用于碱性氨基酸磷酸化修饰的蛋白质组学新方法研究

Phosphorylation of protein basic amino acids residues, meaning phosphohistidine, phosphoarginine and phospholysine, are critical processes involved in the regulation of many important cellular processes, such as signal transduction pathways, protein degradation, stress response and metabolic pathways. However, its role and abundance especially in mammalian cells remain largely unknown as only limited advances have been made in detection of protein basic amino acids phosphorylation and elucidation of its biological function in the past decades. Furthermore, identification of basic amino acids phosphopeptides from complex mixtures is hindered by several fundamental issues, like low stoichiometry and relative instability of phosphoramidate bond leading to acid-catalyzed hydrolysis under standard liquid chromatography conditions. Hence, although excellent methods exist to analyze protein phosphorylation at STY residues, these have been considered ill-suited to the study of basic amino acids phosphorylation. In this study, a novel selective basic amino acids phosphopeptides enrichment strategy and an online high pH reversed phase nanoelectrospray-tandem mass spectrometry method will be developed for basic amino acids phosphopeptides identification, quantification and evaluation. For phosphopeptide enrichment, the immobilized metal affinity chromatography (IMAC) for purification of substoichiometric phosphopeptides is coupled with phosphoserine and phosphothreonine antibodies to deplete high-abundant interfering peptides. In addition, the application of online high pH reversed phase nanoelectrospray-tandem mass spectrometry method could effectively protect basic amino acids phosphopeptides from acidic hydrolysis to improve identification and quantification capacity of basic amino acids phosphopeptides. These techniques will be applied to the deep identification and precise quantitation of basic amino acid phosphoproteome in eukaryotic mammalian cells to provide new solutions for the related biological problems. In conclusion, this work will pave the way for a better understanding of the biological role of basic amino acids phosphorylation.

碱性氨基酸磷酸化修饰，即组氨酸，精氨酸和赖氨酸上的磷酸化修饰，参与信号转导、蛋白质降解、压力应激、代谢等生物学过程，在生命活动中起着重要的调控作用。然而，碱性氨基酸磷酸化修饰绝对丰度低，易发生酸水解，与常规反相色谱的酸性环境不兼容，对研究提出了挑战。而且由于缺乏有效的研究手段，对这些重要的修饰的精确鉴定和生物功能的认识，特别是在真核生物中功能，仍然存在很多空白。因此，本课题拟开发新型碱性氨基酸磷酸化肽段富集方法，采用固定化金属离子亲和色谱富集纯化磷酸化肽段联合广谱丝氨酸、苏氨酸磷酸化抗体去除高峰度干扰肽段；开发在线高pH反相色谱-质谱联用肽段鉴定方法，解决酸性色谱条件易水解的问题；期望可以提高碱性氨基酸磷酸化修饰肽段的鉴定和定量能力。本课题拟将这些技术应用于真核哺乳动物细胞碱性氨基酸磷酸化修饰组的深度鉴定和精确定量，为相关生物学问题的解决提供新的研究方案。

碱性氨基酸磷酸化修饰，即组氨酸，精氨酸和赖氨酸上的磷酸化修饰，在生命活动中起着重要的调控作用。由于缺乏有效的研究手段，对这些修饰的精确鉴定和生物功能认识，特别是真核生物中功能，仍然存在很多空白。本项目针对碱性氨基酸磷酸化修饰的理化特性，开发和建立了在线高pH反相色谱-质谱联用肽段鉴定方法。该方法具有良好的灵敏度，重复性和有效性，与低pH反相色谱-质谱联用肽段鉴定方法形成有效互补。随后，本项目建立了碱性氨基酸磷酸化修饰深度鉴定蛋白质组学方法，并在哺乳动物293T细胞体系内检测到近1000个碱性氨基酸磷酸化修饰位点。最后，通过与TMT化学标记定量方法结合，建立了碱性氨基酸磷酸化修饰精确定量蛋白质组学方法，完成了胰腺神经内分泌肿瘤样本的碱性氨基酸磷酸化修饰组精确定量，发现了在肿瘤组织中差异表达的碱性氨基酸磷酸化位点。我们希望本项目开发的用于碱性氨基酸磷酸化修饰的蛋白质组学新方法可以为相关生物学功能研究提供新型研究手段。