Mass spectrometric analysis of catechol-histidine adducts from insect cuticle

Adducts of catechols and histidine, which are produced by reactions of 1,2- quinones and p-quinone methides with histidyl residues in proteins incorpor ated into the insect exoskeleton, were characterized using electrospray ion ization mass spectrometry (ESMS), tandem electrospray mass spectrometry (ES MS-MS, collision-induced dissociation), and ion trap mass spectrometry (ITM S), Compounds examined included adducts obtained from acid hydrolysates of Manduca sexta (tobacco hornworm) pupal cuticle exuviae and products obtaine d from model reactions under defined conditions. The ESMS and ITMS spectra of 6-(N-3')-histidyldopamine [6-(N-3')-His-DA, pi isomer] isolated from M. sexta cuticle were dominated by a [M + H](+) ion at m/z 308, rather than th e expected m/z 307. High-resolution fast atom bombardment MS yielded an emp irical formula of C14H18N3O5, which was consistent with this compound being 6-(N-1')-histidyl-2-(3,4-dihydroxyphenyl)ethanol [6-(N-1')-His-DOPET] inst ead of a DA adduct. Similar results were obtained when histidyl-catechol co mpounds linked at C-7 of the catechol were examined; the (N-1') isomer was confirmed as a DA adduct, and the (N-3') isomer identified as an (N-1')-DOP ET derivative. Direct MS analysis of unfractionated cuticle hydrolysate rev ealed intense parent and product ions characteristic of 6- and 7-linked add ucts of histidine and DOPET, Mass spectrometric analysis of model adducts s ynthesized by electrochemical oxidative coupling of N-acetyldopamine (NADA) quinone and N-acetylhistidine (NAcH) identified the point of attachment in the two isomers. A prominent product ion corresponding to loss of CO2 from [M + H](+) of 2-NAcH-NADA confirmed this as being the (N-3') isomer. Loss of (H2O + CO) from 6-NAcH-NADA suggested that this adduct was the (N-1') is omer. The results support the hypothesis that insect cuticle sclerotization involves the formation of C-N cross-links between histidine residues in cu ticular proteins, and both ring and side-chain carbons of three catechols: NADA, N-beta-alanyldopamine, and DOPET. (C) 1999 Academic Press.