Metabolomic Profiling from Formalin-Fixed, Paraffin-Embedded Tumor Tissue Using Targeted LC/MS/MS: Application in Sarcoma

The relatively new field of onco-metabolomics attempts to identify relationships between various cancer phenotypes and global metabolite content. Previous metabolomics studies utilized either nuclear magnetic resonance spectroscopy or gas chromatography/mass spectrometry, and analyzed metabolites present in urine and serum. However, direct metabolomic assessment of tumor tissues is important for determining altered metabolism in cancers. In this respect, the ability to obtain reliable data from archival specimens is desirable and has not been reported to date. In this feasibility study, we demonstrate the analysis of polar metabolites extracted directly from ten formalin-fixed, paraffin-embedded (FFPE) specimens, including five soft tissue sarcomas and five paired normal samples. Using targeted liquid chromatography-tandem mass spectrometry (LC/MS/MS) via selected reaction monitoring (SRM), we detect an average of 106 metabolites across the samples with excellent reproducibility and correlation between different sections of the same specimen. Unsupervised hierarchical clustering and principal components analysis reliably recovers a priori known tumor and normal tissue phenotypes, and supervised analysis identifies candidate metabolic markers supported by the literature. In addition, we find that diverse biochemical processes are well-represented in the list of detected metabolites. Our study supports the notion that reliable and broadly informative metabolomic data may be acquired from FFPE soft tissue sarcoma specimens, a finding that is likely to be extended to other malignancies

Serial-omics characterization of equine urine

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

Equine mare urine proteins identified by LC-MS/MS from a serial-omics liquid-liquid extraction.

<p>Equine mare urine proteins identified by LC-MS/MS from a serial-omics liquid-liquid extraction.</p

The workflow of the serial-omics analysis of horse urine from collection and MTBE extraction through high resolution tandem mass spectrometry (LC-MS/MS) analysis.

<p>Each phase of the MTBE extraction is used for either proteomics, metabolomics or lipidomics analyses using untargeted or targeted mass spectrometry in order to gather the—omics profile.</p

Top 60 equine mare urine metabolites by intensity from 474 total identified metabolites by LC-MS/MS from a serial-omics liquid-liquid extraction.

<p>Top 60 equine mare urine metabolites by intensity from 474 total identified metabolites by LC-MS/MS from a serial-omics liquid-liquid extraction.</p

Equine mare urine non-polar lipids identified by LC-MS/MS from a serial-omics liquid-liquid extraction.

<p>Equine mare urine non-polar lipids identified by LC-MS/MS from a serial-omics liquid-liquid extraction.</p

Serial-omics characterization of equine urine - Fig 2

<p><b>A)</b> Omics results from the urinary tract and specimen cup to the number of unique molecules identified from horse urine. A scatterplot showing the molecular weight distribution of lipids, metabolites and proteins. Reprinted from The Merck Manual for Pet Health under a CC BY license, with permission from Merck & Co., original copyright 2017. <b>B)</b> The ratio of metabolite:protein:lipid was approximately 10.0:1.0:0.22. The majority of urine content is small polar molecules (474) followed by a significant amount of proteins (46) and very low number of lipids (10). A short list of some example questions that can be addressed using a serial-omics approach from accessible bodily fluids such as urine, blood, etc.</p

Biological pathways represented by serial-omics of healthy horse mare urine.

<p><b>A)</b> Panther biological processes from proteomics data analysis. <b>B)</b> MetaboAnalyst metabolite enrichemnt sets from polar metabolomics data. <b>C)</b> The few identified lipids were represented by triglycerides and glycerophospholipids. <b>D)</b> Plant metabolites identified as horse diet specific.</p