Structure characterization of functional histidine residues and carbethoxylated derivatives in peptides and proteins by mass spectrometry.

We developed a mass spectrometric method to precisely characterize the structures of the diethyl pyrocarbonate (DEP)-modified amino acid derivatives in intact peptides and proteins. Using acetate-buffered solutions for modification reactions improved the yields of DEP modification. UV quantification of carbethoxylation of angiotensin II was consistent with the degree of mass spectrometrically determined modification. Unequivocal identification of the modification sites in carbethoxylated angiotensin II derivatives was achieved by HPLC separation and mass spectrometric sequencing. With increasing concentrations of DEP, a gradual increase of carbethoxy groups, comprising biscarbethoxylation products, was detected in angiotensin II and in insulin. When using a high molar excess of DEP, histidine carbethoxylation was found together with modifications at alpha-amino groups and tyrosine residues. The sites of carbethoxylation in insulin were identified by MALDI-MS-peptide mapping analyses of the tryptic digestion mixtures from the nonreduced insulin derivatives and after reduction of disulfide bonds, demonstrating that histidine carbethoxylation was sufficiently stable during disulfide bond reduction and tryptic digestion at pH 7.5. The mass spectrometric identification of mono- and biscarbethoxylated histidine residues in insulin is in agreement with surface accessibilities of imidazolyl nitrogen atoms and seems to reflect the microenvironment of the protein tertiary structure. Thus, mass spectrometric peptide mapping analyses of carbethoxylated protein derivatives allowed both the simultaneous identification of histidine carbethoxylation in the presence of other modified groups and the detection of different chemical behavior of histidine residues by the unambiguous identification of mono- and bismodifications.