两种用于液质联用分析的蛋白质酶解处理效果比较

【目的】分析和评估两种蛋白质溶液酶解方法,为蛋白质组学研究中的大规模质谱鉴定分析建立一种稳定的样品制备技术。【方法】分别使用TEAB法(实验组A)和TFE法(实验组B)对牛血清白蛋白(BSA)进行还原、烷基化和溶液酶解,再用LTQ-Orbitrap高分辨率质谱仪采集BSA酶切肽段信息,比较两种方法得到的数据信息,分析肽段数目、可变修饰位点和蛋白质覆盖率。【结果】实验组A(TEAB法)获得36个肽段、39个修饰位点,蛋白质平均覆盖率为59.31%;实验组B(TFE法)获得27个肽段、27个修饰位点,蛋白质平均覆盖率为47.83%,实验组A的数据指标与实验组B差异显著。【结论】采用TEAB法酶切体系能实现蛋白质高效率酶切,达到获得较高肽段覆盖率的目的,可为蛋白质组学后期的质谱分析鉴定奠定实验基础。

Comparison of Two Protein In-solution Enzymatic Digestion Methods for LC-MS Analysis

[Objective] Two methods of proteins in-solution enzymatic digestion were compared and evaluated to establish a reliable sample preparation protocol for large-scale identification in proteomics research.[Methods] Bovine serum albumin (BSA) was reduced and alkylated, and subsequently used for in-solution enzymatic digestion according to the protocols of group A (TEAB method) and group B (TFE method).LTQ-Orbitrap mass spectrometer was utilized to obtaine the signal of enzymatic peptides.The data obtained from two groups were compared.Peptide categories, variable modification site and protein coverage were analyzed.[Results] A total of 36 peptides and 39 modification sites were acquired in group A (TEAB method), and the protein coverage was 59.31%; in group B (TFE method), a total of 27 peptides and 27 modification sites were obtained, and the protein coverage was 47.83%.All the statistical comparisons between groups A and B were significantly different.[Conclusion] TEAB method provided a high efficiency of enzymatic digestion with higher peptide coverage.This method can be served as the experimental basis for MS analysis in proteomics.