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.
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Fakultät für Chemie, Universität Konstanz, 78457 Konstanz, Germany.
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D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D004527:Egg Proteins / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin / Q000737:chemistry
D004527:Egg Proteins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D010455:Peptides; D009113:Muramidase; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate