蛋白质组学样品消化条件的优化

Optimization of Digestion Conditions for Proteomics Samples

随着质谱的飞速发展,基于质谱的“鸟枪法”技术广泛的应用于大规模的蛋白质组学分析。化学反应保护效率过低或者酶切效率过低则会降低鉴定效率,并且在现有的计算方式下会丢失很多肽段信息。因此,蛋白质样品的前处理在现有的蛋白质组学研究中发挥重要作用。本研究对蛋白质的烷基化试剂的反应条件进行优化以提高烷基化效率,同时优化酶解Buffer提高酶切效率以及增加酶量和引入多种酶以提高酶切效率等,最终确定了蛋白质前处理的最优条件,最终使用1μg样品在一次质谱分析鉴定到（2 425±7）个蛋白质,较未优化的方法提高了31%。优化后的蛋白质前处理方法可有效提高现有蛋白质组学的研究效率,可进一步应用于蛋白质的定量及动态分析研究。

With the rapid development in mass spectrometer （MS）, MS-based shotgun technique has been widely used in large-scale proteomics analysis. Low protection efficiency of chemical reactionor low efficiency of enzyme digestion might reduce the efficiency of identification ratio and lose many peptides information under the existing calculation methods. Therefore, protein sample pretreatment plays an increasingly important role in current proteomics research. In this study, the alkylation reagents were optimized to increase the alkylation efficiency, at the same time a series of optimization on the digestion buffer, the enzyme/sample ratio and enzyme combination were carried out to increase the digestion efficiency. Finally, the optimum conditions for protein pretreatment were determined. Inthe optimized protocol, （2 425±7） proteins were identified using 1 μg sample, which was 31% higher than that of the non-optimized method. The results showed that the optimal pretreatment might enhanceresearch efficiency ofproteomics, which could be applied in quantitative and dynamic analysis oforotein.