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Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins

We present a simple ratio method to infer protein composition within cellular structures using proximity labeling approaches but compensating for the diffusion of free radicals. We used tyramide signal amplification (TSA) and label-free mass spectrometry (MS) to compare proteins in nuclear speckles...

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Detalles Bibliográficos
Autores principales: Dopie, Joseph, Sweredoski, Michael J., Moradian, Annie, Belmont, Andrew S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Rockefeller University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480118/
https://www.ncbi.nlm.nih.gov/pubmed/32609799
http://dx.doi.org/10.1083/jcb.201910207
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author Dopie, Joseph
Sweredoski, Michael J.
Moradian, Annie
Belmont, Andrew S.
author_facet Dopie, Joseph
Sweredoski, Michael J.
Moradian, Annie
Belmont, Andrew S.
author_sort Dopie, Joseph
collection PubMed
description We present a simple ratio method to infer protein composition within cellular structures using proximity labeling approaches but compensating for the diffusion of free radicals. We used tyramide signal amplification (TSA) and label-free mass spectrometry (MS) to compare proteins in nuclear speckles versus centromeres. Our “TSA-MS ratio” approach successfully identified known nuclear speckle proteins. For example, 96% and 67% of proteins in the top 30 and 100 sorted proteins, respectively, are known nuclear speckle proteins, including proteins that we validated here as enriched in nuclear speckles. We show that MFAP1, among the top 20 in our list, forms droplets under certain circumstances and that MFAP1 expression levels modulate the size, stability, and dynamics of nuclear speckles. Localization of MFAP1 and its binding partner, PRPF38A, in droplet-like nuclear bodies precedes formation of nuclear speckles during telophase. Our results update older proteomic studies of nuclear speckles and should provide a useful reference dataset to guide future experimental dissection of nuclear speckle structure and function.
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spelling pubmed-74801182021-03-07 Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins Dopie, Joseph Sweredoski, Michael J. Moradian, Annie Belmont, Andrew S. J Cell Biol Tools We present a simple ratio method to infer protein composition within cellular structures using proximity labeling approaches but compensating for the diffusion of free radicals. We used tyramide signal amplification (TSA) and label-free mass spectrometry (MS) to compare proteins in nuclear speckles versus centromeres. Our “TSA-MS ratio” approach successfully identified known nuclear speckle proteins. For example, 96% and 67% of proteins in the top 30 and 100 sorted proteins, respectively, are known nuclear speckle proteins, including proteins that we validated here as enriched in nuclear speckles. We show that MFAP1, among the top 20 in our list, forms droplets under certain circumstances and that MFAP1 expression levels modulate the size, stability, and dynamics of nuclear speckles. Localization of MFAP1 and its binding partner, PRPF38A, in droplet-like nuclear bodies precedes formation of nuclear speckles during telophase. Our results update older proteomic studies of nuclear speckles and should provide a useful reference dataset to guide future experimental dissection of nuclear speckle structure and function. Rockefeller University Press 2020-07-01 /pmc/articles/PMC7480118/ /pubmed/32609799 http://dx.doi.org/10.1083/jcb.201910207 Text en © 2020 Dopie et al. http://www.rupress.org/terms/https://creativecommons.org/licenses/by-nc-sa/4.0/This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Tools
Dopie, Joseph
Sweredoski, Michael J.
Moradian, Annie
Belmont, Andrew S.
Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins
title Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins
title_full Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins
title_fullStr Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins
title_full_unstemmed Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins
title_short Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins
title_sort tyramide signal amplification mass spectrometry (tsa-ms) ratio identifies nuclear speckle proteins
topic Tools
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480118/
https://www.ncbi.nlm.nih.gov/pubmed/32609799
http://dx.doi.org/10.1083/jcb.201910207
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