毛细管区带电泳（CZE）-UV和瞬时毛细管等速电泳-CZE-电喷雾电离-TOF-MS监测重组艾蒿变应原中的脱酰胺作用和羊毛硫氨酸的形成,Analytical Chemistry

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毛细管区带电泳（CZE）-UV和瞬时毛细管等速电泳-CZE-电喷雾电离-TOF-MS监测重组艾蒿变应原中的脱酰胺作用和羊毛硫氨酸的形成
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-09-04 00:00:00 , DOI: 10.1021/acs.analchem.8b02328
Lorenz G. Stock _{1,

2} , Sabrina Wildner _{1,

2} , Christof Regl _{1,

2} , Gabriele Gadermaier _{1,

2} , Christian G. Huber _{1,

2} , Hanno Stutz _{1,

2}

Affiliation

对热应激的响应是与表征重组蛋白的生物学活性和长期稳定性有关的重要参数，重组蛋白在这些条件下可能显示不可逆的，pH依赖性的结构变化。我们选择艾蒿的重组花粉过敏原（艾蒿）rArt v 3.0201作为相关模型，可通过毛细管区带电泳（CZE）-UV和毛细管区带电泳（CZE）-电喷雾电离（ESI）-TOF-MS研究由于热和pH应力引起的结构变化。因此，该重组蛋白在酸性（pH 3.4）和弱碱性（pH 7.3）条件下暴露于95°C长达120分钟。CZE-UV数据显示变应原连续降解，并伴随着几种反应产物的逐渐形成。使用毛细管内瞬时毛细管等速电泳（tCITP）预浓缩通过CZE-ESI-TOF-MS对在较长迁移时间发生的新型变应原变体进行表征，以帮助鉴定次要变体。MS数据显示了响应加热对rArt v 3.0201的各种修改。在增加的迁移时间发现了具有脱酰胺基和硫相关修饰的变体，包括硫的收率和损失。脱硫产生的变应原变体具有多达四个取代初始二硫键的羊毛硫氨酸。另外，质谱揭示了电荷状态分布的变化，这表明伴随的构象变化。此外，鉴定了几种低丰度的氧化变体。随着热应力的延长，各种变体的产品组合增加，并逐渐向具有高羊毛硫氨酸含量的rArt v 3.0201转移。转化动力学和变体组成的复杂性取决于pH，在碱性条件下会增加。脱硫产生的变应原变体具有多达四个取代初始二硫键的羊毛硫氨酸。另外，质谱揭示了电荷状态分布的变化，这表明伴随的构象变化。此外，鉴定了几种低丰度的氧化变体。随着扩展的热应力，变体的产品组合增加，并逐渐向具有高羊毛硫氨酸含量的rArt v 3.0201转移。转化动力学和变体组成的复杂性取决于pH，在碱性条件下会增加。脱硫产生的变应原变体具有多达四个取代初始二硫键的羊毛硫氨酸。另外，质谱揭示了电荷状态分布的变化，这表明伴随的构象变化。此外，鉴定了几种低丰度的氧化变体。随着热应力的延长，各种变体的产品组合增加，并逐渐向具有高羊毛硫氨酸含量的rArt v 3.0201转移。转化动力学和变体组成的复杂性取决于pH，在碱性条件下会增加。随着热应力的延长，各种变体的产品组合增加，并逐渐向具有高羊毛硫氨酸含量的rArt v 3.0201转移。转化动力学和变体组成的复杂性取决于pH，在碱性条件下会增加。随着热应力的延长，各种变体的产品组合增加，并逐渐转向具有高羊毛硫氨酸含量的rArt v 3.0201。转化动力学和变体组成的复杂性取决于pH，在碱性条件下会增加。

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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

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.

更新日期：2018-09-04

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