On-chip protein separation with single-molecule resolution

Abstract Accurate identification of both abundant and rare proteins hinges on the development of single-protein sensing methods. Given the immense variation in protein expression levels in a cell, separation of proteins by weight would improve protein classification strategies. Upstream separation f...

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Autores principales:	Adam Zrehen, Shilo Ohayon, Diana Huttner, Amit Meller
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2572d5e2eeb14f2e86dd3d39972c04ef
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spelling	oai:doaj.org-article:2572d5e2eeb14f2e86dd3d39972c04ef2021-12-02T18:33:51ZOn-chip protein separation with single-molecule resolution10.1038/s41598-020-72463-z2045-2322https://doaj.org/article/2572d5e2eeb14f2e86dd3d39972c04ef2020-09-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-72463-zhttps://doaj.org/toc/2045-2322Abstract Accurate identification of both abundant and rare proteins hinges on the development of single-protein sensing methods. Given the immense variation in protein expression levels in a cell, separation of proteins by weight would improve protein classification strategies. Upstream separation facilitates sample binning into smaller groups while also preventing sensor overflow, as may be caused by highly abundant proteins in cell lysates or clinical samples. Here, we scale a bulk analysis method for protein separation, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), to the single-molecule level using single-photon sensitive widefield imaging. Single-molecule sensing of the electrokinetically moving proteins is achieved by in situ polymerization of the PAGE in a low-profile fluidic channel having a depth of only ~ 0.6 µm. The polyacrylamide gel restricts the Brownian kinetics of the proteins, while the low-profile channel ensures that they remain in focus during imaging, allowing video-rate monitoring of single-protein migration. Calibration of the device involves separating a set of Atto647N-covalently labeled recombinant proteins in the size range of 14–70 kDa, yielding an exponential dependence of the proteins’ molecular weights on the measured mobilities, as expected. Subsequently, we demonstrate the ability of our fluidic device to separate and image thousands of proteins directly extracted from a human cancer cell line. Using single-particle image analysis methods, we created detailed profiles of the separation kinetics of lysine and cysteine -labeled proteins. Downstream coupling of the device to single-protein identification sensors may provide superior protein classification and improve our ability to analyze complex biological and medical protein samples.Adam ZrehenShilo OhayonDiana HuttnerAmit MellerNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-12 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Adam Zrehen Shilo Ohayon Diana Huttner Amit Meller On-chip protein separation with single-molecule resolution
description	Abstract Accurate identification of both abundant and rare proteins hinges on the development of single-protein sensing methods. Given the immense variation in protein expression levels in a cell, separation of proteins by weight would improve protein classification strategies. Upstream separation facilitates sample binning into smaller groups while also preventing sensor overflow, as may be caused by highly abundant proteins in cell lysates or clinical samples. Here, we scale a bulk analysis method for protein separation, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), to the single-molecule level using single-photon sensitive widefield imaging. Single-molecule sensing of the electrokinetically moving proteins is achieved by in situ polymerization of the PAGE in a low-profile fluidic channel having a depth of only ~ 0.6 µm. The polyacrylamide gel restricts the Brownian kinetics of the proteins, while the low-profile channel ensures that they remain in focus during imaging, allowing video-rate monitoring of single-protein migration. Calibration of the device involves separating a set of Atto647N-covalently labeled recombinant proteins in the size range of 14–70 kDa, yielding an exponential dependence of the proteins’ molecular weights on the measured mobilities, as expected. Subsequently, we demonstrate the ability of our fluidic device to separate and image thousands of proteins directly extracted from a human cancer cell line. Using single-particle image analysis methods, we created detailed profiles of the separation kinetics of lysine and cysteine -labeled proteins. Downstream coupling of the device to single-protein identification sensors may provide superior protein classification and improve our ability to analyze complex biological and medical protein samples.
format	article
author	Adam Zrehen Shilo Ohayon Diana Huttner Amit Meller
author_facet	Adam Zrehen Shilo Ohayon Diana Huttner Amit Meller
author_sort	Adam Zrehen
title	On-chip protein separation with single-molecule resolution
title_short	On-chip protein separation with single-molecule resolution
title_full	On-chip protein separation with single-molecule resolution
title_fullStr	On-chip protein separation with single-molecule resolution
title_full_unstemmed	On-chip protein separation with single-molecule resolution
title_sort	on-chip protein separation with single-molecule resolution
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/2572d5e2eeb14f2e86dd3d39972c04ef
work_keys_str_mv	AT adamzrehen onchipproteinseparationwithsinglemoleculeresolution AT shiloohayon onchipproteinseparationwithsinglemoleculeresolution AT dianahuttner onchipproteinseparationwithsinglemoleculeresolution AT amitmeller onchipproteinseparationwithsinglemoleculeresolution
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On-chip protein separation with single-molecule resolution

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