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Robust and scalable single-molecule protein sequencing with fluorosequencing
The need to accurately survey proteins and their modifications with ever higher sensitivities, particularly in clinical settings with limited samples, is spurring development of new single molecule proteomics technologies. Fluorosequencing is one such highly parallelized single molecule peptide sequ...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10516020/ https://www.ncbi.nlm.nih.gov/pubmed/37745461 http://dx.doi.org/10.1101/2023.09.15.558007 |
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author | Mapes, James H. Stover, Julia Stout, Heather D. Folsom, Tucker M. Babcock, Emily Loudwig, Sandra Martin, Christopher Austin, Mariah J. Tu, Fan Howdieshell, Casey J. Simpson, Zachary B. Blom, Thomas Weaver, Daniel Winkler, Daniel Vander Velden, Kent Ossareh, Parham M. Beierle, John M Somekh, Talli Bardo, Angela M. Anslyn, Eric V. Marcotte, Edward M. Swaminathan, Jagannath |
author_facet | Mapes, James H. Stover, Julia Stout, Heather D. Folsom, Tucker M. Babcock, Emily Loudwig, Sandra Martin, Christopher Austin, Mariah J. Tu, Fan Howdieshell, Casey J. Simpson, Zachary B. Blom, Thomas Weaver, Daniel Winkler, Daniel Vander Velden, Kent Ossareh, Parham M. Beierle, John M Somekh, Talli Bardo, Angela M. Anslyn, Eric V. Marcotte, Edward M. Swaminathan, Jagannath |
author_sort | Mapes, James H. |
collection | PubMed |
description | The need to accurately survey proteins and their modifications with ever higher sensitivities, particularly in clinical settings with limited samples, is spurring development of new single molecule proteomics technologies. Fluorosequencing is one such highly parallelized single molecule peptide sequencing platform, based on determining the sequence positions of select amino acid types within peptides to enable their identification and quantification from a reference database. Here, we describe substantial improvements to fluorosequencing, including identifying fluorophores compatible with the sequencing chemistry, mitigating dye-dye interactions through the use of extended polyproline linkers, and developing an end-to-end workflow for sample preparation and sequencing. We demonstrate by fluorosequencing peptides in mixtures and identifying a target neoantigen from a database of decoy MHC peptides, highlighting the potential of the technology for high sensitivity clinical applications. |
format | Online Article Text |
id | pubmed-10516020 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Cold Spring Harbor Laboratory |
record_format | MEDLINE/PubMed |
spelling | pubmed-105160202023-09-23 Robust and scalable single-molecule protein sequencing with fluorosequencing Mapes, James H. Stover, Julia Stout, Heather D. Folsom, Tucker M. Babcock, Emily Loudwig, Sandra Martin, Christopher Austin, Mariah J. Tu, Fan Howdieshell, Casey J. Simpson, Zachary B. Blom, Thomas Weaver, Daniel Winkler, Daniel Vander Velden, Kent Ossareh, Parham M. Beierle, John M Somekh, Talli Bardo, Angela M. Anslyn, Eric V. Marcotte, Edward M. Swaminathan, Jagannath bioRxiv Article The need to accurately survey proteins and their modifications with ever higher sensitivities, particularly in clinical settings with limited samples, is spurring development of new single molecule proteomics technologies. Fluorosequencing is one such highly parallelized single molecule peptide sequencing platform, based on determining the sequence positions of select amino acid types within peptides to enable their identification and quantification from a reference database. Here, we describe substantial improvements to fluorosequencing, including identifying fluorophores compatible with the sequencing chemistry, mitigating dye-dye interactions through the use of extended polyproline linkers, and developing an end-to-end workflow for sample preparation and sequencing. We demonstrate by fluorosequencing peptides in mixtures and identifying a target neoantigen from a database of decoy MHC peptides, highlighting the potential of the technology for high sensitivity clinical applications. Cold Spring Harbor Laboratory 2023-09-16 /pmc/articles/PMC10516020/ /pubmed/37745461 http://dx.doi.org/10.1101/2023.09.15.558007 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator. |
spellingShingle | Article Mapes, James H. Stover, Julia Stout, Heather D. Folsom, Tucker M. Babcock, Emily Loudwig, Sandra Martin, Christopher Austin, Mariah J. Tu, Fan Howdieshell, Casey J. Simpson, Zachary B. Blom, Thomas Weaver, Daniel Winkler, Daniel Vander Velden, Kent Ossareh, Parham M. Beierle, John M Somekh, Talli Bardo, Angela M. Anslyn, Eric V. Marcotte, Edward M. Swaminathan, Jagannath Robust and scalable single-molecule protein sequencing with fluorosequencing |
title | Robust and scalable single-molecule protein sequencing with fluorosequencing |
title_full | Robust and scalable single-molecule protein sequencing with fluorosequencing |
title_fullStr | Robust and scalable single-molecule protein sequencing with fluorosequencing |
title_full_unstemmed | Robust and scalable single-molecule protein sequencing with fluorosequencing |
title_short | Robust and scalable single-molecule protein sequencing with fluorosequencing |
title_sort | robust and scalable single-molecule protein sequencing with fluorosequencing |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10516020/ https://www.ncbi.nlm.nih.gov/pubmed/37745461 http://dx.doi.org/10.1101/2023.09.15.558007 |
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