Mass spectrometry-based structural dissection of fluorescent proteins.

Fluorescent proteins (FPs) are essential for live cell studies using fluorescence microscopy. To date, the molecular basis for FPs' irreversible photobleaching and the nature of the associated photoproducts are a matter of debate. Mass spectrometry, which should be an ideal technique for the structural dissection of FPs, cannot be harnessed efficiently due to their extreme resistance to trypsinolysis, due to the compactness of the barrel structure containing the chromopeptide. We devised a mild endoproteolysis procedure that affords a peptide mass fingerprint almost totally covering the sequence, thus allowing high-resolution mass spectrometric investigations of the protein structure.