Monitoring of Deamidation and Lanthionine Formation in Recombinant Mugwort Allergen by Capillary Zone Electrophoresis (CZE)-UV and Transient Capillary Isotachophoresis-CZE-Electrospray Ionization-TOF-MS.

ABSTRACT

The response to thermal stress is an important parameter relevant for characterizing the biological activity and long-term stability of recombinant proteins, which may show irreversible, pH dependent structural changes under these conditions. We selected the recombinant pollen allergen of mugwort ( Artemisia vulgaris) rArt v 3.0201 as a relevant model to study structural changes due to thermal and pH stress by means of capillary zone electrophoresis (CZE)-UV and capillary zone electrophoresis (CZE)-electrospray ionization (ESI)-TOF-MS. Therefore, this recombinant protein was exposed to 95 °C under acidic (pH 3.4) and slightly alkaline (pH 7.3) conditions for up to 120 min. CZE-UV data showed a continuous degradation of the allergen accompanied by the gradual formation of several reaction products. Characterization of novel allergen variants occurring at longer migration times was done via CZE-ESI-TOF-MS using in-capillary transient capillary isotachophoresis (tCITP) preconcentration to facilitate the identification of minor variants. MS data revealed various modifications of rArt v 3.0201 in response to heating. Variants with deamidations and sulfur-related modifications including both yield and loss of sulfur were identified at increased migration times. Desulfurization produced allergen variants with up to four lanthionines that replaced initial disulfide bonds. In addition, mass spectra revealed shifts in the charge state distribution which indicate concomitant conformational alterations. Moreover, several low-abundant oxidized variants were identified. With extended thermal stress, the portfolio of variants increased and progressively shifted toward rArt v 3.0201 with high lanthionine content. The kinetics of conversion and the complexity of variant composition were pH dependent and increased under alkaline conditions.