Fluorescence complementation via EF-hand interactions.

Fluorescence complementation technology with fluorescent proteins is a powerful approach to investigate molecular recognition by monitoring fluorescence enhancement when non-fluorescent fragments of fluorescent proteins are fused with target proteins, resulting in a new fluorescent complex. Extension of the technology to calcium-dependent protein-protein interactions has, however, rarely been reported. Here, a linker containing trypsin cleavage sites was grafted onto enhanced green fluorescent protein (EGFP). Under physiological conditions, a modified fluorescent protein, EGFP-T1, was cleaved into two major fragments which continue to interact with each other, exhibiting strong optical and fluorescence signals. The larger fragment, comprised of amino acids 1-172, including the chromophore, retains only weak fluorescence. Strong green fluorescence was observed when plasmid DNA encoding complementary EGFP fragments fused to the EF-hand motifs of calbindin D9k (EF1 and EF2) were co-transfected into HeLa cells, suggesting that chromophore maturation and fluorescence complementation from EGFP fragments can be accomplished intracellularly by reassembly of EF-hand motifs, which have a strong tendency for dimerization. Moreover, an intracellular calcium increase upon addition of a calcium ionophore, ionomycin in living cells, results in an increase of fluorescence signal. This novel application of calcium-dependent fluorescence complementation has the potential to monitor protein-protein interactions triggered by calcium signalling pathways in living cells.

N-terminal derivatization of peptides with isothiocyanate analogues promoting Edman-type cleavage and enhancing sensitivity in electrospray ionization tandem mass spectrometry analysis.

N-terminal derivatization of peptides with Edman's reagent, phenyl isothiocyanate (PITC), promotes gas-phase Edman cleavage that yields abundant complementary b(1) and y(n-1) ion pairs by tandem mass spectrometry (MS/MS). The formation of b(1) ions can be utilized as a mass tag to enhance the interpretation of MS/MS spectra and increase the confidence of peptide identification during mass spectrometry analysis. Derivatization of tryptic peptides with another isothiocyanate analogue, 4-sulfophenyl isothiocyanate, also produces signature ions resulting from Edman cleavage and facilitates peptide sequencing on linear or branched peptides. The limitation of these derivatizations, however, is reduced MS signal intensities of modified peptides, due presumably to the tags themselves. Here we have demonstrated that several other isothiocyanate analogues bearing basic moieties can derivatize peptides and significantly improve the MS sensitivity of tagged analytes, while promoting Edman fragmentation and maintaining other sequence fragments as well.