Identification of proteolytic enzymes in human urine

Beside compounds with low molecular weight protein drugs like erythropoietin (EPO) and insulin have been used by athletes to increase the chance of success. Recently, there was discussion about the possible abuse of proteases, because they might efficiently degrade these substances if added to the urine doping control sample. This and the fact, that proteases are easily available (over-the-counter drugs, dishwasher detergent, laundry powder etc.) are reasons why they may be of interest to athletes. In this study more than 100 urine samples of athletes as well as standards of proteases and many blank urine samples have been analysed. The proteolytic activity was determined using fluorescence-based detection kits and 1D-SDS-PAGE. Characteristic proteins were identified with mass spectrometry. Furthermore, blank urine samples were spiked with proteases. As a result endogenous and recombinant erythropoietin was eliminated completely. Beyond, it became evident that the urinary proteome depends on the pH value as well as on both the temperature and the period of storage. Resulting proteome pattern may misleadingly imply the abuse of proteases. Our results indicate that the addition of proteases can be discovered anyway. Many proteases (or their products of cleavage in the case of autolysis) may be identified in low concentrations using mass spectrometry (up to 5µg/mL). After in-gel-digestion with trypsin we focus on the detection of unambiguous nonhuman peptides. Einleitung (geändert)