Proteins fused with fluorescent proteins (FP) are widely used in quantitative biology, biophysics and live cell imaging. However, their fluorescence-based quantification is not straightforward. It is affected by multiple factors: background interference, pH, bleaching, degradation of fused proteins, chromophore maturation kinetics or imaging settings. We have developed a simple and generic LC-MS/MS based method for accurate absolute (molar) quantification of FPs and FP-fused proteins which overcomes these limitations.
The method relies upon a single synthetic protein standard comprising quantotypic peptides from most frequently used FPs e.g. mEGFP, mCherry, mScarlet, Dendra2, mKate2, mNeonGreen, as well as Halo and Snap tags. It enables absolute quantification of more than 70 members from these six FP-families, any of their fusions and proteins labelled with organic chromophores via linkers. Similarly to MS Western approach, an aliquot of metabolically labelled chimera standard is co-digested with a cell lysate and target FP-fused protein is quantified by LC-MS/MS.
The method works for proteins separated by SDS PAGE and for crude protein extracts reaching the attomole to low femtomole sensitivity. Several FPs can be quantified in parallel to determine their molar concentration, expression level and stoichiometric ratio to other (not necessarily FP-fused) proteins.
Archishman Ghosh (Tang group, MPI CBG)
Aliona Bogdanova, Eric Geerstma (PEPC Facility, MPI CBG)
Thomas Wiedemann (MPI for Biochemistry, Martinsried)