Mass spectrometric profiling of glucosamine, glucosamine polymers and their catecholamine adducts - Model reactions and cuticular hydrolysates of Toxorhynchites amboinensis (Culicidae) pupae

Glucosamine (Gln), glucosamine polymers, and their catecholamine adducts we re characterized using positive ion electrospray mass spectrometry (ESMS) a nd tandem mass spectrometry (ESMS-MS). N-acetyldopamine (NADA), a catechola mine found in many insect cuticles, was oxidized using mushroom tyrosinase, and the resulting quinone derivatives were reacted with Gin, (Gln)(3), and polymeric glucosamine (chitosan). Adducts of glucosamine and its trisaccha ride with NADA were readily identified as [M+H](+) ions in ESMS spectra, an d ESMS-MS of selected ions confirmed the condensation of 1-3 NADA residues with Gin. In addition to Gin modification by the quinone derivatives of NAD A, other spectra were consistent with the formation of adducts with N-acety lnoradrenaline and moieties formed by intramolecular cyclization following oxidation. The primary amine of glucosamine was involved in initial adduct formation, but the sites for subsequent additions of oxidized NADA to gluco samine, presumably via hydroxyl groups, could not be identified by ESMS alo ne. The ESMS spectra of chitosan films infused into the spectrometer follow ing solubilization in acidic methanol/water produced spectra similar to tha t of (Gln)(3) up to m/z 502. Ions of gradually decreasing intensity consist ent with (Gln)(x), where x=4-8, were observed. Modification of chitosan fil ms following incubation with NADA plus tyrosinase rendered the films insolu ble in dilute acid, simulating the cross-linking process proposed to occur during insect cuticle sclerotization. Acid hydrolysates of the pupal stage of the mosquito Toxorhynchites amboinensis, using only two pupal exuviae fo r the hydrolyses, were infused into the mass spectrometer without prelimina ry chromatography. Eight amino acids, glucosamine, N-acetylglucosamine, cat echolamines, and a variety of polymers incorporating these compound classes were identified. (C) 1999 Elsevier Science Ltd. All rights reserved.