Distinction of N ‐substituted histidines by electrospray ionization mass spectrometry

The amino acid histidine is one of the primary sites of protein conjugation with catecholamines that occurs during the process of insect cuticle sclerotization.1,2 Oxidative conjugation of the model compounds N-acetylhistidine (NAcHis) with N-acetyldopamine (NADA) via an o-quinone intermediate (NADA quinone) has been studied previously and the site of NAcHis attachment at the aromatic ring of NADA has been elucidated by NMR spectroscopy.3 Analysis of products derived from the acid hydrolysis of insect cuticle provided further evidence for the cross-links between catecholamines and histidyl residues of the cuticular proteins.4,5 A more difficult task is to establish the site of catecholamine attachment at the histidine moiety, for which two di†erent nucleophilic groups, Nq and Nn, can react with N-acetyldopaquinone (Scheme 1). Because of the substitution pattern in the imidazole ring, conjugates formed via nucleophilic attack by the non-equivalent imidazole nitrogen atoms (Nq and Nn) are difficult to distinguish by 2D-NMR without the assistance of molecular modeling and with small quantities of adducts obtained from the reaction mixture by reversed-phase liquid chromatography.4 Here, we report a facile distinction of N-1 (Nq) and N-3 (Nn) substituted histidines using electrospray ionization tandem mass spectrometry (ESI-MS/MS). A mechanistic rationale for the distinct dissociations of the gasphase ions is also discussed. As model compounds, we used N-1 (Nq) and N-3 (Nn) methylated histidines 1 and 2 (purchased from Sigma and Aldrich, respectively, and used as received). The compounds were infused in methanol or aqueous methanol solutions containing 0.5% acetic acid or ammonium acetate. ESI-MS of 1 and 2 resulted in efficient protonation to yield ions 1H` and 2H`, respectively, which appeared at m/z 170 (spectra not shown). Collisionally activated dissociations (CAD) of 1H` and 2H` were investigated in a radiofrequency-only quadrupole collision cell of a Sciex API-III triple-quadrupole tandem mass spectrometer (argon as collision gas, 26 eV laboratory collision energy) and in a quadrupole ion trap (Finnigan LCQ, helium at 10~3 Torr (1 Torr \ 133.3 Pa) as bu†er gas). The N-1 methylated isomer 1H` underwent dominant loss of to form a fragment ion at m/z 124. This dissoH2O] CO ciation had a sharp energy threshold ; the m/z 124 peak was weak at low collision energies, e.g., D1% relative to m/z 170 at D0.5 eV, corresponding to 5% relative collision energy (RCE) in the ion trap instrument. However, at 10% RCE (D1 eV), CAD was almost complete and the m/z 124 ion was by far the predominant species in the spectrum [Fig. 1(a)]. Other primary fragments of 1H` were weak, e.g., m/z 153 (loss of ammonia), 152 (loss of water) and 126 (loss of A second2 ary fragment appeared at m/z 109, which was due to sequential elimination of and To deduce the elemental 2 3 composition of the m/z 109 ion, the [13C,15N]-isotopomer of 1H` at m/z 171 was selected and collisionally dissociated. The relative abundances of the m/z 110 and 109 ions (82.9 and 17.1%, respectively, data not shown) were close to those calculated for loss of (83.3 and 16.7%, respectively), 2 ] 3 but di†ered from those calculated for loss of CO] 2 (75.0 and 25.0%, respectively). By comparison, the ] 3 measured relative abundances of the m/z 125 and 124 ions (87.0 and 13.0%, respectively) agreed well with the calculated values for loss of (87.5 and 12.5%, respectively). H2O] CO The CAD spectrum of the N-3 methylated isomer 2H` differed substantially from that of 1H` [Fig. 1(b)]. Ion 2H` showed loss of ammonia (m/z 153), (m/z 126) and COOH 2 (m/z 125) as important primary dissociations. The m/z 126 ion underwent further dissociations by loss of ammonia (m/z 109) and (m/z 97). The di†erences in primary dissociations 2 observed in the CAD spectra thus allowed unequivocal distinction of the methylation site in the imidazole ring, since the N-1 methylated isomer 1H` lost but the N-3 H2O] CO, isomer did not. CAD spectra obtained on the triple-quadrupole tandem mass spectrometer showed more extensive dissociations, including side-chain cleavages (m/z 95È97) and losses of substituents (m/z 81È83, 68) (Fig. 2). However, the characteristic loss of was dominant in the CAD spectrum of H2O] CO 1H` and virtually absent for 2H`. Similar results were obtained for several isomeric Nacetyldopamine-N-acetylhistidine conjugates and deacetylated derivatives, the latter isolated from acid hydrolyzates of insect