聚多巴胺修饰棉花固定金属离子用于磷酸化多肽的富集

Polydopamine-coated Cotton for Ti^(4+)Immobilization for Selective Enrichment and Isolation of Phosphopeptides

本文通过多巴胺自聚合在天然的棉花纤维表面,构建了仿生聚多巴胺(PDA)膜层,然后利用儿茶酚羟基固定Ti^(4+),设计并合成了一种固定金属亲合色谱(Immobilized Metal Ion Affinity Chromatography,IMAC)材料Cotton@PDA-Ti^(4+),并将其用于磷酸化多肽的富集。该材料机械性能好,化学性能稳定和生物相容性好,且制备过程简单,通过简易的In-pipet-tip固相萃取(SPE)装置使整个富集操作过程简便快速。实验结果表明,Cotton@PDA-Ti^(4+)不仅可以从简单的蛋白酶解物(β-casein)中富集磷酸化多肽,并且在含有大量非磷酸化多肽的复杂体系样品中对磷酸化多肽也表现出良好的选择性。另外,利用Cotton@PDA-Ti^(4+)对磷酸化多肽进行富集也有较高的效率。我们将该材料应用于实际样品,如人体血清以及脱脂牛奶酶解物中磷酸化多肽的富集,均表现出了较好的选择性。说明该方法有可能用于磷酸化蛋白质组的全分析。

In this work, we first designed and synthesized an IMAC material with Ti4＋ immobilized on polydopamine-coated cotton and applied it to phosphopeptides enrichment successfully. The prepared material Cotton@PDA-Ti4＋, exhibited excellent fiber strength,good stability in aqueous or nonaqueous solutions,and great biocompatibility. The material could also be obtained by a simple synthesis method. To shorten the extraction time and simplify the operation,in-pipet-tip SPE format was employed in this study. Our results showed that Cotton@PDA-Ti4＋ adsorbent exhibited high selectivity in the enrichment of phosphopeptides from simple digestions and demonstrated low limit of detection （10 fmol）. Furthermore,Cotton@PDA-Ti4＋ adsorbent was successfully applied for enrichment of phosphopeptides from real biological samples such as human serum and the tryptic digest of nonfat milk. Generally, the preparation strategy will find broad application in large-scale phosphoproteomics analysis.