FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Drug Test Anal / Identification of ex vivo catabolites of peptides with dopin...
Drug testing and analysis2020; 12(6); 771-784; doi: 10.1002/dta.2781

Identification of ex vivo catabolites of peptides with doping potential in equine plasma by HILIC-HRMS.

Abstract: Bioactive peptides pose a great threat to sports integrity. The detection of these peptides is essential for enforcing their prohibition in sports. Identifying the catabolites of these peptides that are formed ex vivo in plasma may improve their detection. In the present study, the stability of 27 bioactive peptides with protection at both termini in equine plasma was examined under different incubation conditions, using HILIC coupled to HRMS. Of the 27 peptides, 13 were stable after incubation at 37°C for 72 hr, but the remaining 14 were less stable. Ex vivo catabolites of these 14 peptides were detected using their theoretical masses generated in silico, their appearance was monitored over the time course of incubation, and their identity was verified by their product ion spectra. Catabolites identified for chemotactic peptide, DALDA, dmtDALDA, deltorphins I and II, Hyp -dermorphin, Lys -dermorphin, and dermorphin analog are novel. A d-amino acid residue at position 2 or 1 of a peptide or next to its C-terminus protected the relevant terminal from degradation by exopeptidases, but such a residue at position 3 did not. A pGlu residue or N-methylation at the N-terminus of a peptide did not protect its N-terminal. Ethylamide at the C-terminus of a peptide provided the C-terminal protection from attacks by carboxypeptidases. The C-terminal Lys amide in DALDA, dmtDALDA, and Lys -dermorphin was susceptible to cleavage by plasma enzymes, which is the first report, to the authors' knowledge. The results from the present study provide insights into the stability of peptides in plasma.
© 2020 John Wiley & Sons, Ltd.
Get Full Text
Publication Date: 2020-03-24 PubMed ID: 32100400DOI: 10.1002/dta.2781Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article is focused on methods for detecting the use of prohibited bioactive peptides in sports by identifying their breakdown products, or catabolites, in the bloodstream. This is accomplished by testing the stability of these peptides and identifying their ex vivo catabolites in equine plasma, using a method combining hydrophilic interaction liquid chromatography (HILIC) and high-resolution mass spectrometry (HRMS).

Introduction and Motivation

  • The paper begins by highlighting the problem of bioactive peptides, compounds that can potentially enhance performance, being used illicitly in sports. To maintain the integrity of these sports, it’s vital to detect and prohibit the use of these substances.
  • However, detecting these peptides is not straightforward. They break down in the bloodstream, and it’s their catabolites, or products of their breakdown, that are often detected. Providing a method to identify these catabolites may improve the detection of peptide doping in sports.

Methods

  • The researchers took an experimental approach, testing the stability of 27 different bioactive peptides after they were incubated with equine plasma. This was done under several different incubation conditions. The tools used in this study were hydrophilic interaction liquid chromatography (HILIC), which helps separate complex mixtures, coupled with high-resolution mass spectrometry (HRMS). This technique allows the peptides and their catabolites to be precisely identified.
  • The theoretical masses of the peptides and their catabolites were generated in silico, or using computational software, and these were used for detection. They monitored their appearance over time and verified their identity through product ion spectra, a kind of mass spectrometry that differentiates compounds by their mass and charge.

Results

  • Thirteen of the 27 evaluated peptides were found to remain stable after being incubated at a temperature of 37°C for 72 hours. However, the other 14 peptides were less stable. Their catabolites formed under these conditions were detected using their previously generated theoretical masses.
  • They also discovered that certain structural elements within the peptides affected their stability. For example, d-amino acid residues present at certain positions provide protection against degradation by exopeptidases, with the position effecting the level of protection. Moreover, a pGlu residue or N-methylation at the N-terminus of a peptide did not offer any protection to its N-terminal, but an ethylamide at the C-terminus did protect against degradation by carboxypeptidases. This information may help in future designing of peptide drugs for better stability.

Conclusions

  • The research not only provides a method for identifying catabolites of bioactive peptides but also offers insights into how the stability of these peptides in the bloodstream can be affected by their structures.
  • The novel findings about specific peptide catabolites and the protection offered by certain peptide structures will be useful for the development and refinement of drug detection and enforcement programs within sports organizations.

Cite This Article

APA
Guan F, Fay S, Li X, You Y, Robinson MA. (2020). Identification of ex vivo catabolites of peptides with doping potential in equine plasma by HILIC-HRMS. Drug Test Anal, 12(6), 771-784. https://doi.org/10.1002/dta.2781

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 12
Issue: 6
Pages: 771-784

Researcher Affiliations

Guan, Fuyu
  • Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, Kennett Square, PA, USA.
  • Pennsylvania Equine Toxicology and Research Laboratory, West Chester, PA, USA.
Fay, Savannah
  • Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, Kennett Square, PA, USA.
  • Pennsylvania Equine Toxicology and Research Laboratory, West Chester, PA, USA.
Li, Xiaoqing
  • Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, Kennett Square, PA, USA.
  • Pennsylvania Equine Toxicology and Research Laboratory, West Chester, PA, USA.
You, Youwen
  • Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, Kennett Square, PA, USA.
  • Pennsylvania Equine Toxicology and Research Laboratory, West Chester, PA, USA.
Robinson, Mary A
  • Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, Kennett Square, PA, USA.
  • Pennsylvania Equine Toxicology and Research Laboratory, West Chester, PA, USA.

MeSH Terms

  • Amino Acid Sequence
  • Animals
  • Biotransformation
  • Chromatography, High Pressure Liquid
  • Computer Simulation
  • Doping in Sports / methods
  • Growth Hormone-Releasing Hormone / blood
  • Horses / metabolism
  • Magnetic Resonance Spectroscopy
  • Mass Spectrometry
  • Oligopeptides / blood
  • Opioid Peptides / blood
  • Peptides / blood
  • Solid Phase Extraction
  • Substance Abuse Detection / methods

Grant Funding

  • N/A / Pennsylvania Department of Agriculture State Horse Racing Commission

References

This article includes 40 references
  1. Stuehrwohldt N, Schaller A. Regulation of plant peptide hormones and growth factors by post-translational modification.. Plant Biol (Berlin) 2019;21:49-63.
  2. Camanni F, Ghigo E, Arvat E. Growth hormone-releasing peptides and their analogs.. Front Neuroendocrinol 1998;19(1):47-72.
  3. Janecka A, Perlikowska R, Gach K, Wyrebska A, Fichna J. Development of opioid peptide analogs for pain relief.. Curr Pharm Des 2010;16(9):1126-1135.
  4. Liu WX, Wang R. Endomorphins: potential roles and therapeutic indications in the development of opioid peptide analgesic drugs.. Med Res Rev 2012;32(3):536-580.
  5. van den Broek I, Blokland M, Nessen MA, Sterk S. Current trends in mass spectrometry of peptides and proteins: application to veterinary and sports-doping control.. Mass Spectrom Rev 2015;34(6):571-594.
  6. Negri L, Melchiorri P, Lattanzi R. Pharmacology of amphibian opiate peptides.. Peptides 2000;21(11):1639-1647.
  7. Cox HD, Hughes CM, Eichner D. Detection of GHRP-2 and GHRP-6 in urine samples from athletes.. Drug Test Anal 2015;7(5):439-444.
  8. Guan F, Uboh CE, Soma LR, Robinson M, Maylin GA, Li X. Detection, quantification, and identification of dermorphin in equine plasma and urine by LC-MS/MS for doping control.. Anal Bioanal Chem 2013;405(14):4707-4717.
  9. Thomas A, Hoeppner S, Geyer H. Determination of growth hormone-releasing peptides (GHRP) and their major metabolites in human urine for doping controls by means of liquid chromatography-mass spectrometry.. Anal Bioanal Chem 2011;401(2):507-516.
  10. Timms M, Hall N, Levina V, Vine J, Steel R. A high-throughput LC-MS/MS screen for GHRP in equine and human urine, featuring peptide derivatization for improved chromatography.. Drug Test Anal 2014;6(10):985-995.
  11. Wang CC, Hartmann-Fischbach P, Krueger TR, Wells TL, Feineman AR, Compton JC. Fast and sensitive analysis of dermorphin and HYP6-dermorphin in equine plasma using liquid chromatography tandem mass spectrometry.. Drug Test Anal 2014;6(4):342-349.
  12. Steel R, Timms M, Levina V, Vine J. A high throughput screen for 17 Dermorphin peptides in equine and human urine and equine plasma.. Drug Test Anal 2014;6(9):909-921.
  13. Guan FY, Robinson MA. Comprehensive solid-phase extraction of multitudinous bioactive peptides from equine plasma and urine for doping detection.. Anal Chim Acta 2017;985:79-90.
  14. Thomas A, Delahaut P, Krug O, Schänzer W, Thevis M. Metabolism of growth hormone releasing peptides.. Anal Chem 2012;84(23):10252-10259.
  15. Semenistaya E, Zvereva I, Thomas A, Thevis M, Krotov G, Rodchenkov G. Determination of growth hormone releasing peptides metabolites in human urine after nasal administration of GHRP-1, GHRP-2, GHRP-6, hexarelin, and ipamorelin.. Drug Test Anal 2015;7(10):919-925.
  16. Esposito S, Deventer K, Geldof L, Van Eenoo P. In vitro models for metabolic studies of small peptide hormones in sport drug testing.. J Pept Sci 2015;21(1):1-9.
  17. Powell MF. Peptide stability in drug development: in vitro peptide degradation in plasma and serum.. Annu Rep Med Chem 1993;28:285-294.
  18. Pollaro L, Heinis C. Strategies to prolong the plasma residence time of peptide drugs.. Med Chem Commun 2010;1(5):319-324.
  19. John H, Maronde E, Forssmann W-G, Meyer M, Adermann K. N-terminal acetylation protects glucagon-like peptide GLP-1-(7-34)-amide from DPP-IV-mediated degradation retaining cAMP- and insulin-releasing capacity.. Eur J Med Res 2008;13:73-78.
  20. Ma X, Pang Z, Zhou J. Acetylation and amination protect angiotensin 1-7 from physiological hydrolyzation and therefore increases its antitumor effects on lung cancer.. Mol Pharm 2018;15(6):2338-2347.
  21. Guan F, You Y, Li X, Robinson MA. A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using HILIC coupled to high resolution mass spectrometry.. Drug Test Anal 2019;11(9):1308-1325.
  22. Powell MF, Stewart T, Jr Otvos L. Peptide stability in drug development. II effect of single amino acid substitution and glycosylation on peptide reactivity in human serum.. Pharmaceutical Research: An Official Journal of the American Association of Pharmaceutical Scientists 1993;10:1268-1273.
  23. Bradley G, Naude RJ, Muramoto K, Yamauchi F, Oelofsen W. Ostrich (Struthio camelus) carboxypeptidase B: purification, kinetic properties and characterization of the pancreatic enzyme.. Int J Biochem Cell Biol 1996;28(5):521-529.
  24. Mock WL, Xu DH. Catalytic activity of carboxypeptidase B and of carboxypeptidase Y with anisylazoformyl substrates.. Bioorg Med Chem Lett 1999;9(2):187-192.
  25. Lyons PJ, Fricker LD. Carboxypeptidase O is a glycosylphosphatidylinositol-anchored intestinal peptidase with acidic amino acid specificity.. J Biol Chem 2011;286(45):39023-39032.
  26. Seidler J, Zinn N, Boehm ME, Lehmann WD. De novo sequencing of peptides by MS/MS.. Proteomics 2010;10(4):634-649.
  27. Yan Y, Kusalik AJ, Wu F-X. Recent developments in computational methods for de novo peptide sequencing from tandem mass spectrometry (MS/MS).. Protein Pept Lett 2015;22(11):983-991.
  28. Liang Y, Neta P, Yang X, Stein SE. Collision-induced dissociation of deprotonated peptides. Relative abundance of side-chain neutral losses, residue-specific product ions, and comparison with protonated peptides.. J Am Soc Mass Spectrom 2018;29(3):463-469.
  29. Reubsaet JLE, Beijnen JH, Bult A, Van Maanen RJ, Marchal JAD, Underberg WJM. Analytical techniques used to study the degradation of proteins and peptides: chemical instability.. J Pharm Biomed Anal 1998;17(6-7):955-978.
  30. Heinrich N, Albrecht E, Sandow J. Disposition of 3H-labeled buserelin continuously infused into rats.. Eur J Drug Metab Pharmacokinet 1996;21(4):345-350.
  31. Sasaki Y, Chiba T, Ambo A, Suzuki K. Degradation of deltorphins and their analogs by rat brain synaptosomal peptidases.. Chem Pharm Bull 1994;42(3):592-594.
  32. Negri L, Improta G. Distribution and metabolism of dermorphin in rats.. Pharmacol Res Commun 1984;16(12):1183-1191.
  33. Sasaki Y, Hosono M, Matsui M. On the degradation of dermorphin and D-Arg2-dermorphin analogs by a soluble rat brain extract.. Biochem Biophys Res Commun 1985;130(3):964-970.
  34. Scalia S, Salvadori S, Marastoni M. Reversed-phase HPLC study on the in vitro enzymic degradation of dermorphin.. Peptides 1986;7(2):247-251.
  35. Samii A, Bickel U, Stroth U, Pardridge WM. Blood-brain barrier transport of neuropeptides: analysis with a metabolically stable dermorphin analog.. Am J Physiol 1994;267:E124-E131.
  36. Szeto HH, Lovelace JL, Fridland G. In vivo pharmacokinetics of selective μ-opioid peptide agonists.. J Pharmacol Exp Ther 2001;298:57-61.
  37. Taylor A. Aminopeptidases - structure and function.. FASEB J 1993;7(2):290-298.
  38. Nandan A, Nampoothiri KM. Molecular advances in microbial aminopeptidases.. Bioresour Technol 2017;245(Pt B):1757-1765.
  39. Tanco S, Lorenzo J, Garcia-Pardo J. Proteome-derived peptide libraries to study the substrate specificity profiles of carboxypeptidases.. Mol Cell Proteomics 2013;12(8):2096-2110.
  40. Petrera A, Lai ZW, Schilling O. Carboxyterminal protein processing in health and disease: key actors and emerging technologies.. J Proteome Res 2014;13(11):4497-4504.

Citations

This article has been cited 1 times.
  1. Gómez-Guerrero NA, González-López NM, Zapata-Velásquez JD, Martínez-Ramírez JA, Rivera-Monroy ZJ, García-Castañeda JE. Synthetic Peptides in Doping Control: A Powerful Tool for an Analytical Challenge.. ACS Omega 2022 Nov 1;7(43):38193-38206.
    doi: 10.1021/acsomega.2c05296pubmed: 36340120google scholar: lookup
Subscribe to our newsletter
Join 99,357 horse owners like you who receive our email updates on horse health and nutrition.