Absolute quantification of protein copy number in single cells with immunofluorescence microscopy calibrated using single molecule microarrays
File(s)
Accepted version
Author(s)
Chatzimichail, Stelios
Supramaniam, pashiini
Salehi-Reyhani, Sayed Ali
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.
Date Issued
2021-04-20
Date Acceptance
2021-04-08
Citation
Analytical Chemistry, 2021, 93 (17), pp.6656-6664
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
Engineering and Physical Sciences Research Council
Identifier
https://pubs.acs.org/doi/10.1021/acs.analchem.0c05177
Grant Number
EP/S001603/3
Subjects
Analytical Chemistry
0301 Analytical Chemistry
0399 Other Chemical Sciences
Publication Status
Published
Date Publish Online
2021-04-20