A03 Non-targeted analysis of the plasma proteome between early and late rugby playing career

Abstract

Plasma proteomics can offer objective biological insight into the physiological responses of athletes. Rugby players may be repeatedly exposed to high-impact collisions that could predispose them to a higher risk of neurodegenerative conditions. The potential mechanisms underlying the heightened risk are unclear and discovery proteomics could provide unbiased insight into the pathways involved. Plasma extracellular vesicles (EV) are a novel system of intercellular communication which have been implicated in the propagation of misfolded proteins in the brain and could carry putative diagnostic biomarkers. This project investigates whether differences in plasma EV proteome exist between early and late career rugby players. Twenty-four male participants were recruited, including 8 academy players (18 ± 1 years), 8 rugby players (33 ± 5 years) with 10-year professional rugby career and 8 CrossFit athletes (32 ± 5 years) with no history of collision-related sports injuries. Participants gave their informed consent to the ethically approved procedures. Venous blood samples were collected in EDTA blood sample tubes, according to standard operating procedures. Hyper-porous strong-anion exchange magnetic microparticles (MagReSyn® SAX) were used to capture membrane-bound particles (i.e. EV) from plasma. EV-enriched samples were digested with trypsin and analysed using nano-flow liquid chromatography and high-resolution tandem mass spectrometry (Thermo, U3000 and Q-Exactive mass spectrometer). Label free quantitation was performed using Progenesis QI-P (Waters Corp) and proteins were identified by Mascot (Matrix Sci) searches of the UniProt Knowledge base. Statistical differences were investigated by one-way analysis of variance (with correction for multiple testing) in R (v4.3.2). Functional enrichment analysis was performed using ShinyGO (0.80). In total, 449 proteins were confidently identified (false discovery rate; FDR <1%) and gene ontology profiling confirmed 414 of these proteins were of EV origin (Jensen COMPARTMENTS). One-way ANOVA highlighted 192 significantly (P < 0.05, q < 0.02) different proteins across the 3 participant groups, of which 24 proteins were specific to professional rugby players. Functional enrichment analysis revealed key pathways related to Vitamin digestion and absorption, complement and coagulation cascade, inflammatory response, neuron death and negative regulation of cell death (FDR=5%).In conclusion, proteomic profiling is able to detect robust differences between the plasma EV proteome of long-serving professional rugby players compared to younger rugby players or age-matched athletes that did not have a history of collision-related sports injuries. Our findings shed new light on processes affected by a professional rugby playing career, further exploitation of this type of analysis could be used to discover sport-specific biomarkers.