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An efficient and robust HPLC method to determine the sialylation levels of human epithelial cells
Sialyltransferase, an enzyme responsible for attaching sialic acid to the cell surface, is reported to play a key role in cancer, making sialyltransferase a potential therapeutic target in drug development. Several methods have been developed to quantify sialic acids in biological samples however li...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8765648/ https://www.ncbi.nlm.nih.gov/pubmed/35041670 http://dx.doi.org/10.1371/journal.pone.0257178 |
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author | Kim, Hyo Jeong Schweiker, Stephanie Powell, Katie Levonis, Stephan |
author_facet | Kim, Hyo Jeong Schweiker, Stephanie Powell, Katie Levonis, Stephan |
author_sort | Kim, Hyo Jeong |
collection | PubMed |
description | Sialyltransferase, an enzyme responsible for attaching sialic acid to the cell surface, is reported to play a key role in cancer, making sialyltransferase a potential therapeutic target in drug development. Several methods have been developed to quantify sialic acids in biological samples however limitations exists and quantification in complex cell matrices lack investigation. Hence, this paper outlines a simple method to detect and quantify sialic acids in cancer cells for evaluating sialyltransferase activity of potential therapeutic compounds. An efficient method was developed using a reverse-phase ion-pairing HPLC-UV using triisopropanolamine as the ion-pairing agent with a C18 column. Neu5Ac was successfully eluted with the retention time 6.344 min with a flow rate of 0.4 mL/min. The proposed method was validated appropriately according to the AOAC guidelines (2013). This work demonstrates that the proposed method is not only relatively simple but also cost and time effective compared to pre-existing methods to successfully determine both free and protein-bound Neu5Ac in a complex cancer cell matrix. Furthermore, by applying the proposed method, a statistically significant decrease was observed for both HeLa and HuCCT1 cell lines with the application of deoxycholic acid–a known sialyltransferase inhibitor. Hence, the proposed method seems promisingly applicable to evaluate the effectiveness of potential sialyltransferase inhibitors. |
format | Online Article Text |
id | pubmed-8765648 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-87656482022-01-19 An efficient and robust HPLC method to determine the sialylation levels of human epithelial cells Kim, Hyo Jeong Schweiker, Stephanie Powell, Katie Levonis, Stephan PLoS One Research Article Sialyltransferase, an enzyme responsible for attaching sialic acid to the cell surface, is reported to play a key role in cancer, making sialyltransferase a potential therapeutic target in drug development. Several methods have been developed to quantify sialic acids in biological samples however limitations exists and quantification in complex cell matrices lack investigation. Hence, this paper outlines a simple method to detect and quantify sialic acids in cancer cells for evaluating sialyltransferase activity of potential therapeutic compounds. An efficient method was developed using a reverse-phase ion-pairing HPLC-UV using triisopropanolamine as the ion-pairing agent with a C18 column. Neu5Ac was successfully eluted with the retention time 6.344 min with a flow rate of 0.4 mL/min. The proposed method was validated appropriately according to the AOAC guidelines (2013). This work demonstrates that the proposed method is not only relatively simple but also cost and time effective compared to pre-existing methods to successfully determine both free and protein-bound Neu5Ac in a complex cancer cell matrix. Furthermore, by applying the proposed method, a statistically significant decrease was observed for both HeLa and HuCCT1 cell lines with the application of deoxycholic acid–a known sialyltransferase inhibitor. Hence, the proposed method seems promisingly applicable to evaluate the effectiveness of potential sialyltransferase inhibitors. Public Library of Science 2022-01-18 /pmc/articles/PMC8765648/ /pubmed/35041670 http://dx.doi.org/10.1371/journal.pone.0257178 Text en © 2022 Kim et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://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 Kim, Hyo Jeong Schweiker, Stephanie Powell, Katie Levonis, Stephan An efficient and robust HPLC method to determine the sialylation levels of human epithelial cells |
title | An efficient and robust HPLC method to determine the sialylation levels of human epithelial cells |
title_full | An efficient and robust HPLC method to determine the sialylation levels of human epithelial cells |
title_fullStr | An efficient and robust HPLC method to determine the sialylation levels of human epithelial cells |
title_full_unstemmed | An efficient and robust HPLC method to determine the sialylation levels of human epithelial cells |
title_short | An efficient and robust HPLC method to determine the sialylation levels of human epithelial cells |
title_sort | efficient and robust hplc method to determine the sialylation levels of human epithelial cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8765648/ https://www.ncbi.nlm.nih.gov/pubmed/35041670 http://dx.doi.org/10.1371/journal.pone.0257178 |
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