Establishing personalized blood protein reference ranges using non-invasive microsampling and targeted proteomics: Implications for anti-doping strategies

To prevent doping practices in sports, the World Anti-Doping Agency implemented the Athlete Biological Passport (ABP) program, monitoring biological variables over time to indirectly reveal effects of doping rather than detect the doping substance or method itself. In the context of this program and as part of the Partnership for Clean Competition, we developed a highly multiplexed mass spectrometry-based proteomics assay for 319 peptides corresponding to 250 proteins, including proteins associated with blood doping practices. We determined the ‘baseline’ expression profiles of these potential biomarkers in capillary blood (dried blood spots, DBS) using multiple reaction monitoring (MRM). Combining DBS micro-sampling with highly multiplexed MRM assays is the best suited technology to enhance the effectiveness of the ABP program, as it represents a cost-effective and robust alternative analytical method with high specificity and selectivity of targets in the attomole range. DBS were collected from 10 healthy athletes over a period of 140 days (28 time points per participant). These comprehensive findings provide a personalized blood proteome "fingerprint" showcasing that the proteome is unique to an individual, and likely comparable to a DNA-fingerprint. The results can serve as a baseline for future studies investigating doping-related perturbations.