Serial-omics characterization of equine urine

Abstract

Horse urine is easily collected and contains molecules readily measurable using mass spectrometry that can be used as biomarkers representative of health, disease or drug tampering. This study aimed at analyzing microliter levels of horse urine to purify, identify and quantify proteins, polar metabolites and non-polar lipids. Urine from a healthy 12 year old quarter horse mare on a diet of grass hay and vitamin/mineral supplements with limited pasture access was collected for serial-omics characterization. The urine was treated with methyl tert-butyl ether (MTBE) and methanol to partition into three distinct layers for protein, non-polar lipid and polar metabolite content from a single liquid-liquid extraction and was repeated two times. Each layer was analyzed by high performance liquid chromatography—high resolution tandem mass spectrometry (LC-MS/MS) to obtain protein sequence and relative protein levels as well as identify and quantify small polar metabolites and lipids. The results show 46 urine proteins, many related to normal kidney function, structural and circulatory proteins as well as 474 small polar metabolites but only 10 lipid molecules. Metabolites were mostly related to urea cycle and ammonia recycling as well as amino acid related pathways, plant diet specific molecules, etc. The few lipids represented triglycerides and phospholipids. These data show a complete mass spectrometry based—omics characterization of equine urine from a single 333 μL mid-stream urine aliquot. These omics data help serve as a baseline for healthy mare urine composition and the analyses can be used to monitor disease progression, health status, monitor drug use, etc.

Metabolomics in clinical and forensic toxicology, sports anti‐doping and veterinary residues

“…Yuan et al used an orthogonal ionisation approach to investigate the health status of the equine athlete when targeting compounds such as proteins, lipids and small polar metabolites. 134 The one-horse urine study revealed 46 proteins, 10 lipids and 474 small polar metabolites, which are indicative of a healthy mare. These findings can be used to track the health status of horses, specifically mares, and for future reference to other administration or health studies as a comparative measure.…”

“…12 Common metabolomics studies of the equine athlete look at its exercise physiology, and therefore, metabolites are measured for energy production and utilisation. 12,134 Investigations into the health status of an equine athlete will provide information on biomarkers of disease and healthy athletes. Yuan et al used an orthogonal ionisation approach to investigate the health status of the equine athlete when targeting compounds such as proteins, lipids and small polar metabolites.…”

“…The horse has shown advanced aerobic and muscular capabilities that has been isolated through breeding 12 . Common metabolomics studies of the equine athlete look at its exercise physiology, and therefore, metabolites are measured for energy production and utilisation 12,134 …”

“…Investigations into the health status of an equine athlete will provide information on biomarkers of disease and healthy athletes. Yuan et al used an orthogonal ionisation approach to investigate the health status of the equine athlete when targeting compounds such as proteins, lipids and small polar metabolites 134 . The one‐horse urine study revealed 46 proteins, 10 lipids and 474 small polar metabolites, which are indicative of a healthy mare.…”

DIA mass spectrometry characterizes urinary proteomics in neonatal and adult donkeys

“…The establishment of normal urinary protein profiles is critical for further investigation of diagnosing and monitoring the progression of diseases 4 . There has been normal urinary proteomics investigated in many species including human 3 , cattle 5 and horses 6 . In healthy cows, 1564 urinary proteins in total are identified and the majority proteins are categorized into cytoplastic, catalytic activity and metabolism 5 .…”