Determination of diethylpyrocarbonate-modified amino acid residues in alpha 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 tyrosine residues were followed by UV spectrophotometry. The resulting modified OMD was analyzed using enzyme digestion, reverse-phase HPLC, electrospray ionization-mass spectrometry (ESI/MS), and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry (MALDI-TOF/MS). The inherent problem of instability of DEPC-modified histidine residues was overcome by adjusting the time scale of the postreaction processing of modified OMD. There were observed differences in reactivity of histidine 97 and histidine 100 that were consistent throughout the pH range 6-8. Furthermore, several lysine residues were modified and the amount of modification increased over the pH range 6-8. These experiments show that HPLC-ESI/MS and MALDI-TOF/MS analysis coupled with enzyme digestion provide the necessary information to describe the reaction of DEPC with OMD. In addition, the results provide the carbethoxy-histidine stability and histidine reactivity information of DEPC-modified OMD necessary for the design of experiments to characterize the drug binding properties of OMD.
Dage
J L
JL
University of Cincinnati, Ohio 45221, USA.
Sun
H
H
Halsall
H B
HB
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D009961:Orosomucoid / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
C112130:OMD-GP1 orosomucid; D009961:Orosomucoid; D014443:Tyrosine; D006639:Histidine; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D014357:Trypsin; D008239:Lysine; D004047:Diethyl Pyrocarbonate