Fine-tuning the specificity of boronate affinity monoliths toward glycoproteins through pH manipulation.
Fine-tuning the specificity of boronate affinity monoliths toward glycoproteins through pH manipulation.
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DOI：
10.1039/c2an36048a
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发表时间：
2013-01
期刊：
影响因子：
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通讯作者：
Yue‐dong Lu;Zijun Bie;Yunchun Liu;Zhen Liu
中科院分区：
文献类型：
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作者：
Yue‐dong Lu;Zijun Bie;Yunchun Liu;Zhen Liu
硼酸酯亲和功能材料由于其选择性分离和富集糖蛋白和糖肽的能力，最近引起了越来越多的关注。作为凝集素更便宜、更稳定的竞争对手，人们普遍认为硼酸对糖蛋白具有相对更广泛的特异性。为了更好地理解和有效地利用硼酸盐亲和力，有必要确定硼酸是否对单个或子类糖蛋白表现出凝集素样的窄特异性。在这里，我们报告了一个pH值的操作策略微调的特异性硼酸亲和整料对两个子类的糖蛋白，唾液酸化和非唾液酸化糖蛋白。当结合pH >硼酸的pK（a）一个pH单位或更多时，硼酸盐亲和整料优先结合含有中性糖的糖蛋白，并且由于静电排斥而排除含有唾液酸的糖蛋白。当结合pH <pK（a）一个pH单位或更多时，由于硼酸对唾液酸残基的特殊结合亲和力，硼酸盐亲和整料与唾液酸化糖蛋白结合。首先使用硼酸盐亲和提取与nano-ESI-Orbitrap MS/MS检测的离线组合验证了对唾液酸和中性糖的替代特异性。对唾液酸化和nonsialylated糖蛋白的替代特异性，然后证明通过离线硼酸盐亲和提取与MALDI-TOF MS的组合。最后，开发的方法被施加到替代提取完整的唾液酸化和nonsialylated糖蛋白加标在人血清中。
Boronate affinity functionalized materials have recently drawn increasing attention due to their capability to selectively isolate and enrich glycoproteins and glycopeptides. As cheaper and more stable competitors to lectins, boronic acids are generally believed to yield a relatively wider spectrum specificity to glycoproteins. For better understanding and effective utilization of boronate affinity, it is necessary to establish if boronic acids exhibit lectin-like narrow specificity towards individual or a sub-class of glycoproteins. Here we report a pH manipulation strategy for fine-tuning the specificity of boronate affinity monoliths towards two sub-classes of glycoproteins, sialylated and nonsialylated glycoproteins. When the binding pH > the pK(a) of the boronic acid by one pH unit or more, the boronate affinity monolith preferentially binds to glycoproteins containing neutral sugars and excludes sialic acid containing glycoproteins due to electrostatic repulsion. When the binding pH < the pK(a) by one pH unit or more, the boronate affinity monolith binds to sialylated glycoproteins due to the exceptional binding affinity of the boronic acid towards sialic acid residues. The alternative specificity towards sialic acid and neutral sugar was first verified using an off-line combination of boronate affinity extraction with nano-ESI-Orbitrap MS/MS detection. The alternative specificity towards sialylated and nonsialylated glycoproteins was then demonstrated by means of off-line combination of boronate affinity extraction with MALDI-TOF MS. Finally, the developed approach was applied to the alternative extraction of intact sialylated and nonsialylated glycoproteins spiked in human serum.