数字农科院2.0

Pipeline For Targeted Meta-Proteomic Analyses To Assess The Diversity Of Cattle Rumen Microbial Urease

文献类型： 外文期刊

作者： Zhang, XY; Zhao, SG; He, Y; Zheng, N; Yan, XH; Wang, JQ

作者机构：

关键词： Urease; Trypsin; Glu-C; Lys-C; In-Gel; In-Solution; Meta-Proteomics

期刊名称： FRONTIERS IN MICROBIOLOGY

ISSN： 1664-302X

年卷期： 2020 年 11 卷

页码：

收录情况： JCR（2021版）

摘要： In the rumen of cattle, urease produced by ureolytic bacteria catalyzes the hydrolysis of urea to ammonia, which plays an important role in nitrogen metabolism and animal production. A high diversity of rumen bacterial urease genes was observed in our previous study;; however, information on urease protein diversity could not be determined due to technical limitations. Here, we developed a targeted meta-proteomic pipeline to analyze rumen urease protein diversity. Protein extraction (duration of cryomilling in liquid nitrogen), protein digestion state (in-solution or in-gel), and the digestion enzyme used (trypsin or Glu-C/Lys-C) were optimized, and the digested peptides were analyzed by LC-MS/MS. Four minutes was the best duration for cryomilling and yielded the highest urease activity. Trypsin digestion of in-gel proteins outperformed other digestion methods and yielded the greatest number of identifications and superior peptide performance in regards to the digestion efficiency and high-score peptide. The annotation of peptides by PEAKS software revealed diversity among urease proteins, with the predominant proteins being fromProchlorococcus,Helicobacter, and uncultured bacteria. In conclusion, trypsin digestion of in-gel proteins was the optimal method for the meta-proteomic pipeline analyzing rumen microbial ureases. This pipeline provides a guide for targeted meta-proteomic analyses in other ecosystems.

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