MSACL 2026 Abstract

Discovery and Targeted MS-Based Validation of Protein Biomarkers of rhEPO Misuse Independent of Hypoxic Exposure During Altitude Training

Vincent R. Richard (1), Peter Kubiniok (1, 2), Timon Geib (1), Laleh Ebrahimi (1,3), Foroughsadat Absar (1). Eileen Tudorica (1), Constantinos Blidjios (1), Vincent Lacasse (1,6), Thomas C. Bonne (4), Jacob Bejder (4), Nikolai B. Nordsborg (4), Christoph H. Borchers (1,5,6,7)
(1) Segal Cancer Proteomics Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada, (2) Quantivum Inc., Montreal, QC, (3) Division of Experimental Medicine, McGill University, Montreal, QC, Canada, (4). Department of Nutrition Exercise and Sports, University of Copenhagen, Copenhagen, Denmark, (5) Gerald Bronfman Department of Oncology, Jewish General Hospital, Montreal, QC, Canada, (6) Department of Pathology, McGill University, Montreal, QC, Canada, (7) Department of Experimental Medicine, McGill University, Montreal, QC, Canada

Vincent Richard, PhD (Presenter) Jewish General Hospital - Lady Davis Institute

Presenter Bio: Research scientist at the Jewish General Hospital Lady Davis Institute (McGill University). Proteomics service platform manager for the Segal Cancer Proteomics Centre. Hands-on experience with MS-based proteomics, metabolomics, lipidomics, and clinical analysis of small molecules.

Vincent Richard is a research associate and manager of the proteomics service platform of the Segal Cancer Proteomics Centre (SCPC) with extensive experience (10+ years, 30+ publications) in the use and development of analytical techniques related to mass spectrometry-based proteomics, metabolomics, and lipidomics. Vincent completed a PhD program in the department of Cell & Molecular Biology under the mentorship of Dr. Vladimir Titorenko at Concordia University (Montreal, QC) where he was awarded the prestigious Fredrick Banting and Charles Best Doctoral Student award from the Canadian Institutes of Health Research (CIHR), having ranked within the top 5% of all applicants. Vincent’s doctoral research utilized sub-cellular fractionation and high resolution MS-based lipidomics among other techniques to study processes regulating ageing and organismal lifespan. In 2015 Vincent joined the SCPC to conduct post-doctoral research related to quantitative mass spectrometry based-proteomics and clinical mass spectrometry having developed techniques for 2D-LC-MS/MS and immunoaffinity enrichment based targeted quantification of blood plasma proteins, and various other cancer related proteins (PDL1) using stable isotope labelled peptide standards (SIS). Additional research during this time centered on methods to quantify small molecule targets from dried blood spots for clinical analysis (plasma metanephrines, Geneva cocktail targets). Since 2019 Vincent has worked as a core facility manager responsible for facilitating and conducting numerous collaborative service projects for the LDI and the broader research community leading to co-authorship in high impact journals (Cell Metabolism, Breast Cancer Research, Molecular & Cellular Proteomics, etc).

Relevant Financial Disclosures (within past 24 months, reported on Jun 10, 2026) No relevant financial relationship(s) to disclose.

INTRODUCTION:
Recombinant human erythropoietin (rhEPO) is an erythropoiesis-stimulating hormone and a banned substance by both the United States Anti-Doping Agency (USADA) and the World Anti-Doping Agency (WADA). rhEPO is illegally used to improve athletic performance in terms of aerobic capacity (VO2 max), endurance, and recovery times. Current detection methods are costly, challenging, and struggle to detect more sophisticated blood doping modalities including rhEPO microdosing, and concomitant high-altitude training which induces hypoxia-related blood adaptations similar to rhEPO use. Our goal was to determine whether rhEPO induces erythrocyte protein alterations which can be used as sensitive and selective biomarkers for the detection of rhEPO misuse, independent of high-altitude training induced hypoxia, and to develop and validate MRM-based quantitative proteomic assays to detect rhEPO misuse.

METHODS:
This study utilized 849 athlete-derived erythrocyte samples from the University of Copenhagen Department of Nutrition Exercise and Sports biobank. Subjects were assigned to rhEPO (20 IU/kg) or placebo groups and trained at either sea-level or at high-altitude with weekly blood collection during two 4 week baseline, experimental, or follow-up periods. Protein extraction and tryptic digestion was conducted in a fully automated workflow utilizing an Agilent Bravo to perform protein aggregation capture. Discovery proteomics was conducted using an Evosep One – Bruker timsTOF HT platform operated in diaPASEF mode. DIA data was searched using DIA-NN v2.0 with an erythrocyte specific spectral library. This data was used to select peptides for targeted validation using an Agilent 6495D system with synthetic isotopically labelled standards.

RESULTS:
To improve the reproducibility and throughput of our sample processing workflow, we developed an automated protein extraction and PAC-based digestion method utilizing an Agilent Bravo, allowing two 96-well plates of samples to be completely processed in 40 minutes prior to overnight incubation for tryptic digestion, with minimal user intervention. All 849 samples, as well as 15 process controls, were processed in under four hours of hands-on time by a single operator. Although currently configured for erythrocyte samples, this approach can also be used to process plasma or dried blood specimens in a reproducible and high-throughput manner.

To identify putative biomarkers of rhEPO misuse, we compared untargeted longitudinal proteome profiles of either rhEPO or placebo groups that trained at either sea level or high altitude, from 39 athletes sampled weekly over 24 weeks. diaPASEF data were searched against a project-specific spectral library containing over 17,000 peptides from more than 2,000 proteins. In total, we quantified 1,673 proteins in the study cohort, with 900 consistently quantified across all samples. Proteins showing rhEPO-specific changes in expression were identified using a permutation-based linear regression model to establish longitudinal trends in protein expression, followed by fuzzy clustering-based refinement as well as supported vector machine (SVM) modeling, resulting in a list of 121 differentially expressed target proteins. Proteins were ranked according to resultant p-values, and a final panel of 40 protein targets for MRM assay development and validation was established, allowing sensitive and precise quantitation of these markers in a separate validation cohort of 191 samples with a median LLOQ in the mid-attomole range and assay precision below 15% CV. Targeted validation was performed by MRM identifying a sub-set of 5 protein biomarkers that could be used to the accurately predict rhEPO use independent of hypoxic exposure during altitude training (AUC (LOSO) – 0.76).

NOVEL ASPECT:
This study is the first to identify specific biomarkers of rhEPO microdosing using longitudinal discovery (DIA-PASEF) and MRM-based targeted quantitative proteomics in the context of high altitude training for sports.