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bioRxiv · 10.1101/2025.09.19.677389

Absolute Quantification of Fluorescent Protein Fusions by Proteomics

Shevchenko, A.·Ghosh, A.·Schuhmann, A.·Bogdanova, A.·Thomas, H.·Rao, V.·Geertsma, E. R.·Tang, T.-Y. D.
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Abstract

Fusions with fluorescence proteins (FPs) play a pivotal role in experimental biology because of their sensitive and spatially precise visualization by spectroscopy. However, observed fluorescence is not always proportional to their molar abundance. Only a fraction of the fusion protein containing the mature fluorescence chromophore is detectable by spectroscopy and there is no generic method for estimating its molar abundance. We developed a fluorescence-independent mass-spectrometry based approach for accurate absolute sub-femtomole quantification of FP-fusions that also estimates the protein faction having fully matured chromophore. The method exploits an isotopically labelled 68 kDa recombinant protein standard containing peptide proxies for 6 prototypical FPs (mCherry, mScarlet-I, mKate2, EGFP, mNeonGreen and Dendra2) and two self-labelling (Halo- and SNAP-) tags. It enables quantification of proteins fused to any of more than 70 FPs or self-labelling tags. It is versatile, robust, precise and can be used broadly for the absolute quantification of fluorescent fusions in in-vivo and in-vitro cellular systems. As case study we combined mass spectrometry with fluorescence spectroscopy to study expression kinetics of FP fusions in cell-free systems. Molar concentrations of the expressed fusion protein, its fraction containing the mature fluorescent chromophore and of the fluorescent protein were integrated into a mathematical model to obtain kinetic rates of translation, chromophore maturation and folding.

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Shevchenko, A., Ghosh, A., Schuhmann, A., Bogdanova, A., Thomas, H., Rao, V., Geertsma, E. R., Tang, T.-Y. D.. 2025-09-21. Absolute Quantification of Fluorescent Protein Fusions by Proteomics. https://doi.org/10.1101/2025.09.19.677389

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