High desolvation temperature facilitates the ESI-source H/D exchange at non-labile sites of hydroxybenzoic acids and aromatic amino acids

Abstract

Hydrogen/deuterium exchange coupled with high-resolution mass spectrometry has become a powerful analytical approach for structural investigations of complex organic matrices. Here we report the feasibility of the site-specific H/D exchange of non-labile hydrogens directly in the electrospray ionization (ESI) source, which was facilitated by an increase in the desolvation temperature from 200 °C up to 400 °C. We have found that the exchanges at non-labile sites were observed only for the model compounds capable of keto-enol tautomeric transformations (e.g., 2,3-, 2,4-dihydroxybenzoic acids, gallic acid, DOPA), and only when water was used as a solvent. We hypothesized that the detected additional exchanges were induced by the presence of hydroxyls in the sprayed water droplets generated in the negative ESI mode. It was indicative of the exchange reactions taking place in the sprayed droplets rather than in the gas phase. To support this hypothesis, the H/D exchange experiments were run in deuterated water under base-catalyzed conditions for three model compounds, which showed the most intensive exchanges in the MS experiments: DOPA, 2,4-DHB, and 5-acetylsalicylic acid. (2)H NMR spectroscopy has confirmed keto-enolic transformations of the model compounds leading to the specific labeling of the corresponding non-labile sites. We believe that the proposed technique will be useful for structural investigations of natural complex mixtures (e.g. proteins, humic substances) using site-specific H/D exchange.

Hydrogen/deuterium exchange in mass spectrometry

“…The influence of capillary temperature on ESI HDX in case of proteins and peptides was investigated. High desolvation temperatures may lead to exchange of non‐labile protons during in‐ESI source HDX of benzoic acid derivatives and aromatic amino acids . The influence of capillary and source temperatures were also investigated on APCI/APPI HDX for nitrogen, oxygen, and sulfur compounds .…”

“…The fundamental question of in‐ESI source H/D exchange where on the way from ESI emitter to mass spectrometer reactions occur. In previously discussed work gas‐phase exchange was considered, which is almost independent on solvent and depends on deuterated agent . However, exchange can take place in charged microdroplets in solution‐like phase, in this case depends on substrate, solvent composition, pH and temperature.…”

“…However, exchange can take place in charged microdroplets in solution‐like phase, in this case depends on substrate, solvent composition, pH and temperature. In attempts to ascertain whether H/D occurs in gas or solution phase authors conduct in‐ESI source HDX on model set of hydroxybenzoic acids and aromatic amino acids with variation of solvent and desolvation capillary temperature. It was found that by using methanol as ESI solvent it is possible to enable exchange of only labile protons in D 2 O vapor.…”

“…The distinct dependence on the solvent composition indicates the solution‐like reaction rather than gas‐phase. Generalization of data allowed author to suggest the new mechanism of HDX in charged microdroplets based on keto‐enol tautomerism, represented in Figure …”

CID fragmentation, H/D exchange and supermetallization of Barnase-Barstar complex

“…Recently it was suggested to perform the H/D exchange reaction directly in the ESI source 28, 29 . This approach was applied to study proteins 30–32 , oligosaccharides 33 , natural complex mixtures 34–37 and small organic molecules 38 .…”