Establishing Personalized Blood Protein Reference Ranges Using Noninvasive Microsampling and Targeted Proteomics: Implications for Antidoping 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 the effects of doping rather than detect the doping substance or the method itself. In the context of this program, a highly multiplexed mass spectrometry-based proteomics assay for 319 peptides corresponding to 250 proteins was developed, including proteins associated with blood-doping practices. "Baseline" expression profiles of these potential biomarkers in capillary blood (dried blood spots (DBS)) were established using multiple reaction monitoring (MRM). Combining DBS microsampling 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 data were collected from 10 healthy athlete volunteers over a period of 140 days (28 time points per participant). These comprehensive findings provide a personalized targeted blood proteome "fingerprint" showcasing that the targeted 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.