类芽孢杆菌(Paenibacillus sp.)蛋白质组学研究中三种蛋白提取方法的比较分析

Comparison of three protein extraction methods for the proteomics research on Paenibacillus sp.

摘要:

【目的】革兰氏阳性类芽孢杆菌（Paenibacillus sp.）本身细胞壁的结构特点导致其菌体全蛋白不易获得。本研究选取了3种破碎方法——溶菌酶联合超声破碎法（方法一）、溶菌酶联合SDS热处理破碎法（方法二）、液氮联合超声破碎法（方法三）进行革兰氏阳性菌的细胞破碎，以期获得适于样品菌株基于质谱技术进行蛋白质组学研究的制备方法。【方法】在蛋白样品的制备过程中，对3种不同破碎方法的蛋白提取得率和SDS-PAGE检测分析结果进行比较；随后将3种蛋白样品制备方法的样品用质谱技术进行鉴定，分析不同蛋白样品基于质谱技术鉴定蛋白的差异。【结果】在蛋白样品的制备提取过程中，不同破碎方法的蛋白提取率大致相同。用单因素方差比较3种提取方法质谱鉴定蛋白数的差异性，方法三鉴定的蛋白数最多（2 638个），其次是方法一（2 452个），方法二鉴定的蛋白数最少（2 003个）。进一步用韦恩图分析比较不同提取方法的蛋白鉴定通量差异，综合考虑蛋白提取效率的结果以及液氮研磨法提取蛋白的缺点，最终选取溶菌酶联合超声破碎法（方法一）提取菌株全蛋白作为该菌基于质谱分析其蛋白质组学研究中最适合的方法。最后，对质谱鉴定菌株蛋白包括分子量、等电点、疏水性的基本性质进行分析，发现3种破碎方法质谱鉴定的蛋白与模式菌株多黏类芽孢杆菌（Paenibacillus polymyxa）基因组中预测蛋白的各个组分分布占比基本一致，都保证了菌株蛋白质组数据信息的完整性。【结论】基于质谱技术开展革兰氏阳性类芽孢杆菌（Paenibacillus sp.）的蛋白质组学研究，溶菌酶联合超声破碎法是提取该菌株全蛋白最适合的方法。

关键词:革兰氏阳性菌;蛋白裂解液;蛋白质组学;多肽

Abstract:

[Objective] The cell wall structure characteristics make it a challenge to obtain the whole proteins of Paenibacillus sp. as a Gram-positive bacterium. We employed three representative lysis methods:lysozyme combined with ultrasonic treatment (method 1), lysozyme combined with SDS heat treatment (method 2), and liquid nitrogen combined with ultrasonic treatment (method 3), to obtain the samples for mass spectrometry-based proteomics research on Paenibacillus sp. [Methods] We compared the protein extraction efficiency of the three methods based on protein yield and SDS-PAGE. Furthermore, we analyzed the obtained protein samples by mass spectrometry. [Results] The protein extraction efficiency of the three methods was similar. The number of proteins identified in the sample prepared with method 3 was the highest (2 638 proteins), followed by that with method 1 (2 452 proteins), and that with method 2 was the lowest (2 003 proteins). The Venn diagram was established to compare the protein identification flux of the samples prepared with different methods. Considering the protein extraction efficiency and the shortcomings of method 3, we finally chose method 1 as the most suitable method to extract the proteins for mass spectrometry-based proteomic analysis. Finally, the basic properties of proteins identified by mass spectrometry, including molecular weight, isoelectric point, and hydrophobicity, were analyzed. The proportion of each component of the proteins obtained by the three methods was the same as that of the predicted proteins of the model strain Paenibacillus polymyxa. [Conclusion] For the proteomics research on Gram-positive Paenibacillus sp. by mass spectrometry, lysozyme combined with ultrasonic treatment is the most suitable method for sample preparation.

Key words:Gram-positive bacteria;protein lysates;proteomics;polypeptide