基于混合模板印迹策略的磷酸化肽分子印迹材料的制备及其在免疫分析中的应用

Phosphoprotein is one of the most important biomolecules in the human physiological process, the detailed analysis of the phosphorylated proteins can help us establish a fundamental understanding of biological processes and signaling networks at the molecular level. Nowadays, phosphoproteome analysis is predominantly performed by MS. The complexity and low stoichiometry of protein phosphorylation pose a serious challenge for phosphoproteome analysis. Moreover, MS often fails to identify phosphopeptides in the complex peptide mixtures because of the severe signal suppression effect of the non-phosphopeptides. Therefore, it has become a crucial step to specifically isolate subsets of phosphopeptides from a complex peptide mixture. To date, phosphopeptides can be enriched by a number of approaches, such as immunoprecipitation, ion exchange chromatography and chemical coupling. However, there are still some drawbacks in these approaches: the immunoprecipitation is only applicable to phosphotyrosine, the ion exchange chromatography is time-consuming and the chemical coupling often exhibits sequence bias for peptides containing aspartic and glutamic. Thus, the development of novel strategy for phosphopeptides enrichment is still of great importance. This project aims at developing a multitempaltes controllable surface oriented imprinting approach by virtue of the phosphopeptides as the templates. The obtained material could recognize not only the intact target but also its characteristic fragments. On the basis of these approaches, molecularly imprinted material based enrichment will be developed and their combination with biological mass spectrometry will also be developed for phosphoprotein biomarkers-based early screening of cancer diseases. The project can not only provide advanced solution for the detection of low-abundance and unstable disease-related proteins but also facilitate the applications of molecular imprinting technology in real world such as immunoassay and phosphoproteome analysis.

磷酸化蛋白是一类人体生理过程中极其重要的生物分子，蛋白质的磷酸化在细胞生命活动中起着重要的调控作用。在磷酸化蛋白组学研究中,磷酸化肽的质谱鉴定面临着组分复杂、相对低丰度以及来自非磷酸化肽的质谱信号干扰等各种困难。因此需要发展磷酸化肽的选择性富集策略以满足磷酸化蛋白组学研究中的需求。虽然目前已有多种磷酸化肽的选择性富集方法，但仍然还有各自的局限性。有鉴于此，本项目拟提出混合模板-可控定向表面印迹技术，以混合磷酸化肽为模板分子，制备得到可以识别靶标蛋白和其特征片段的全新仿生识别材料，建立基于分子印迹材料的新型免疫富集法，并探索与生物质谱联用方法的建立。本项目的研究可望为复杂生物样品中低丰度和不稳定的疾病相关蛋白检测提供新策略，促进分子印迹材料在免疫分析、蛋白质组学等方面的实际应用。

本项目针对磷酸化蛋白质组学分析中的关键科学问题-磷酸化肽的选择性富集，围绕着具有特异性识别能力亲和材料的设计与合成，开展了系列研究，包括模板分子磷酸化肽段的选择性富集，可控印迹过程的条件优化与拓展，原位酶解-混合模板印迹策略的开发以及亲和材料-生物质谱联用平台的建立。合成了针对不同模板分子的分子印迹材料，并实现了复杂样品中的目标分子的选择性富集与高灵敏质谱检测。合成的单分散单层的介孔二氧化钛纳米片，可以实现复杂样品中磷酸化肽的选择性富集，为磷酸化蛋白分子印迹材料制备过程中的印迹模板分子的获取提供了新方法。同时，合成的磷酸化肽分子印迹材料可以实现复杂样本中疾病相关磷酸化蛋白（Tau蛋白）及其酶解产物的选择性分析。我们进一步拓展在此基础上发展出的可控印迹体系的应用范围，实现了小分子（沙丁胺醇）和大分子（干扰素α）的亲和材料制备。此外，我们还结合了金属有机框架化合物的独特性质，通过合成的硼亲和亲属有机框架化合物实现了亲和富集与质谱检测的一体化，极大的提高方法分析性能并简化了实验步骤。本项目目前已发表SCI论文6篇，另有2篇论文在整理中。