基于液相-质谱的MPPZ稳定同位素标记蛋白质组定量方法的建立

Quantitative proteomics is an important research field in post-genomics era. There are two strategies for proteome quantification: label-free methods and stable isotope labeling methods which have become the most important strategy for quantitative proteomics at present. Chemical labeling based quantification methods offer the complementary choice for proteome quantification。Since proteins and peptides contain a lot of functional groups, such as sulfhydryl groups, amino groups and carboxyl groups, by chemical labeling, the stable isotopes are introduced via the reaction of suitable chemical reagents with such groups. Various kinds of chemical baleling methods have been developed, which are suitable for the analysis of different kinds of samples. However, duo to the great diversity and the extreme complexity of proteome samples, the accuracy, precision, dynamic range, and throughput for quantification methods still need to be improved..In this project, we plant to synthesis of five MPPZ stable isotope labeling reagents and will establish a strategy for quantitative proteome analysis through MPPZ stable isotopic labeling. Several major influencing factor will examine. The investigation on the dynamic range and quantitative accuracy will perform by calculating the ratio of the peak intensity in mass spectra with several MPPZ-peptide couples.

定量蛋白质组学已经成为后基因时代的重要研究方向之一。目前该领域的研究主要采用无标记定量方法和稳定同位素标记定量法。其中，基于质谱的稳定同位素标记蛋白质组定量方法发展非常迅速，已为生命科学研究提供了重要的技术支撑。化学标记引入同位素的一种非常有价值的蛋白质组相对定量方法。由于蛋白质/肽段含有大量的官能团，如巯基、氨基和羧基，因此通过化学反应在这些官能团上引入稳定同位素标签。已开发多种化学标记方法，并应用于不同蛋白组定量分析。然而，由于蛋白质组样品的多样性和复杂性，亟需开发准确度高、动态范围宽、高通量方法。.在本研究中，拟合成新的五种MPPZ同位素标记试剂，将建立一种基于液相色谱-质谱的MPPZ稳定同位素标记，定量蛋白质组学研究方法，优化影响标记效率的各种条件。对多对MPPZ标记肽段在液相色谱-质谱中的动态范围及定量准确度进行考察。

建立了一种基于LC-MS的MPPZ稳定同位素标记蛋白质组定量方法。合成了稳定同位素标记试剂d0/d4/d8-MPPZ，通过核磁共振光谱、质谱等对其结构进行了表征。MPPZ与多肽反应生成多肽-MPPZ衍生物。优化了影响标记效率的各种条件。在碳酸氢铵缓冲溶液（pH = 8.0）中， 40°C条件下 MPPZ与多肽反应2h时，标记即可完全。采用亲水色谱柱，以水（含0.1% 甲酸）-乙腈（含0.1% 甲酸）或10 mM 甲酸铵溶液-乙腈作为流动相，分离了多肽-MPPZ衍生物。对d0/d8-MPPZ标记肽段在LC-MS中的动态范围及定量准确度进行了考察。结果表明，在10倍动态范围内，线性关系良好（r = 0.9993）,理论值和观测值的偏差为0.4%。