteome in clinical samples. Frontiers Media S.A. 2021-04-26 /pmc/articles/PMC8107388/ /pubmed/33981677 http://dx.doi.org/10.3389/fchem.2021.676100 Text en Copyright © 2021 Fan, Liu, Zheng, Qin and Qian. 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 | Chemistry Fan, Zhiya Liu, Tong Zheng, Fei Qin, Weijie Qian, Xiaohong An Ultrafast N-Glycoproteome Analysis Method Using Thermoresponsive Magnetic Fluid-Immobilized Enzymes |
title | An Ultrafast N-Glycoproteome Analysis Method Using Thermoresponsive Magnetic Fluid-Immobilized Enzymes |
title_full | An Ultrafast N-Glycoproteome Analysis Method Using Thermoresponsive Magnetic Fluid-Immobilized Enzymes |
title_fullStr | An Ultrafast N-Glycoproteome Analysis Method Using Thermoresponsive Magnetic Fluid-Immobilized Enzymes |
title_full_unstemmed | An Ultrafast N-Glycoproteome Analysis Method Using Thermoresponsive Magnetic Fluid-Immobilized Enzymes |
title_short | An Ultrafast N-Glycoproteome Analysis Method Using Thermoresponsive Magnetic Fluid-Immobilized Enzymes |
title_sort | ultrafast n-glycoproteome analysis method using thermoresponsive magnetic fluid-immobilized enzymes |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107388/ https://www.ncbi.nlm.nih.gov/pubmed/33981677 http://dx.doi.org/10.3389/fchem.2021.676100 |
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