基于PMMA修饰亲水聚多巴胺包覆磁性石墨烯材料的低丰度蛋白/肽段的MALDI-TOF-MS分析新方法

Here we study out a sandwich structured PMMA（Poly (methyl methacrylate)）functionalized magnetic graphene materials via polydopamine coating, and expect to enrich low-abundance Proteins/peptides with them for MALDI-TOF-MS analysis. The materials would combine the merit of PMMA, polydopamine, grapheme and Fe3O4, and could enrich low-concentration Proteins/peptides, effectively, rapidly, and conveniently. Graphene would first combine with Fe3O4 spheres via a simple hydrothermal reaction. Then the hydrophilic polydopamine layer coated magnetic grapheme would be synthesized by self-polymerization reaction. After that, a polymerizable silane coupling agent, 3-methacryloxypropyltrimethoxysilane (MPS), would be modified on both layers of polydopamine to form the intermediate. An aqueous phase radical polymerization of methyl methacrylate (MMA) would added in the mixture to obtain the ultimate product PMMA functionalized polydopamine coated magnetic graphene materials. Thanks to the polydopamine coating and graphene frame, the materials would possess favorable environmental stability, biocompatibility, dispersibility and super surface in water. The PMMA polymer on the surface of the microspheres have linear hydrophobic chains, which would be used as hydrophobic probes toward Proteins/peptides and the co-concentration of salts could be avoided at the same time. The enrichment process would be fast without reiterative centrifugation because of the Fe3O4 particles in the materials. The resulting peptides loaded on PMMA functionalized magnetic grapheme materials could be directly analyzed by MALDI-TOF-MS without elution. This work can be extended to design core-shell polydopamine coated magnetic graphene with other polymers as shell for bioseparation applications.

本研究拟将磁性分离、PMMA富集和聚多巴胺包覆磁性石墨烯辅助分析等优势结合，发展一种基于PMMA修饰的亲水聚多巴胺包覆磁性石墨烯材料的低丰度蛋白/肽段的MALDI-TOF-MS分析新方法。本研究将首先通过水热合成法在石墨烯表面生成具有超顺磁性的Fe3O4粒子，得到磁性石墨烯；之后采用自聚合反应在石墨烯表面包覆一层聚多巴胺，然后在材料的聚多巴胺层外进行PMMA等修饰；将PMMA修饰的聚多巴胺包覆磁性石墨烯用于富集生物样品中的低丰度肽段/蛋白，同时达到脱盐目的；利用磁铁分离方法将富集了蛋白/肽段的材料从样品中分离出；最后，吸附了蛋白/肽段的材料不需溶剂洗脱，可直接用MALDI-TOF-MS进行高通量、高灵敏的质谱分析，亦可用溶剂洗脱后经LC-MS分析。本研究中拟修饰的外层功能分子PMMA可拓展到其它的疏水链，如C8烷基、C18烷基、油酸基团以及交联聚苯乙烯、醋酸乙烯酯共聚物等。

在国家自然科学基金的支持下，我们开展了基于PMMA功能化的磁性石墨烯材料的蛋白组学低丰度富集新技术新方法的研究。PMMA功能化的磁性石墨烯材料被设计及合成，并通过疏水相互作用被应用于低丰度蛋白/肽段富集。用PMMA功能化的磁性石墨烯材料富集低丰度标准肽段水溶液，质谱检测信噪比能提高约100倍；材料对MYO酶解液富集肽段覆盖率能达到90%，回收率达到98%，对BSA蛋白酶解液也具有很好的富集效果；材料能用于从人尿样和人血清样品中直接富集内源性的肽段。.此外，我们还开展了基于沸石咪唑酯框架结构的磁性材料低丰度富集研究。通过室温下的快速、温和的溶胶凝胶反应合成了磁性沸石咪唑酯框架结构材料。由于Zn2+与组氨酸的低配位、多孔结构、高比表面积和孔内疏水性表面，合成的纳米复合材料在对BSA和HAS酶解肽段的富集上表现优异。在BSA酶解肽段与BSA蛋白比例达到1：200（摩尔比）时，仍具有很好的排阻BSA蛋白的效果，为简化复杂生物样品和富含组氨酸肽段的选择性富集提供了新视角。我们还开展了基于两性离子亲水磁性介孔材料的内源性糖肽的富集研究。合成了兼具纳米磁球、介孔二氧化硅层和两性亲水半胱氨酸的复合材料，材料能排阻大分子蛋白质，并同时在孔道内吸附内源性糖肽，从而实现对糖肽的高效富集。.总的来说，在国家自然科学基金的支持下，本课题在蛋白组学分析样品预处理方面做了较多研究工作。我们已在国际核心期刊上发表SCI论文1篇（Talanta 167 (2017) 392–397），另有两篇研究论文已成稿，待发表。另，我们撰写了一部学术专著《磁性微纳米材料在蛋白质组学中的应用》，作者：邓春晖、陈和美，由复旦大学出版社于2017年12月出版发行。.

内容获取失败，请点击重试