IDENTIFYING SITES OF PROTEIN MODIFICATION BY USING MATRIX-ASSISTED LASER-DESORPTION IONIZATION-TIME-OF-FLIGHT MASS-SPECTROMETRY AND ONLINE-MEMBRANE-PRECONCENTRATION CAPILLARY-ELECTROPHORESIS TANDEM-MASS-SPECTROMETRY

A strategy for rapidly identifying the number and sites of chemical or posttranslational modification of proteins is described. The use of m atrix-assisted laser desorption/ionization-time-of-flight-mass spectro metry to determine the molecular weight of the adducted protein as wel l as map the proteolytic digest of peptides offers a rapid method to s creen for the possible site of adduction. To unequivocally determine t he amino acid sequence of the peptide bearing the adduct as well as st ructurally characteize the covalent modification, the peptide mixture is subjected to membrane preconcentration-capillary electrophoresis-ma ss spectrometry and tandem mass spectrometry (mPC-CE-MS/MS). The high resolving separation capability of capillary electrophoresis (CE) affo rd a chromatograhic step that lends itself to separation of complex mi xtures of peptides with minimal sample loss. The membrane preconcentra tion-CE cartridge allows sample loading volumes 10,000-fold greater th an conventional CE. In this work the binding site of the fluorescent l abel acrylodan to the intestinal fatty binding protein is characterize d and shown to be covalently bound at lysine-27, by using mPC-CE-MS/MS . (C) 1997 American Society for Mass Spectrometry