An enhanced label‐free proteomics approach for deep‐diving into equine plasma proteome, including the discovery of protein biomarkers for strenuous exercise

Plasma proteins have been a valuable source of biomarkers for clinical uses and for monitoring of the illicit use of prohibited substances or practices in equine sports. We have previously reported the first use of label-free proteomics in profiling equine plasma proteome. This study aimed to refine the method by systematically evaluating various plasma fractionation methods and the use of narrower precursor mass ranges in data-independent acquisition (DIA) mass spectrometry (MS). Tandem fractionations of equine plasma with octanoic acid precipitation followed by solid-phase extraction (SPE) with C4 cartridges provided the largest increase in the number of new proteins identified. The use of two narrow precursor mass ranges of m/z 400-600 and 600-800 in DIA not only identified most proteins detectable by using a single mass range of m/z 350-1500 but also identified ~27% more proteins. The improved method was applied to analyse the plasma proteome of 'postrace' samples which, unlike other samples, had been collected from racehorses soon after racing. Multivariate data analysis has identified upregulation of 14 proteins and downregulation of six proteins in postrace plasma compared with the non-postrace plasma samples. Literature review of these proteins has provided evidence of exercise-induced haemolysis and changes in antioxidant enzyme activities, kinin system, insulin signalling and energy metabolism after strenuous exercise. The improved method has enabled a deeper profiling of the equine plasma proteome and identified the proteins associated with normal physiological changes after racing which are potential confounding factors in the development of a biomarker approach for doping control.