Absolute quantification of fluorescent proteins by mass spectrometry

Objectives: Fusions with fluorescent proteins (FPs) are widely used for targeted visualization of proteins in a variety of biological contexts. While microscopy monitors the localization, dynamics and interactions of FP-labelled proteins, their molar abundance remains unknown. Proteins fluorescence is affected by bleaching, slow chromophore maturation or autofluorescence and is sensitive to experimental conditions. To overcome these problems we developed fluorescence-independent method for the absolute (molar) quantification of a variety of FPs using LC-MS/MS and a single protein chimera standard.

Method: We selected and validated quantitypic peptides from the sequences of six popular FPs (mScarlet-1, mKate2, mCherry, EGFP, mNeonGreen and Dendra2) and from two small self-labelling proteins HaloTag and Snap-Tag. The gene encoding for concatenated peptide sequences together with for five peptides from reference protein (BSA) was designed in-silico. Corresponding chimera protein named "qFP8" was expressed in ΔargA ΔlysA auxotrophic strain of E.coli in the media supplemented with 13C6,15N4-L-arginine and 13C6-L-lysine. An aliquot of E.coli lysate containing isotopically labelled chimera standard was either co-digested with the analysed material, or digested separately with known amount of BSA, and then spiked into the sample lysate and analysed by LC-MS/MS. Absolute quantification was performed based on the extracted abundance (FragPipe, SkyLine software) of peptides derived from chimeric standard protein and corresponding FPs (Fig.1). First, chimeric standard was quantified by BSA and then the amount of FP was calculated using corresponding isotopically labelled peptide proxies. The amount of FP-fused protein is equimolar to the FP.

Results: We demonstrated that 30 proxy peptides included into the chimeric protein standard enabled targeted quantification of more than 70 known FPs, their fusions and proteins labelled with organic dyes (e.g. Alexa) via Snap-Tag and HaloTag at the low femtomole to attomole level. Each FP was independently quantified with 3 to 5 peptides with inter-peptide CV < 20%. Several FPs could be quantified simultaneously in a single experiment directly from whole cell or tissue lysate down to single cell level. Since the concentration of chimera is exactly known, it can be used as generic internal standard for absolute quantification of any protein detectable at MS1 spectra.

We employed this method to monitor the expression of FPs and FP-labelled proteins in bacterial and human cells, and in cell-free in-vitro translation-transcription systems. Combination of MS and fluorescence readouts were applied to model expression kinetics of FP-labelled human ID-proteins.