DETERMINATION OF DIETHYLPYROCARBONATE-MODIFIED AMINO-ACID-RESIDUES INALPHA(1)-ACID GLYCOPROTEIN BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY AND MATRIX-ASSISTED-LASER-DESORPTION IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRY/

The chemical modification reagent diethylpyrocarbonate (DEPC) was used to modify alpha(1)-acid glycoprotein (orosomucoid, OMD) under various conditions, The extents of DEPC modification of the histidine and tyr osine residues were followed by UV spectrophotometry. The resulting mo dified OMD was analyzed using enzyme digestion, reverse-phase HPLC, el ectrospray ionization-mass spectrometry (ESUMS), and matrix-assisted l aser desorption ionization time-of-flight-mass spectrometry (MALDI-TOF /MS). The inherent problem of instability of DEPC-modified histidine r esidues was overcome by adjusting the time scale of the postreaction p rocessing of modified OMD. There were observed differences in reactivi ty of histidine 97 and histidine 100 that were consistent throughout t he pH range 6-8. Furthermore, several lysine residues were modified an d the amount of modification increased over the pH range 6-8, These ex periments show that HPLC-ESI/MS and MALDI-TOF/MS analysis coupled with enzyme digestion provide the necessary information to describe the re action of DEPC with GRID, In addition, the results provide the carbeth oxy-histidine stability and histidine reactivity information of DEPC-m odified OMD necessary for the design of experiments to characterize th e drug binding properties of OMD. (C) 1998 Academic Press.