广式酱油发酵周期长、微生物种群多样、代谢物复杂，缺乏微生物蛋白组学分析的样本制备方法。该研究通过比较改良的TCA浓缩法和超滤法两种蛋白纯化方法，利用SDS-PAGE凝胶电泳和LTQ Orbitrap Velos Pro ETD质谱检测酱油微生物宏蛋白组样本制备效果。实验结果表明：TCA浓缩法制备的蛋白样品，其SDS-PAGE凝胶图谱有26个清晰条带，比超滤法多13个；两种法所制备的蛋白经胰蛋白酶酶切后的质谱图可分别鉴定到的2 495和1 682个肽段，说明TCA浓缩法制备的蛋白样品酶切更充分；不同发酵时期（0、60、120 d）的酱油经TCA浓缩法制备的蛋白样品，烷基化、酶切后，通过LTQ Orbitrap Velos Pro ETD质谱检测和Proteome Discoverer 2.5软件分析，可分别鉴定到1 035、1 104、1 046种蛋白质。通过TCA浓缩法制备蛋白组分析样本，操作简便，实验速度快，蛋白质鉴定率高，适用于广式酱油微生物的蛋白质组学的检测分析，为利用蛋白组学分析广式酱油的微生物发酵分子机制打下了一定的基础。

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.

中山市科学技术局项目（CXTD2020006）