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。