Absolute protein quantification using fluorescence measurements with FPCountR

Abstract

This paper presents a generalisable method for the calibration of fluorescence readings on microplate readers, in order to convert arbitrary fluorescence units into absolute units. FPCountR relies on the generation of bespoke fluorescent protein (FP) calibrants, assays to determine protein concentration and activity, and a corresponding analytical workflow. We systematically characterise the assay protocols for accuracy, sensitivity and simplicity, and describe an ‘ECmax’ assay that outperforms the others and even enables accurate calibration without requiring the purification of FPs. To obtain cellular protein concentrations, we consider methods for the conversion of optical density to either cell counts or alternatively to cell volumes, as well as examining how cells can interfere with protein counting via fluorescence quenching, which we quantify and correct for the first time. Calibration across different instruments, disparate filter sets and mismatched gains is demonstrated to yield equivalent results. It also reveals that mCherry absorption at 600 nm does not confound cell density measurements unless expressed to over 100,000 proteins per cell. FPCountR is presented as pair of open access tools (protocol and R package) to enable the community to use this method, and ultimately to facilitate the quantitative characterisation of synthetic microbial circuits.

Mutational Profiling of SARS-CoV-2 PLpro in human cells reveals requirements for function, structure, and drug escape

“…4a), allowing PLpro abundance to be monitored by flow cytometry. Similar methods have been used to measure protein abundance in other systems (Matreyek et al 2018; Thorn 2017; Csibra and Stan 2022). We expressed this library in parental 293T cells and sorted cells from high and low mClover3 gates, recovered mRNA to count variants, and calculated ratiometric scores to determine the abundance of each variant.…”

Engineering orthogonal ribosomes for real-time monitoring using fluorescence

“…All subsequent experiments were carried out in DH10B cells using our pSEVA361 based reporters. This reporter system has the advantage of having been thoroughly characterised from our earlier work, and is part of a library of fluorescent protein reporters (Csibra & Stan 2022).…”

“…Protein and RNA fluorescence was monitored using green (ex 485/20 nm, em 535/25 nm) and red (ex 560/20 nm, em 610/20 nm) filter sets, with minor variations depending on the instrument used. Plate reader measurements were calibrated for red fluorescence using mCherry lysates, and OD with microspheres, as previously described (Csibra & Stan 2022).…”

Structural Insights into the Binding of Red Fluorescent Protein mCherry-Specific Nanobodies

“…Since the initial extraction and identification of green fluorescence protein (GFP) from jellyfish several decades ago [ 1 ], fluorescence proteins (FPs) have proven invaluable tools for biological research owing to their exceptional properties. Besides GFP, various FPs were widely used across diverse scientific fields, such as bioimaging [ 2 ], cell tracking, protein localization studies [ 3 ], gene expression analysis [ 4 , 5 ], and the detection of protein-protein interactions [ 6 ]. Moreover, in the natural environment, FPs expressed by living organisms can be utilized to gauge the dynamic quantity and activity of fused proteins based on the intensity of fluorescence [ 7 ].…”