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Mass spectrometric detection of KRAS protein mutations using molecular imprinting

Cancer is a disease of cellular evolution where single base changes in the genetic code can have significant impact on the translation of proteins and their activity. Thus, in cancer research there is significant interest in methods that can determine mutations and identify the significant binding s...

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Autores principales: Norman, Rachel L., Singh, Rajinder, Muskett, Frederick W., Parrott, Emma L., Rufini, Alessandro, Langridge, James I., Runau, Franscois, Dennison, Ashley, Shaw, Jacqui A., Piletska, Elena, Canfarotta, Francesco, Ng, Leong L., Piletsky, Sergey, Jones, Donald J. L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8675027/
https://www.ncbi.nlm.nih.gov/pubmed/34854867
http://dx.doi.org/10.1039/d1nr03180e
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author Norman, Rachel L.
Singh, Rajinder
Muskett, Frederick W.
Parrott, Emma L.
Rufini, Alessandro
Langridge, James I.
Runau, Franscois
Dennison, Ashley
Shaw, Jacqui A.
Piletska, Elena
Canfarotta, Francesco
Ng, Leong L.
Piletsky, Sergey
Jones, Donald J. L.
author_facet Norman, Rachel L.
Singh, Rajinder
Muskett, Frederick W.
Parrott, Emma L.
Rufini, Alessandro
Langridge, James I.
Runau, Franscois
Dennison, Ashley
Shaw, Jacqui A.
Piletska, Elena
Canfarotta, Francesco
Ng, Leong L.
Piletsky, Sergey
Jones, Donald J. L.
author_sort Norman, Rachel L.
collection PubMed
description Cancer is a disease of cellular evolution where single base changes in the genetic code can have significant impact on the translation of proteins and their activity. Thus, in cancer research there is significant interest in methods that can determine mutations and identify the significant binding sites (epitopes) of antibodies to proteins in order to develop novel therapies. Nano molecularly imprinted polymers (nanoMIPs) provide an alternative to antibodies as reagents capable of specifically capturing target molecules depending on their structure. In this study, we used nanoMIPs to capture KRAS, a critical oncogene, to identify mutations which when present are indicative of oncological progress. Herein, coupling nanoMIPs (capture) and liquid chromatography-mass spectrometry (detection), LC-MS has allowed us to investigate mutational assignment and epitope discovery. Specifically, we have shown epitope discovery by generating nanoMIPs to a recombinant KRAS protein and identifying three regions of the protein which have been previously assigned as epitopes using much more time-consuming protocols. The mutation status of the released tryptic peptide was identified by LC-MS following capture of the conserved region of KRAS using nanoMIPS, which were tryptically digested, thus releasing the sequence of a non-conserved (mutated) region. This approach was tested in cell lines where we showed the effective genotyping of a KRAS cell line and in the plasma of cancer patients, thus demonstrating its ability to diagnose precisely the mutational status of a patient. This work provides a clear line-of-sight for the use of nanoMIPs to its translation from research into diagnostic and clinical utility.
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spelling pubmed-86750272022-01-11 Mass spectrometric detection of KRAS protein mutations using molecular imprinting Norman, Rachel L. Singh, Rajinder Muskett, Frederick W. Parrott, Emma L. Rufini, Alessandro Langridge, James I. Runau, Franscois Dennison, Ashley Shaw, Jacqui A. Piletska, Elena Canfarotta, Francesco Ng, Leong L. Piletsky, Sergey Jones, Donald J. L. Nanoscale Chemistry Cancer is a disease of cellular evolution where single base changes in the genetic code can have significant impact on the translation of proteins and their activity. Thus, in cancer research there is significant interest in methods that can determine mutations and identify the significant binding sites (epitopes) of antibodies to proteins in order to develop novel therapies. Nano molecularly imprinted polymers (nanoMIPs) provide an alternative to antibodies as reagents capable of specifically capturing target molecules depending on their structure. In this study, we used nanoMIPs to capture KRAS, a critical oncogene, to identify mutations which when present are indicative of oncological progress. Herein, coupling nanoMIPs (capture) and liquid chromatography-mass spectrometry (detection), LC-MS has allowed us to investigate mutational assignment and epitope discovery. Specifically, we have shown epitope discovery by generating nanoMIPs to a recombinant KRAS protein and identifying three regions of the protein which have been previously assigned as epitopes using much more time-consuming protocols. The mutation status of the released tryptic peptide was identified by LC-MS following capture of the conserved region of KRAS using nanoMIPS, which were tryptically digested, thus releasing the sequence of a non-conserved (mutated) region. This approach was tested in cell lines where we showed the effective genotyping of a KRAS cell line and in the plasma of cancer patients, thus demonstrating its ability to diagnose precisely the mutational status of a patient. This work provides a clear line-of-sight for the use of nanoMIPs to its translation from research into diagnostic and clinical utility. The Royal Society of Chemistry 2021-11-29 /pmc/articles/PMC8675027/ /pubmed/34854867 http://dx.doi.org/10.1039/d1nr03180e Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by/3.0/
spellingShingle Chemistry
Norman, Rachel L.
Singh, Rajinder
Muskett, Frederick W.
Parrott, Emma L.
Rufini, Alessandro
Langridge, James I.
Runau, Franscois
Dennison, Ashley
Shaw, Jacqui A.
Piletska, Elena
Canfarotta, Francesco
Ng, Leong L.
Piletsky, Sergey
Jones, Donald J. L.
Mass spectrometric detection of KRAS protein mutations using molecular imprinting
title Mass spectrometric detection of KRAS protein mutations using molecular imprinting
title_full Mass spectrometric detection of KRAS protein mutations using molecular imprinting
title_fullStr Mass spectrometric detection of KRAS protein mutations using molecular imprinting
title_full_unstemmed Mass spectrometric detection of KRAS protein mutations using molecular imprinting
title_short Mass spectrometric detection of KRAS protein mutations using molecular imprinting
title_sort mass spectrometric detection of kras protein mutations using molecular imprinting
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8675027/
https://www.ncbi.nlm.nih.gov/pubmed/34854867
http://dx.doi.org/10.1039/d1nr03180e
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