Sample Preparation for Microbial Proteomics Analysis of Cantonese Soy Sauce
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Abstract:
The long fermentation period, diverse microbial populations, and complex metabolites of Cantonese soy sauce have prevented the development of a standardized sample preparation method for microbial proteomics analysis. In the present study, two modified protein purification methods were compared, namely trichloroacetic acid (TCA) precipitation and ultrafiltration. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and LTQ Orbitrap Velos Pro ETD mass spectrometry were used to determine the efficacy of the two sample preparation methods for metaproteomics analysis of soy sauce microbes. Twenty-six distinct bands were observed in the SDS-PAGE patterns of protein samples prepared by TCA precipitation, and 13 were observed in samples prepared by ultrafiltration. 2 495 and 1 682 respective peptides were identified from the mass spectra obtained after trypsin digestion of protein samples prepared by the two methods, indicating that protein samples prepared by TCA precipitation were digested to a greater extent. Protein samples were prepared from soy sauce at different fermentation stages (0, 60, and 120 days) using the TCA precipitation method. After alkylation and enzyme digestion, these samples were subjected to protein analysis using LTQ Orbitrap Velos Pro ETD mass spectrometry and Proteome Discoverer 2.5 software, from which 1 035, 1 104, and 1 046 proteins were respectively identified. These results indicate that the preparation of samples for proteomics analysis by TCA precipitation provides the advantages of simple operation, rapid experimentation, and a high protein identification rate. It is thus suitable for use in proteomics testing and analysis of microbes in Cantonese soy sauce. The results of this study provide a scientific basis for proteomics analysis of the molecular mechanisms of microbial fermentation in Cantonese soy sauce.
