Peptide profiling in cow urine reveals molecular signature of physiology-driven pathways and in-silico predicted bioactive properties

Abstract

Peptidomics allows the identification of peptides that are derived from proteins. Urinary peptidomics has revolutionized the field of diagnostics as the samples represent complete systemic changes happening in the body. Moreover, it can be collected in a non-invasive manner. We profiled the peptides in urine collected from different physiological states (heifer, pregnancy, and lactation) of Sahiwal cows. Endogenous peptides were extracted from 30 individual cows belonging to three groups, each group comprising of ten animals (biological replicates n = 10). Nano Liquid chromatography Mass spectrometry (nLC-MS/MS) experiments revealed 5239, 4774, and 5466 peptides in the heifer, pregnant and lactating animals respectively. Urinary peptides of <10 kDa size were considered for the study. Peptides were extracted by 10 kDa MWCO filter. Sequences were identified by scanning the MS spectra ranging from 200 to 2200 m/z. The peptides exhibited diversity in sequences across different physiological states and in-silico experiments were conducted to classify the bioactive peptides into anti-microbial, anti-inflammatory, anti-hypertensive, and anti-cancerous groups. We have validated the antimicrobial effect of urinary peptides on Staphylococcus aureus and Escherichia coli under an in-vitro experimental set up. The origin of these peptides was traced back to certain proteases viz. MMPs, KLKs, CASPs, ADAMs etc. which were found responsible for the physiology-specific peptide signature of urine. Proteins involved in extracellular matrix structural constituent (GO:0005201) were found significant during pregnancy and lactation in which tissue remodeling is extensive. Collagen trimers were prominent molecules under cellular component category during lactation. Homophilic cell adhesion was found to be an important biological process involved in embryo attachment during pregnancy. The in-silico study also highlighted the enrichment of progenitor proteins on specific chromosomes and their relative expression in context to specific physiology. The urinary peptides, precursor proteins, and proteases identified in the study offers a base line information in healthy cows which can be utilized in biomarker discovery research for several pathophysiological studies.

Peptidome Profiling of <em>Bubalus Bubalis</em> Urine and Assessment of its Antimicrobial Activity against Mastitis-Causing Pathogens

“…By scanning MS/MS spectra ranging from 200 to 2200 m/z we identified a total of 8165 peptides, with molecular weight ranging from 700-5000 Da. We obtained a similar pattern of molecular weight distributions in our previous findings [26]. The predominating amino acid residues in the sequences were Ala, Gly, Leu, Pro, and Ser.…”

“…Referring to our investigation in cows, we found more than 80.6% similarity between the proteases of buffaloes and cows (Fig. 4B) [26]. Immune functions ontology showed the enrichment antibacterial peptide production (31.25%) a function relevant in our search of antimicrobial peptides in urinary peptidome.…”

“…In line with our previous findings, we observed a similar pattern of amino acid distribution in all predicted bioactivities except anticancer sequences. In the case of the buffalo anticancer sequence, Cys, Gly and Pro were predominating residues whereas, in cow anticancer sequences, only Gly and Pro were abundantly distributed throughout the sequence length while Cys residue was completely absent [26].…”

Camel (Camelus spp.) Urine Bioactivity and Metabolome: A Systematic Review of Knowledge Gaps, Advances, and Directions for Future Research

“…Although the use of web-based platforms for the prediction of bioactivity and the associated beneficial effects of chemicals can support the process of new drugs’ discovery, it is highly recommended to additionally perform evaluations of key biochemical parameters (i.e., permeability) and design functional assays (in vitro/in vivo) given the intrinsic limitations of prediction rates that can sometimes arise from in silico results when approached separately [ 32 , 86 ].…”

“…Thus, further investigation is needed to perform a detailed characterization of the chemical composition of this biofluid and to assess how its metabolic profile and properties are affected by different extrinsic and intrinsic factors. As stated by Kumar et al [ 32 ], urine is constituted by different secretions that aid the protection of the urogenital tract against environmental pathogens/threats and organic failures. Once the biological activity of whole urine is tested and confirmed under experimental conditions based on standardized principles established through international conventions, the proceeding steps involve the characterization of its chemical composition and the subsequent isolated prediction and testing of the bioactivity, through in-silico and in vitro/in vivo experiments, respectively, of each of the molecules encountered to classify them into functional groups [ 33 , 34 ].…”