Absolute quantification of fluorescent protein fusions by mass spectrometry

Fusions with fluorescent 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 concentration. Only a fraction of the fusion protein that contains the mature fluorescence chromophore is detectable by spectroscopy and there is no accurate and generic method for estimating its molar abundance. We have developed a fluorescence-independent mass spectrometry-based method for absolute (molar) sub-femtomole quantification of FP-fusions that also estimates the fraction of fully matured chromophore. The method exploits an isotopically labeled 68 kDa recombinant protein standard expressed in E. coli and used without further purification. This chimeric protein contains multiple peptide proxies for six prototypical FPs (mCherry, mScarlet-I, mKate2, EGFP, mNeonGreen, and Dendra2) and two self-labelling (Halo- and SNAP-) tags and supports the quantification of proteins fused to any of 615 common FPs and tags. The method can be used broadly for the absolute quantification of fluorescent fusions in vivo and in vitro and is complementary to fluorescence measurements. We further combined mass spectrometry with fluorescence spectroscopy to study expression kinetics of FP fusions in cell-free systems. Molar concentrations of the expressed fusion, its fraction with mature chromophore, and of the fluorescing protein were integrated into a mathematical model to obtain kinetic rates of translation, chromophore maturation, and folding.