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Absolute quantification of protein copy number in single cells with immunofluorescence microscopy calibrated using single molecule microarrays

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Title: Absolute quantification of protein copy number in single cells with immunofluorescence microscopy calibrated using single molecule microarrays
Authors: Chatzimichail, S
Supramaniam, P
Salehi-Reyhani, SA
Item Type: Journal Article
Abstract: Great strides toward routine single cell analyses have been made over the last decade, particularly in the field of transcriptomics. For proteomics, amplification is not currently possible and has necessitated the development of ultra-sensitive platforms capable of performing such analyses on single cells. These platforms are improving in terms of throughput and multiplexability but still fall short in relation to more established methods such as fluorescence microscopy. However, microscopy methods rely on fluorescence intensity as a proxy for protein abundance and are not currently capable of reporting this in terms of absolute copy number. Here, a microfluidic implementation of single molecule microarrays for single cell analysis is assessed in its ability to calibrate fluorescence microscopy data. We show that the equivalence of measurements of the steady-state distribution of protein abundance to single molecule microarray data can be exploited to pave the way for absolute quantitation by fluorescence and immunofluorescence microscopy. The methods presented have been developed using GFP but are extendable to other proteins and other biomolecules of interest.
Issue Date: 20-Apr-2021
Date of Acceptance: 8-Apr-2021
URI: http://hdl.handle.net/10044/1/87382
DOI: 10.1021/acs.analchem.0c05177
ISSN: 0003-2700
Publisher: American Chemical Society
Start Page: 6656
End Page: 6664
Journal / Book Title: Analytical Chemistry
Volume: 93
Issue: 17
Copyright Statement: © 2021 American Chemical Society. This document is the Accepted Manuscript version of a Published Work that appeared in final form in Annals of Chemistry, after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.analchem.0c05177
Sponsor/Funder: Engineering and Physical Sciences Research Council
Funder's Grant Number: EP/S001603/3
Keywords: Analytical Chemistry
0301 Analytical Chemistry
0399 Other Chemical Sciences
Publication Status: Published
Online Publication Date: 2021-04-20
Appears in Collections:Department of Surgery and Cancer
Faculty of Medicine