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

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.