Serial-omics characterization of equine urine and mane hair by LC-MS/MS

Introduction 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. Mane hair from a 11 year old appendix gelding was extracted with MTBE for metabolomics, lipidomics and proteomics and -omics integration. These data help serve as a baseline for healthy equine composition and can be used to monitor disease progression, health status, drug use, etc. Methods 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) and triple quadrupole SRM based technology to obtain protein sequence and relative protein levels as well as identify and quantify small polar metabolites and lipids. The same analysis was attempted with mane hair from a 11 yr old appendix gelding.  Preliminary Data 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. The top layer contained lipid molecules and these profiled using a QExactive HF in DDA LC-MS/MS mode with polarity switching - very few lipids were obtained from urine, as expected. The middle layer was the aqueous polar metabolite layer was analyzed by both untargeted metabolomics and targeted metabolomics-this fraction yielded the largest number of identified molecules from urine. The bottom pellet contained protein and was analyzed by high resolution LC-MS/MS in positive mode and searched against the horse Uniprot database. It is important to note that the majority of global urine -omics studies have taken place from human and mouse urine samples while most horse urine studies have focused on specific targeted compounds. However, we expect that many mammals should have a somewhat similar urine profile as far as it concerns the major metabolites and proteins. However, diet also plays a crucial role as well as differences in the digestion system between horses and humans. As a result, we found a significant number of plant related metabolites due to a diet of hay and vegetation. These analyses demonstrate a baseline -omics analysis from horse urine and can be used as a reference for future results. This technique can be applied in discovering the presence of a diseased or drug administered-horse from the norm by comparing alterations in the metabolites, lipids and proteins.  Novel Aspect First Serial-omics (multi-omics:metabolomics, lipidomics, proteomics) analyses from a single sample of equine urine and hair for biomarker identification and profiling