Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins
Abstract
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.
Additional Information
© 2020 Dopie et al. 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/). Submitted: 29 October 2019. Revised: 3 April 2020. Accepted: 13 May 2020. The authors thank all members of the Belmont laboratory for suggestions and advice. A.S. Belmont acknowledges support from National Institutes of Health R01 grant GM58460. The California Institute of Technology Proteome Exploration Laboratory is supported by the Beckman Institute and National Institutes of Health grant 1S10OD02001301. Light microscopy was performed in the Department of Molecular and Cellular Biology Light Microscopy Facility, funded with support from the Carver Foundation. The authors declare no competing financial interests. Author contributions: J. Dopie and A.S. Belmont conceived of and designed the research plan. J. Dopie designed the experimental plan and performed all experiments. A. Moradian and M.J. Sweredoski provided advice on preparation of samples for MS, performed MS, provided analysis of MS data, and advised on the ratio analysis approach. The manuscript was written by J. Dopie and A.S. Belmont with input from A. Moradian and M.J. Sweredoski regarding the MS.Attached Files
Published - jcb_201910207.pdf
Supplemental Material - jcb_201910207_tables1.xlsx
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Additional details
- PMCID
- PMC7480118
- Eprint ID
- 104181
- Resolver ID
- CaltechAUTHORS:20200701-103605335
- GM58460
- NIH
- Caltech Beckman Institute
- 1S10OD02001301
- NIH
- Carver Foundation
- Created
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2020-07-01Created from EPrint's datestamp field
- Updated
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2021-11-16Created from EPrint's last_modified field