Cargando…
Synthesis and Application of a Clickable Epoxomicin‐Based Probe for Proteasome Activity Analysis
The proteasome is a multisubunit protein complex responsible for the degradation of proteins, making it essential in myriad cellular processes. Several reversible and irreversible peptide substrates inspired by known proteasome inhibitors have been developed to visualize it and monitor its activity;...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9354099/ https://www.ncbi.nlm.nih.gov/pubmed/35849029 http://dx.doi.org/10.1002/cpz1.490 |
_version_ | 1784762990405353472 |
---|---|
author | Salazar‐Chaparro, Andres F. Halder, Saayak Trader, Darci J. |
author_facet | Salazar‐Chaparro, Andres F. Halder, Saayak Trader, Darci J. |
author_sort | Salazar‐Chaparro, Andres F. |
collection | PubMed |
description | The proteasome is a multisubunit protein complex responsible for the degradation of proteins, making it essential in myriad cellular processes. Several reversible and irreversible peptide substrates inspired by known proteasome inhibitors have been developed to visualize it and monitor its activity; however, they have limited commercial availability or possess fluorophores that overlap with other known chemical probes, limiting their simultaneous use. The protocols presented here describe the synthesis of a clickable epoxomicin‐based probe followed by the copper‐catalyzed installment of an azide‐containing fluorophore, and the application of the synthesized peptide in proteasome activity assays by SDS‐PAGE and flow cytometry. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Solid‐phase synthesis of clickable peptide fragment (2) Basic Protocol 2: In‐solution coupling of epoxy‐ketone moiety to fragment (2) Basic Protocol 3: Copper‐catalyzed click reaction of (3) with fluorophore of choice Basic Protocol 4: Monitoring proteasome activity by SDS‐PAGE in HEK‐293T cells Alternate Protocol: Monitoring proteasome activity by flow cytometry in HEK‐293T cells |
format | Online Article Text |
id | pubmed-9354099 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-93540992022-10-14 Synthesis and Application of a Clickable Epoxomicin‐Based Probe for Proteasome Activity Analysis Salazar‐Chaparro, Andres F. Halder, Saayak Trader, Darci J. Curr Protoc Protocol The proteasome is a multisubunit protein complex responsible for the degradation of proteins, making it essential in myriad cellular processes. Several reversible and irreversible peptide substrates inspired by known proteasome inhibitors have been developed to visualize it and monitor its activity; however, they have limited commercial availability or possess fluorophores that overlap with other known chemical probes, limiting their simultaneous use. The protocols presented here describe the synthesis of a clickable epoxomicin‐based probe followed by the copper‐catalyzed installment of an azide‐containing fluorophore, and the application of the synthesized peptide in proteasome activity assays by SDS‐PAGE and flow cytometry. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Solid‐phase synthesis of clickable peptide fragment (2) Basic Protocol 2: In‐solution coupling of epoxy‐ketone moiety to fragment (2) Basic Protocol 3: Copper‐catalyzed click reaction of (3) with fluorophore of choice Basic Protocol 4: Monitoring proteasome activity by SDS‐PAGE in HEK‐293T cells Alternate Protocol: Monitoring proteasome activity by flow cytometry in HEK‐293T cells John Wiley and Sons Inc. 2022-07-18 2022-07 /pmc/articles/PMC9354099/ /pubmed/35849029 http://dx.doi.org/10.1002/cpz1.490 Text en © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. |
spellingShingle | Protocol Salazar‐Chaparro, Andres F. Halder, Saayak Trader, Darci J. Synthesis and Application of a Clickable Epoxomicin‐Based Probe for Proteasome Activity Analysis |
title | Synthesis and Application of a Clickable Epoxomicin‐Based Probe for Proteasome Activity Analysis |
title_full | Synthesis and Application of a Clickable Epoxomicin‐Based Probe for Proteasome Activity Analysis |
title_fullStr | Synthesis and Application of a Clickable Epoxomicin‐Based Probe for Proteasome Activity Analysis |
title_full_unstemmed | Synthesis and Application of a Clickable Epoxomicin‐Based Probe for Proteasome Activity Analysis |
title_short | Synthesis and Application of a Clickable Epoxomicin‐Based Probe for Proteasome Activity Analysis |
title_sort | synthesis and application of a clickable epoxomicin‐based probe for proteasome activity analysis |
topic | Protocol |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9354099/ https://www.ncbi.nlm.nih.gov/pubmed/35849029 http://dx.doi.org/10.1002/cpz1.490 |
work_keys_str_mv | AT salazarchaparroandresf synthesisandapplicationofaclickableepoxomicinbasedprobeforproteasomeactivityanalysis AT haldersaayak synthesisandapplicationofaclickableepoxomicinbasedprobeforproteasomeactivityanalysis AT traderdarcij synthesisandapplicationofaclickableepoxomicinbasedprobeforproteasomeactivityanalysis |