Proteases in Doping Control Analysis

Abstract

Urine manipulation in sports drug testing has become a serious problem for doping control laboratories, and recent scandals in elite endurance sports have revealed the problem of urine manipulation presumably using proteases, which will impede the detection of drugs such as erythropoietin (EPO) or other peptide hormones. Using commonly accepted analytical strategies, a protocol was developed enabling the determination of elevated protease activities in doping control specimens followed by the visualization of protein degradation and identification of proteases such as chymotrypsin, trypsin and papain. Therefore, protease detection kits based on fluorescein isothiocyanate-labeled casein were employed, and protease concentrations greater than 15 µg/mL of urine entailed subsequent 1-dimensional gel electrophoretic visualization of urinary proteins. The presence of 20 µg of proteases per mL of urine caused a complete degradation of proteins usually observed in urinary matrices (“trace of burning”), while respective proteases were still detected in spiked urine samples after 10 days of storage at + 4 and - 20 °C. Identification of target proteases at respective molecular weights was accomplished using bottom-up sequencing approaches based on in-gel digestion of separated enzymes followed by capillary liquid chromatography - Orbitrap tandem mass spectrometry.

doping - mass spectrometry - proteomics - sport - urine

References

• 1 Breidbach A, Catlin D, Green G, Tregub I, Truong H, Gorzek J. Detection of recombinant human erythropoietin in urine by isoelectric focusing.  Clin Chem. 2003;  49 901-907
• 2 Burrows D L, Nicolaides A, Rice P J, Dufforc M, Johnson D A, Ferslew K E. Papain: a novel urine adulterant.  J Anal Toxicol. 2005;  29 275-295
• 3 Fledersbacher S. Die Tour ohne Doping? Unmöglich. [TV broadcasting] Mainz; ZDF 2006
• 4 Homer K A, Beighton D. Fluorometric determination of bacterial protease activity using fluorescein isothiocyanate-labeled proteins as substrates.  Anal Biochem. 1990;  191 133-137
• 5 Jelkmann W. Molecular biology of erythropoietin.  Intern Med. 2004;  43 649-659
• 6 Khan A, Grinyer J, Truong S T, Breen E J, Packer N H. New urinary EPO drug testing method using two-dimensional gel electrophoresis.  Clin Chim Acta. 2005;  358 119-130
• 7 Khan A, Packer N H. Simple urinary sample preparation for proteomic analysis.  J Proteome Res. 2006;  5 2824-2838
• 8 Lasne F, de Ceaurriz J. Recombinant erythropoietin in urine.  Nature. 2000;  405 635
• 9 Lasne F, Martin L, Crepin N, de Ceaurriz J. Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones.  Anal Biochem. 2002;  311 119-126
• 10 Oh J, Pyo J H, Jo E H, Hwang S I, Kang S C, Jung J H, Park E K, Kim S Y, Choi J Y, Lim J. Establishment of a near-standard two-dimensional human urine proteomic map.  Proteomics. 2004;  4 3485-3497
• 11 Pieper R, Gatlin C L, McGrath A M, Makusky A J, Mondal M, Seonarain M, Field E, Schatz C R, Estock M A, Ahmed N, Anderson N G, Steiner S. Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots.  Proteomics. 2004;  4 1159-1174
• 12 Raab W P. Enzymes and isoenzymes in urine. Dubach U Enzymes in Urine and Kidney. Bern; Huber 1968: 17-69
• 13 Ravela S, Valmu L, Stenman U-H. Presentation at the 17th International Mass Spectrometry Conference. Prague; 2006
• 14 Sahli W. Über das Vorkommen von Pepsin und Trypsin in normalen menschlichen Harn.  Pflügers Arch ges Physiol. 1885;  36 209-214
• 15 See W A, Smith J L. Activated proteolytic enzymes in the urine of whole organ pancreas transplant patients with duodenocystostomy.  Transplant Proc. 1991;  23 1615-1616
• 16 See W A, Smith J L. Urinary levels of activated trypsin in whole-organ pancreas transplant patients with duodenocystostomies.  Transplantation. 1991;  52 630-633
• 17 See W A, Smith J L. Urinary trypsin levels observed in pancreas transplant patients with duodenocystostomies promote in vitro fibrinolysis and in vivo bacterial adherence to urothelial surfaces.  Urol Res. 1992;  20 409-413
• 18 Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.  Anal Chem. 1996;  68 850-858
• 19 Scholer A. The effect of urine manipulation on substance abuse testing.  T + K. 2004;  71 127-137
• 20 Spahr C S, Davis M T, McGinley M D, Robinson J H, Bures E J, Beierle J, Mort J, Courchesne P L, Chen K, Wahl R C, Yu W, Luethy R, Patterson S D. Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry I. Profiling an unfractionated tryptic digest.  Proteomics. 2001;  1 93-107
• 21 Thevis M, Schänzer W. Identification and characterization of peptides and proteins in doping control analysis.  Curr Proteomics. 2005;  2 191-208
• 22 Twining S S. Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes.  Anal Biochem. 1984;  143 30-34
• 23 Vartio T. Proteolytic activity of human serum and urine at various pH's.  Ann Med Exp Biol Fenn. 1961;  39 115-119
• 24 World Anti-Doping Agency . The 2006 prohibited list.  . 2006; 
• 25 Wu A H, Bristol B, Sexton K, Cassella-McLane G, Holtman V, Hill D W. Adulteration of urine by “Urine Luck”.  Clin Chem. 1999;  45 1051-1057