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In-depth characterization and comparison of the N-glycosylated proteome of two-dimensional- and three-dimensional-cultured breast cancer cells and xenografted tumors

Native intact N-glycopeptide analysis can provide access to the comprehensive characteristics of N-glycan occupancy, including N-glycosites, N-glycan compositions, and N-glycoproteins for complex samples. The sample pre-processing method used for the analysis of intact N-glycopeptides usually depend...

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Autores principales: Mao, Yonghong, Zhao, Yang, Zhang, Yong, Yang, Hao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728280/
https://www.ncbi.nlm.nih.gov/pubmed/33301522
http://dx.doi.org/10.1371/journal.pone.0243789
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author Mao, Yonghong
Zhao, Yang
Zhang, Yong
Yang, Hao
author_facet Mao, Yonghong
Zhao, Yang
Zhang, Yong
Yang, Hao
author_sort Mao, Yonghong
collection PubMed
description Native intact N-glycopeptide analysis can provide access to the comprehensive characteristics of N-glycan occupancy, including N-glycosites, N-glycan compositions, and N-glycoproteins for complex samples. The sample pre-processing method used for the analysis of intact N-glycopeptides usually depends on the enrichment of low abundance N-glycopeptides from a tryptic peptide mixture using hydrophilic substances before LC-MS/MS detection. However, the number of identified intact N-glycopeptides remains inadequate to achieve an in-depth profile of the N-glycosylation landscape. Here, we optimized the sample preparation workflow prior to LC-MS/MS analysis by systematically comparing different analytical methods, including the use of different sources of trypsin, combinations of different proteases, and different enrichment materials. Finally, we found that the combination of Trypsin (B)/Lys-C digestion and zwitterionic HILIC (Zic-HILIC) enrichment significantly improved the mass spectrometric characterization of intact N-glycopeptides, increasing the number of identified intact N-glycopeptides and offering better analytical reproducibility. Furthermore, the optimized workflow was applied to the analysis of intact N-glycopeptides in two-dimensional (2D) and three-dimensional (3D)-cultured breast cancer cells in vitro and xenografted tumors in mice. These results indicated that the same breast cancer cells, when cultured in different microenvironments, can show different N-glycosylation patterns. This study also provides an interesting comparison of the N-glycoproteome of breast cancer cells cultured in different growth conditions, indicating the important role of N-glycosylated proteins in cancer cell growth and the choice of the cell culture model for studies in tumor biology and drug evaluation.
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spelling pubmed-77282802020-12-17 In-depth characterization and comparison of the N-glycosylated proteome of two-dimensional- and three-dimensional-cultured breast cancer cells and xenografted tumors Mao, Yonghong Zhao, Yang Zhang, Yong Yang, Hao PLoS One Research Article Native intact N-glycopeptide analysis can provide access to the comprehensive characteristics of N-glycan occupancy, including N-glycosites, N-glycan compositions, and N-glycoproteins for complex samples. The sample pre-processing method used for the analysis of intact N-glycopeptides usually depends on the enrichment of low abundance N-glycopeptides from a tryptic peptide mixture using hydrophilic substances before LC-MS/MS detection. However, the number of identified intact N-glycopeptides remains inadequate to achieve an in-depth profile of the N-glycosylation landscape. Here, we optimized the sample preparation workflow prior to LC-MS/MS analysis by systematically comparing different analytical methods, including the use of different sources of trypsin, combinations of different proteases, and different enrichment materials. Finally, we found that the combination of Trypsin (B)/Lys-C digestion and zwitterionic HILIC (Zic-HILIC) enrichment significantly improved the mass spectrometric characterization of intact N-glycopeptides, increasing the number of identified intact N-glycopeptides and offering better analytical reproducibility. Furthermore, the optimized workflow was applied to the analysis of intact N-glycopeptides in two-dimensional (2D) and three-dimensional (3D)-cultured breast cancer cells in vitro and xenografted tumors in mice. These results indicated that the same breast cancer cells, when cultured in different microenvironments, can show different N-glycosylation patterns. This study also provides an interesting comparison of the N-glycoproteome of breast cancer cells cultured in different growth conditions, indicating the important role of N-glycosylated proteins in cancer cell growth and the choice of the cell culture model for studies in tumor biology and drug evaluation. Public Library of Science 2020-12-10 /pmc/articles/PMC7728280/ /pubmed/33301522 http://dx.doi.org/10.1371/journal.pone.0243789 Text en © 2020 Mao 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
Mao, Yonghong
Zhao, Yang
Zhang, Yong
Yang, Hao
In-depth characterization and comparison of the N-glycosylated proteome of two-dimensional- and three-dimensional-cultured breast cancer cells and xenografted tumors
title In-depth characterization and comparison of the N-glycosylated proteome of two-dimensional- and three-dimensional-cultured breast cancer cells and xenografted tumors
title_full In-depth characterization and comparison of the N-glycosylated proteome of two-dimensional- and three-dimensional-cultured breast cancer cells and xenografted tumors
title_fullStr In-depth characterization and comparison of the N-glycosylated proteome of two-dimensional- and three-dimensional-cultured breast cancer cells and xenografted tumors
title_full_unstemmed In-depth characterization and comparison of the N-glycosylated proteome of two-dimensional- and three-dimensional-cultured breast cancer cells and xenografted tumors
title_short In-depth characterization and comparison of the N-glycosylated proteome of two-dimensional- and three-dimensional-cultured breast cancer cells and xenografted tumors
title_sort in-depth characterization and comparison of the n-glycosylated proteome of two-dimensional- and three-dimensional-cultured breast cancer cells and xenografted tumors
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728280/
https://www.ncbi.nlm.nih.gov/pubmed/33301522
http://dx.doi.org/10.1371/journal.pone.0243789
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