Detection of proteases and their proteolysis and autolysis products in urine by LC-MS

The use of peptide hormones for performance enhancement is documented by positive doping control samples with e.g. human chorionic gonadotropin or erythropoietin and by confession of athletes or confiscation of peptide hormone products. With the development of doping control methods, dopers start searching for manipulation and masking strategies to circumvent the detection of prohibited substances in urine samples. One way to eliminate peptide hormones from doping control samples is the use of proteases that degrade all proteins in urine and beside the proteolytic activity usually also autolyse. A method was established and validated to detect proteases and their degradation products in urine by SDS-PAGE as a screening procedure and LC-MS(/MS) for confirmation and for identifying a specific protease in the sample. Two doping control urine samples were shown to be positive for bacillolysin and four seized products for bacillolysin, subtilisin and aspergillopepsin. Introduction Proteases are enzymes that are capable of circumventing the detection of peptide hormones from urine samples and the addition to urine is therefore prohibited by the World AntiDoping Agency (WADA) in the category “chemical and physical manipulation”. Proteases degrade proteins and different proteolytic enzymes have a different specificity in fragmenting proteins. Additionally, proteases with varying specificity and efficiency also have a different capability to autolyse and produce small peptides originating from the protease itself. Proteases are of vegetable or animal origin and in the past 20 years a large variety of xenobiotic proteases with specific properties in respect to activity at different pH and temperature ranges, different specificity and different efficiency in proteolyis and autolysis have been recombinantly produced. Washing powder is one of the usual protease products which demonstrates that products with proteases can easily be obtained by cheating