Serial-omics characterization of equine urine.

Min Yuan,Susanne B Breitkopf,John M Asara,Andrea Motta

doi:10.1371/journal.pone.0186258

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.

Highlights

Over the last two decades, mass spectrometry has been used extensively to characterize the protein and small molecule content in biological samples [1]
When methyl tert-butyl ether (MTBE), methanol and water are added to urine, the protein precipitates to the bottom while the aqueous polar metabolites form a middle layer and the non-polar lipids form the top layer
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

Summary

Introduction

Over the last two decades, mass spectrometry has been used extensively to characterize the protein and small molecule content in biological samples [1]. Mass spectrometry including gas chromatography and liquid chromatography has been used extensively to profile molecules in urine, both from animals and humans [2, 3]. Methods have been developed and evaluated for preparing horse urine samples for small molecule analysis including precipitation and liquidliquid extraction methods [4]. High resolution mass spectrometry and speed of fragmentation scanning has allowed proteins to be profiled from many diseases tissues and mammalian proteomes including relative protein levels and post-translational modifications of proteins [1, 5,6,7]. Lipidomics profiling has recently become popular in present day—omics.

Objectives

Methods

Results

Conclusion