A systematic assessment of structural heterogeneity and IgG/IgE-binding of ovalbumin
A systematic assessment of structural heterogeneity and IgG/IgE-binding of ovalbumin
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DOI：
10.1039/d0fo02980g
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发表时间：
2021-07-13
期刊：
影响因子：
6.1
通讯作者：
Kaltashov, Igor A.
中科院分区：
文献类型：
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作者：
Yang, Wenhua;Tu, Zongcai;Kaltashov, Igor A.
卵白蛋白（OVA）是鸡蛋中主要的过敏原之一，由于一系列翻译后修饰（PTMs）而表现出广泛的结构异质性。然而，分析天然OVA的结构异质性具有挑战性，异质性与OVA的IgG/ ige结合之间的关系尚不清楚。本研究采用盐梯度离子交换色谱法（IXC）和电喷雾质谱在线检测（ESI MS）对OVA的结构异质性进行了评价，同时采用抑制- elisa法对OVA的IgG/IgE结合特性进行了评价。鉴定了130多种不同的OVA蛋白形态（包括无聚糖种和32对等压种）。乙酰化、磷酸化、氧化和琥珀酰亚胺修饰的蛋白质形式降低了IgG/IgE的结合能力，而结构修饰较少的蛋白质形式具有较高的IgG/IgE结合能力。含唾液酸聚糖修饰的OVA亚型具有最高的IgG/IgE结合能力。结果表明，盐梯度洗脱的在线原生IXC/MS可用于快速评估蛋白质的结构异质性。进一步了解IgG/IgE结合能力与卵细胞结构之间的关系，为开发降低蛋白质致敏性的生物技术或食品加工方法提供了基础。
Ovalbumin (OVA), one of the major allergens in hen egg, exhibits extensive structural heterogeneity due to a range of post-translational modifications (PTMs). However, analyzing the structural heterogeneity of native OVA is challenging, and the relationship between heterogeneity and IgG/IgE-binding of OVA remains unclear. In this work, ion exchange chromatography (IXC) with salt gradient elution and on-line detection by native electrospray ionization mass spectrometry (ESI MS) was used to assess the structural heterogeneity of OVA, while inhibition-ELISA was used to assess the IgG/IgE binding characteristics of OVA. Over 130 different OVA proteoforms (including glycan-free species and 32 pairs of isobaric species) were identified. Proteoforms with acetylation, phosphorylation, oxidation and succinimide modifications had reduced IgG/IgE binding capacities, whereas those with few structural modifications had higher IgG/IgE binding capacities. OVA isoforms with a sialic acid-containing glycan modification had the highest IgG/IgE binding capacity. Our results demonstrate that on-line native IXC/MS with salt gradient elution can be used for rapid assessment of the structural heterogeneity of proteins. An improved understanding of the relationship between IgG/IgE binding capacity and OVA structure provides a basis for developing biotechnology or food processing methods for reducing protein allergenicity reduction.