Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Summary

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Abstract

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Introduction

The term "metabolomics" can encompass many things. For example, a metabolomics experiment can be performed using a variety of analytical platforms such as NMR and both gas and/or liquid chromatography coupled with mass spectrometry. Furthermore, metabolomics experiments can be performed in a targeted or non-targeted manner, or a combination of both. A targeted metabolomics experiment will involve directed analysis of a panel of molecules important to the biological question at hand (e.g. small molecules involved in the TCA cycle will allow for accurate quantitation of that pathway). In this situation, the biological hypothesis is dictating the choice of ....

Protocol

1. Sample Organization

1. To create a plate map for sample preparation on a spreadsheet, in the first column enter a sample list in order of loading. In a second column enter the 96 well plate locations using correct nomenclature for your autosampler software.
2. Save one well in each plate for QC samples.
3. If your LC autosampler can handle two plates at a time, separate the sample list into batches of 190 and save this information in a second worksheet.
4. Within each of these batches, use the randomize function [note: "=rand()"in Excel] to randomize the injection order. Now copy and paste your injection order into the LC-MS sample que....

Results

The basic analytical steps of a non-targeted metabolite profiling experiment by UPLC-MS are outlined in Figure 1. The raw data for each sample can be visualized as a base peak chromatogram. Figure 2 shows an example base peak chromatogram of a serum sample analyzed by gradient option (a) in the tutorial. Following statistical analysis as described above, metabolite identification is attempted for all statistically significant molecular features. Confident identification (level 1) req.......

Discussion

This tutorial is meant to serve as a starting point for conducting large scale non-targeted metabolite profiling by UPLC-MS. The workflow is focused on metabolites that can be extracted with an aqueous methanol solvent, retained on a C8 or C18 UPLC column, and detected as positive ions. In the situation where there is not a pre-determined bias towards a specific metabolite class and a hypothesis generating global profile is desired, this protocol is valuable as it will result in the detection of a large percentage of ser.......

Materials

Name	Company	Catalog Number	Comments
Name of Reagent/Material	Company	Catalog Number	Comments
96 well plates - 500 μl wells	VWR	40002-020	These are used for sample preparation
96 well plate mats	VWR	89026-514	These are used for sample preparation
96 well plates - 350 μl wells	Waters Corporation	WAT058943	These are used for sample injection
96 well plate mats	Waters Corporation	186000857	These are used for sample injection
96 well plate heat seals	Waters Corporation	186002789	These can be used for sample injection or long term storage
96 well plate heat sealer	Waters Corporation	186002786
LC-MS grade methanol	Fluka	34966
LC-MS grade acetonitrile	Fluka	34967
LC-MS grade aater	Fluka	39253
LC-MS grade formic acid	Fluka	56302
Multichannel electronic pipettor	VWR	89000-674
Pipett tips	Eclipse (purchased through Light Labs)	B-5061/B-4061
Chilled centrifuge - Allegra X-12R	Beckman Coulter	N/A - contact Beckman Coulter
Acquity Ultra performance Liquid Chromatography (UPLC) System	Waters Corporation	N/A - contact Waters Corporation
UPLC C8 column (gradient option a)	Waters Corporation	186002876
UplC T3 column (gradient option b)	Waters Corporation	186003536
Xevo G2 Q-TOF Mass spectrometer	Waters Corporation	N/A - contact Waters Corporation