Abstract
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 from Prochlorococcus, 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.
Highlights
In the diets of ruminants, urea is commonly used as a costefficient replacement for animal and vegetable proteins as nitrogen sources (Kertz, 2010; Lin et al, 2012)
The results showed that cryomilling in liquid nitrogen for 4 min maximized urease activity (955.73 nmol/min/mg) and protein concentration (137.73 μg/mL) compared to cryomilling for 3 or 5 min (Figure 1)
Four different digestion methodologies were assessed to select the optimum method for targeted meta-proteomic analyses of rumen microbial urease. These approaches focused on assessing different digestion states and digestion enzymes used, in which tandem Glu-C/Lys-C digestion was a potential replacement for trypsin digestion to perform the greatest number of urease peptides by virtual digestion of different digestion enzymes (Supplementary Figure S2)
Summary
In the diets of ruminants, urea is commonly used as a costefficient replacement for animal and vegetable proteins as nitrogen sources (Kertz, 2010; Lin et al, 2012). To better understand urea metabolism in the rumen, some ureolytic bacteria were isolated and identified using a culturedependent method, and different ureolytic bacteria demonstrated varying urease activities (Cook, 1976; Lauková and Koniarová, 1995; Patra, 2018). Using high-throughput sequencing of the bacterial 16S rRNA genes, Jin et al (2016) revealed that rumen ureolytic bacteria were abundant in the genera of Pseudomonas, Haemophilus, Neisseria, Streptococcus, Actinomyces, Bacillus, and unclassified Succinivibrionaceae. They identified a high degree of urease gene sequence diversity by high-throughput sequencing of the urease gene, ureC, from ruminant microbial populations. It is not certain that a given DNA will express an active protein, and the functionality of the protein encoded needs to be further confirmed
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