Identification of fluorescein-5'-isothiocyanate-modification sites in proteins by electrospray-ionization mass spectrometry.

Model peptides and proteins, such as hen eggwhite lysozyme, have been modified with fluorescein-5'-isothiocyanate (FITC) to yield the corresponding fluorescein-thiocarbamoyl (FTC) conjugates (N, N'-disubstituted thiourea and dithiourethane adducts). The extent of FITC incorporation, i.e., number of modified residues, has been identified by direct molecular weight determination using matrix-assisted laser desorption-ionization and electrospray-ionization mass spectrometry (MALDI-MS; ESI-MS). A specific fragmentation by cleavage of the FTC moiety from modified residues occurs by nozzle-skimmer dissociation in ESI mass spectra at increased declustering potential. This fragmentation pathway is easily obtained and renders ESI-MS an efficient tool for the characterization of FITC-modified proteins, and identification of modification sites in FTC-peptide mixtures.