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Resolving Molecular Heterogeneity with Single-Molecule Centrifugation
[Image: see text] For many classes of biomolecules, population-level heterogeneity is an essential aspect of biological function—from antibodies produced by the immune system to post-translationally modified proteins that regulate cellular processes. However, heterogeneity is difficult to fully char...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9936575/ https://www.ncbi.nlm.nih.gov/pubmed/36716175 http://dx.doi.org/10.1021/jacs.2c11450 |
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author | Luo, Yi Chang, Jeffrey Yang, Darren Bryan, J. Shepard MacIsaac, Molly Pressé, Steve Wong, Wesley P. |
author_facet | Luo, Yi Chang, Jeffrey Yang, Darren Bryan, J. Shepard MacIsaac, Molly Pressé, Steve Wong, Wesley P. |
author_sort | Luo, Yi |
collection | PubMed |
description | [Image: see text] For many classes of biomolecules, population-level heterogeneity is an essential aspect of biological function—from antibodies produced by the immune system to post-translationally modified proteins that regulate cellular processes. However, heterogeneity is difficult to fully characterize for multiple reasons: (i) single-molecule approaches are needed to avoid information lost by ensemble-level averaging, (ii) sufficient statistics must be gathered on both a per-molecule and per-population level, and (iii) a suitable analysis framework is required to make sense of a potentially limited number of intrinsically noisy measurements. Here, we introduce an approach that overcomes these difficulties by combining three techniques: a DNA nanoswitch construct to repeatedly interrogate the same molecule, a benchtop centrifuge force microscope (CFM) to obtain thousands of statistics in a highly parallel manner, and a Bayesian nonparametric (BNP) inference method to resolve separate subpopulations with distinct kinetics. We apply this approach to characterize commercially available antibodies and find that polyclonal antibody from rabbit serum is well-modeled by a mixture of three subpopulations. Our results show how combining a spatially and temporally multiplexed nanoswitch-CFM assay with BNP analysis can help resolve complex biomolecular interactions in heterogeneous samples. |
format | Online Article Text |
id | pubmed-9936575 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-99365752023-02-18 Resolving Molecular Heterogeneity with Single-Molecule Centrifugation Luo, Yi Chang, Jeffrey Yang, Darren Bryan, J. Shepard MacIsaac, Molly Pressé, Steve Wong, Wesley P. J Am Chem Soc [Image: see text] For many classes of biomolecules, population-level heterogeneity is an essential aspect of biological function—from antibodies produced by the immune system to post-translationally modified proteins that regulate cellular processes. However, heterogeneity is difficult to fully characterize for multiple reasons: (i) single-molecule approaches are needed to avoid information lost by ensemble-level averaging, (ii) sufficient statistics must be gathered on both a per-molecule and per-population level, and (iii) a suitable analysis framework is required to make sense of a potentially limited number of intrinsically noisy measurements. Here, we introduce an approach that overcomes these difficulties by combining three techniques: a DNA nanoswitch construct to repeatedly interrogate the same molecule, a benchtop centrifuge force microscope (CFM) to obtain thousands of statistics in a highly parallel manner, and a Bayesian nonparametric (BNP) inference method to resolve separate subpopulations with distinct kinetics. We apply this approach to characterize commercially available antibodies and find that polyclonal antibody from rabbit serum is well-modeled by a mixture of three subpopulations. Our results show how combining a spatially and temporally multiplexed nanoswitch-CFM assay with BNP analysis can help resolve complex biomolecular interactions in heterogeneous samples. American Chemical Society 2023-01-30 /pmc/articles/PMC9936575/ /pubmed/36716175 http://dx.doi.org/10.1021/jacs.2c11450 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Luo, Yi Chang, Jeffrey Yang, Darren Bryan, J. Shepard MacIsaac, Molly Pressé, Steve Wong, Wesley P. Resolving Molecular Heterogeneity with Single-Molecule Centrifugation |
title | Resolving Molecular
Heterogeneity with Single-Molecule
Centrifugation |
title_full | Resolving Molecular
Heterogeneity with Single-Molecule
Centrifugation |
title_fullStr | Resolving Molecular
Heterogeneity with Single-Molecule
Centrifugation |
title_full_unstemmed | Resolving Molecular
Heterogeneity with Single-Molecule
Centrifugation |
title_short | Resolving Molecular
Heterogeneity with Single-Molecule
Centrifugation |
title_sort | resolving molecular
heterogeneity with single-molecule
centrifugation |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9936575/ https://www.ncbi.nlm.nih.gov/pubmed/36716175 http://dx.doi.org/10.1021/jacs.2c11450 |
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