title: Proteolysis and autolysis of proteases and the detection of degradation products in doping control.
abstract: The determination of protein- or peptide-based performance-enhancing pharmaceuticals has gained increasing attention in sports drug testing in recent decades. Proteases possess a high potential for use in the manipulation of regular urine specimens, interfering with established doping control procedures for proteins or peptides. Hence, the present approach was developed to identify degradation products directly from proteases with exogenous origin in urine samples by means of SDS-PAGE and capillary liquid chromatography-Orbitrap (tandem) mass spectrometry. The mass spectrometric identification was accomplished by database search considering the accurate monoisotopic precursor mass with data dependent MS/MS analysis and no setting for specific cleavage site(s) or enzyme(s). The main characteristics of the method were shown in an exemplary way for the proteases Papain, Bacillolysin, Trypsin and Subtilisin with sufficient results for specificity, limit of detection, working range, precision and recovery after incubation. All experiments were performed with fortified urine samples under consideration of assumed realistic conditions for adulteration by cheating sportsmen.
authors.affiliation: Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Thomas
authors.forename: Maxie
authors.initials: M
authors.lastname: Kohler
authors.forename: Katja
authors.initials: K
authors.lastname: Walpurgis
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods; D004300:Doping in Sports; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000032:analysis* / Q000378:metabolism*; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014556:Urine / Q000737:chemistry*
chemical_list: D010455:Peptides; D010447:Peptide Hydrolases