Identification and characterization of urinary peptides using Q-TOF mass spectrometry in cow


Cow urine has been found to be the most effective secretion of animal origin with innumerable therapeutic values. It has potent antimicrobial activity which may be attributed to the presence of urinary peptides in it. Lack of evidence for the presence of such antimicrobial peptides (AMPs) in cow urine lead to the present study. Using a technique of membrane filtration, ion exchange chromatography, acid-urea poly-acrylamide gel electrophoresis (AU-PAGE), reverse phase high performance liquid chromatography (RP-HPLC) and ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-QTOF-MS/MS), we successfully extracted and characterized cationic peptides from the urine of healthy, normal cycling indigenous cow. We could identify two urinary peptides which were reported to contain antimicrobial properties. These identified peptides were 97 and 99 amino acids long Neutrophil defensin-4 and β-defensin-127 of defensin class. These peptides may be responsible for the innate immunity and serve as an alternative to the conventional antibiotics in the time to come.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2


  1. Brogden KA, Ackermann M, McCray PB, Tack BF (2003) Antimicrobial peptides in animals and their role in host defences. Int J Antimicrob Agents 22:465–478.

    CAS  Article  PubMed  Google Scholar 

  2. Candiano G, Santucci L, Petretto A, Bruschi M, Dimuccio V, Urbani A, Bagnasco S, Ghiggeri GM (2010) 2D-electrophoresis and the urine proteome map: where do we stand? J Proteom 73:829–844.

    CAS  Article  Google Scholar 

  3. Ericksen B, Wu Z, Lu W, Lehrer RI (2005) Antibacterial activity and specificity of the six human {alpha}-defensins. Antimicrob Agents Chemother 49:269–275.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Ganz T (2003) Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 3:710–720.

    CAS  Article  PubMed  Google Scholar 

  5. Ganz T, Selsted ME, Szklarek D, Harwig SS, Daher K, Bainton DF, Lehrer RI (1985) Defensins. Natural peptide antibiotics of human neutrophils. J Clin Investig 76:1427–1435.

    CAS  Article  PubMed  Google Scholar 

  6. Hancock REW, Scott MG (2000) The role of antimicrobial peptides in animal defenses. Proc Natl Acad Sci 97:8856–8861.

    CAS  Article  PubMed  Google Scholar 

  7. Liu R, Zheng W, Li J, Wang L, Wu H, Wang X, Shi L (2015) Rapid identification of bioactive peptides with antioxidant activity from the enzymatic hydrolysate of Mactra veneriformis by UHPLC-Q-TOF mass spectrometry. Food Chem 167:484–489.

    CAS  Article  PubMed  Google Scholar 

  8. Mesihää S (2015) Mass spectrometric characterization of urinary fibrinogen-derived peptides in prostate cancer and renal cell carcinoma. University of Oulu, Oulu

    Google Scholar 

  9. Pandey GS (2009) Bhav Prakash Nighantu (Indian Materia Medica) of Sri Bhavamisra (c.1600–1600 AD). In: Ath Mutravargh. Chaukhamba Bharati Academy, Varanasi, p 778

  10. Park CH, Valore EV, Waring AJ, Ganz T (2001) Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem 276:7806–7810.

    CAS  Article  PubMed  Google Scholar 

  11. Pech D (2010) Determination of amino acid sequence of hemelipoglycoprotein from tick Dermacentor marginatus by mass spectrometry. University of South Bohemia, České Budějovice

    Google Scholar 

  12. Porter E, Valore EV, Anouseyan R, Salzman NH (2015) Detection of antimicrobial (poly)peptides with acid urea polyacrylamide gel electrophoresis followed by Western immunoblot. Methods Mol Biol 1225:105–115.

    CAS  Article  PubMed  Google Scholar 

  13. Ramautar R, Heemskerk AAM, Hensbergen PJ, Deelder AM, Busnel J-M, Mayboroda OA (2012) CE-MS for proteomics: advances in interface development and application. J Proteom 75:3814–3828.

    CAS  Article  Google Scholar 

  14. Schutte BC, Mitros JP, Bartlett JA, Walters JD, Jia HP, Welsh MJ, Casavant TL, McCray PB (2002) Discovery of five conserved beta-defensin gene clusters using a computational search strategy. Proc Natl Acad Sci USA 99:2129–2133.

    CAS  Article  PubMed  Google Scholar 

  15. Simon SL, Lamoureux L, Plews M, Stobart M, LeMaistre J, Ziegler U, Graham C, Czub S, Groschup M, Knox JD (2008) The identification of disease-induced biomarkers in the urine of BSE infected cattle. Proteome Sci 6:23.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Spencer JD, Schwaderer AL, DiRosario JD, McHugh KM, McGillivary G, Justice SS, Carpenter AR, Baker PB, Harder J, Hains DS (2011) Ribonuclease 7 is a potent antimicrobial peptide within the human urinary tract. Kidney Int 80:174–180.

    CAS  Article  PubMed  Google Scholar 

  17. Spencer JD, Hains DS, Porter E, Bevins CL, DiRosario J, Becknell B, Wang H, Schwaderer AL (2012) Human alpha defensin 5 expression in the human kidney and urinary tract. PLoS ONE 7:e31712.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Tang Y (1999) A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated -defensins. Science 286:498–502.

    CAS  Article  PubMed  Google Scholar 

  19. Valore EV, Park CH, Quayle AJ, Wiles KR, McCray PB, Ganz T (1998) Human β-defensin-1: an antimicrobial peptide of urogenital tissues. J Clin Investig 101:1633–1642.

    CAS  Article  PubMed  Google Scholar 

  20. Wilde CG, Griffith JE, Marra MN, Snable JL, Scott RW (1989) Purification and characterization of human neutrophil peptide 4, a novel member of the defensin family. J Biol Chem 264:11200–11203

    CAS  PubMed  Google Scholar 

  21. Wu Z, Cocchi F, Gentles D, Ericksen B, Lubkowski J, Devico A, Lehrer RI, Lu W (2005) Human neutrophil alpha-defensin 4 inhibits HIV-1 infection in vitro. FEBS Lett 579:162–166.

    CAS  Article  PubMed  Google Scholar 

  22. Xu C, Shu S, Xia C, Wang P, Sun Y, Xu C, Li C (2015) Mass spectral analysis of urine proteomic profiles of dairy cows suffering from clinical ketosis. Vet Q 35:133–141.

    Article  PubMed  Google Scholar 

  23. Zasloff M (2007) Antimicrobial peptides, innate immunity, and the normally sterile urinary tract. J Am Soc Nephrol 18:2810–2816.

    CAS  Article  PubMed  Google Scholar 

Download references


The authors acknowledge and thank Dr. Tomas Ganz, Ph.D., M.D., Distinguished Professor of Medicine and Pathology, Dept. of Medicine, David Geffen School of Medicine, Los Angeles (UCLA), and Sandor Life Sciences, Bengaluru, India for providing the precious technical support.


This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author information




AS: conceptualization, methodology, software, validation, analysis. RN: visualization, supervision. AK: writing- original draft preparation, reviewing and editing. AP: data curation.

Corresponding author

Correspondence to Ambika Sharma.

Ethics declarations

Conflict of interest

All the authors confirm that this article content has no conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Sharma, A., Nigam, R., Kumar, A. et al. Identification and characterization of urinary peptides using Q-TOF mass spectrometry in cow. J Proteins Proteom (2020).

Download citation


  • Urinary peptides
  • Neutrophil defensin-4
  • β-defensin-127
  • Cow