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 modif ied with fluorescein-5'-isothiocyanate (FITC) to yield the corresponding fl uorescein-thiocarbamoyl (FTC) conjugates (N,N'-disubstituted thiourea and d ithiourethane adducts). The extent of FITC incorporation, i.e., number of m odified residues, has been identified by direct molecular weight determinat ion using matrix-assisted laser desorption-ionization and electrospray-ioni zation mass spectrometry (MALDI-MS; ESI-MS). A specific fragmentation by cl eavage of the FTC moiety from modified residues occurs by nozzle-skimmer di ssociation in ESI mass spectra at increased declustering potential. This fr agmentation 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.