基于质量亏损的准等重标记定量蛋白质组学新方法研究

It is important to develop proteome quantification methods with high accuracy, precision, throughput, wide dynamic range and deep quantification coverage for biological and medical research. The project intends to develop novel quantification methods based on the subtle mass defect differences between 13C/12C, 15N/14N and 2H/1H. Firstly, pseudo-isobaric a1 ion pairs can be resolved by high MS/MS resolution and used for proteome quantification; secondly, 6-plex a1-ion-pair based quantification methods will be developed by calculated incorporation of formaldehyde, sodium cyanoborohydride and their isotopologues to improve the quantification throughput; thirdly, novel labeling reagents will be synthesized and used for Lys-N digests labeling. Product ion can be correctly annotated based on the different mass differences from different types of product ions. Thus, deep quantification coverage can be obtained by interpreting MS/MS spectra using de novo sequencing in combination with database search methods; lastly, all the developed methods will be used to analyze the differentially expressed proteins from human liver cancer cell lines with high and low metastasis rates. The developed mass defect based proteome quantification methods in this project are promising to significantly improve the quantification accuracy, precision, throughput, dynamic range and coverage and further provide credible proteomics data sets for discovering function proteins in cell metastasis.

展具有高准确度、高精密度、高通量、宽动态范围和深度覆盖的蛋白质组定量方法，对生物和医学研究领域具有重要意义。本项目拟利用13C/12C、15N/14N和2H/1H质量亏损值的不同发展蛋白质组定量方法。首先，高分辨二级谱将准等重的a1离子分辨而成对出现，利用成对的a1离子实现蛋白质组的相对定量；其次，通过组合甲醛、氰基硼氢化钠及其同位素体建立基于a1离子的6重标记定量方法，提高蛋白质组定量分析的通量；第三，通过合成新型标记试剂并将该试剂用于Lys-N酶解产物标记，利用二级谱中碎片离子质量差异的不同实现谱图中碎片离子类型的注释，进而结合从头测序和数据库检索实现蛋白质组的深度定性定量分析；最后将上述方法用于人肝癌高低转移能力细胞系差异蛋白质分析。本项目设计的基于质量亏损的新方法有望显著提高蛋白质组定量分析的准确度、精密度、通量、动态范围和覆盖度，进而为深入研究癌细胞转移机理提供差异表达蛋白质信息

根据计划书要求，本课题发展了三种蛋白质组定量方法，即基于a1离子对的蛋白质组定量方法、基于放大母离子分离窗口的蛋白质组定量方法以及基于质量亏损的细胞培养稳定同位素标记（SILAC）蛋白质组定量方法，实现了蛋白质组样品的高准确度定量分析。基于a1离子对的蛋白质组定量方法，定量结果与理论值的相对偏差低于7%，比值的标准偏差小于0.25，分析通量为6重，动态范围为20倍。基于放大母离子分离窗口的蛋白质组定量方法可以实现与a1离子对的定量方法近似的定量结果，而大大简化了分析流程。将上述方法应用到A549细胞上皮细胞-间充质转化过程蛋白质的变化趋势分析，发现了多种已经报道的与EMT过程相关的蛋白，同时发现脂肪酸beta氧化过程在EMT过程的潜在作用。基于质量亏损的SILAC方法与常规SILAC和NeuCode SILAC方法相比，比值的标准偏差降低一倍以上，表明更多的蛋白质可以实现高准确的定量。应用到人肝癌高低转移能力细胞系的蛋白质组差异分析发现了多种与细胞转移相关的标识蛋白。新发展的方法在蛋白质组高精准度的优势将为深入探讨蛋白质在生理病理中的作用发挥重要作用。