基于SH2超亲体的酪氨酸磷酸化肽段富集新方法研究

Protein tyrosine phosphorylation controls many aspects of signaling in multicellular organisms. pTyr peptides, which are estimated to account for <1% of the total phosphopeptides in a cell, are typically enriched using antibodies. However, at a scale affordable to most academic labs, the antibody-based AP (affinity purification)-MS/MS approach tends to preferentially identify the most abundant pTyr peptides, overlooking numerous less abundant, but physiologically relevant pTyr peptides. The inadequacy of current AP-MS/MS approach for pTyr identification calls for innovative strategies for pTyr enrichment. We have recently found that an SH2 domain-derived pTyr-superbinder is a promising affinity reagent for the enrichment of tyrosine phosphopeptides. When using at an optimal amount, the superbinder compared favorably to conventional anti-phosphotyrosine antibodies. But the present experimental workflow for the enrichment of pTyr peptides by superbinder is tedious and time consuming with relatively lager sample loss, limited its application in tissue sample or unstimulated cancer cell sample where pTyr proteins are of much lower abundance. To overcome this circumstance, this project is designed to develop improved technology and strategy for the enrichment of pTyr peptides utilizing the SH2 domain-derived pTyr-superbinder. This would include the immobilization of the superbinder to CNBr activated sepharose beads without losing its enzyme activity, the fractionation of superbinder enriched pTyr peptides via different binding affinity, the combined use of pTyr-superbinders with complementary specificity (such as the Src and Grb2 superbinder). All these technologies will be combined together for the systematic and sensitive identification of pTyr sites in tissue sample or unstimulated cancer cells.

在酪氨酸激酶和磷酸酶作用下的酪氨酸可逆磷酸化在一系列信号转导通路中发挥着重要的作用。当前酪氨酸磷酸化蛋白质组学研究的覆盖度和灵敏度不足，制约其下游的生物学研究的开展，亟需开发相关新技术、新方法。目前，酪氨酸磷酸化抗体是唯一有效的磷酸化酪氨酸蛋白质富集手段。但抗体价格昂贵，不利于在实验室中大量使用。最近我们发现氨基酸突变的SH2模体-即SH2超亲体可以应用于酪氨酸磷酸化蛋白质或肽段的富集，其富集能力达到或优于常规商品化抗体。本项目拟进一步发展基于SH2超亲体的酪氨酸磷酸化肽段富集新方法研究。通过将SH2超亲体进行活性共价固载，克服实验流程繁琐，与质谱兼容性不佳的缺点。通过发展创新的分级策略以及混合使用特异性互补的SH2超亲体，提高酪氨酸磷酸化蛋白质组的分析覆盖率。与抗体相比，SH2超亲体可在实验室中大量表达纯化，成本低廉，本项目的完成将有望使该方法在酪氨酸磷酸化的研究方面获得广泛应用。

尽管仅占所有蛋白质磷酸化水平的1‰，酪氨酸磷酸化蛋白质在细胞信号转导方面具有重要的调控功能。使用抗体进行酪氨酸蛋白质或肽段的富集存在价格昂贵、特异性低、重复性差等一系列缺点。本项目发展了基于廉价高效的SH2超亲体的酪氨酸磷酸化肽段富集新方法与新技术，以提高酪氨酸磷酸化蛋白质组的覆盖度和灵敏度。首先，为简化实验流程并降低样品损失，将共价固载的SH2超亲体与Ti4+-IMAC 联用，使酪氨酸磷酸化肽段的鉴定数目与之前方法相比提高了41%，富集特异性达到90.1%。将该策略与稳定同位素二甲基标记方法联用，应用于EGF刺激的HeLa细胞样品中的酪氨酸磷酸化分析，实现了对262个高可信酪氨酸磷酸化位点的定量分析。针对基于SH2超亲体的酪氨酸磷酸化富集策略中往往需要消耗大量的样品起始量，不容易实现微量样品分析的问题，我们将SH2超亲体修饰到毛细管整体柱的基质上制备得到SH2微反应器。由于结合了毛细管整体柱的高灵敏度和SH2超亲体对酪氨酸磷酸化高亲和性的特点，该SH2微反应器可以大大提高微量样品中酪氨酸磷酸化分析的灵敏度，将其用于过钒酸钠刺激过的HeLa细胞样品分析时，从仅仅100 μg样品中就能鉴定到796个高可信度的酪氨酸磷酸化位点。针对缺乏SH2结构域与修饰底物间特异性相互作用的高通量研究方法的问题，建立了一种基于SH2超亲体和高选择性竞争洗脱的酪氨酸磷酸化蛋白质组学的分析方法。该方法以与SH2超亲体间有强亲和作用力的肽段pYEEI为基础设计竞争性试剂，将结合在SH2超亲体上的酪氨酸磷酸化肽段根据亲和力大小依次洗脱下来。该方法在获得酪氨酸磷酸化蛋白质组深入覆盖信息的同时，还可得到磷酸化酪氨酸残基周围分布的氨基酸序列与结构域之间亲和力的关联信息。综上所述，我们发展的基于SH2超亲体的酪氨酸磷酸化肽段富集策略可以为酪氨酸磷酸化依赖的信号网络的机制研究提供强有力的技术支持。