Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Summary

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Abstract

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Introduction

Due to recent technological advances in the field of HRMS, untargeted, hypothesis-generating metabolomics approaches have become a feasible approach to analysis of complex samples.¹ Mass spectrometers capable of 100,000 resolution facilitating routine low part per million (ppm) mass accuracy have become widely available from multiple vendors.^2,3 This mass accuracy allows greater specificity and confidence in a preliminary assignment of analyte identity, isotopic pattern recognition, and adduct identification.⁴ When coupled with an appropriate extraction procedure and high-performance LC or UPLC, complex mixtures can be analyzed with ad....

Protocol

1. Sample Extraction from Cells

1. For a 10 cm plate of cells: collect 1.5 ml of lifted cell suspension in media into a pre-labeled 10 ml glass centrifuge tube. For adherent lines, cells should be lifted with gentle scraping in 1.5 ml of media kept on ice. Optional: If internal standards are used, add an appropriate aliquot at this step.
   Comment: Quenching of cellular metabolism is crucial for certain metabolites. For analysis of time-sensitive metabolites, procedures such as cold methanol extraction should be considered.¹⁷
2. Add 6 ml of chloroform (CHCl₃)/methanol (CH₃OH) (2:1, v/v) to each o....

Results

The results presented show selected data from a 6-hr treatment of SH-SY5Y glioblastoma cells with the pesticide and mitochondrial complex I inhibitor rotenone. For brevity, only the organic phase positive mode data is presented. The samples were processed and analyzed as described above (Figure 1, Table 1, Table 2) and loaded onto two differential analysis platforms for label-free quantification, SIEVE and XCMS online. Although a large number of hits (Figure 2, Figure 3).......

Discussion

Untargeted metabolomics offers a powerful tool for investigating endogenous or xenobiotic biotransformations, or capturing a metabolic profile from a sample of interest. The output of the technique scales with the resolution and sensitivity of the technology used to separate and analyze the sample, the ability to deal with the large datasets generated, and the ability to mine the dataset for useful information (e.g. accurate mass database searching). Recently, this has been facilitated by advances in high resolu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent
Phosphate Buffered Saline	Mediatech	21-031-CM
Water (H2O)	Fisher Scientific	W7-4	(optima)
Acetonitrile (CH3CN)	Fisher Scientific	A996-4	(optima)
Methanol (CH3OH)	Fisher Scientific	A454-4	(optima)
Isopropanol	Fisher Scientific	A464-4	(optima)
Chloroform (CH3Cl)	Sigma-Aldrich	366927	Hazard
Dichloromethane (CH2Cl2)	Acros Organics	61030-1000	To replace chloroform
Diethyl Ether	Sigma-Aldrich	346136	To replace chloroform
Formic Acid (FA)	Fisher Scientific		(optima)
NH4OH	Fisher Scientific	A470-250	(optima)
Ammonium formate (HCOONH4)	Sigma-Aldrich	78314
MicroSpin C18 Columns	Nest Group Inc	SS18V
Pasteur Pipettes	Fisher Scientific	13-678-200
10 ml Glass Centrifuge Tubes	Kimble Chase	73785-10
10 ml Plastic Centrifuge Tubes	CellTreat	CLS-4301-015
LC Vials (glass)	Waters	60000751CV
LC Inserts (glass)	Waters	WAT094171
LC Vials (plastic)	Waters	186002640
0.22 μm Filters	Corning	8169	nylon
2 ml Eppendorf Tubes	BioExpress	C-3229-1	Low Retention
Equipment
High Resolution Mass Spectrometer	Thermo Scientific	LTQ XL-Orbitrap
HPLC/UPLC	Waters	nanoACQUITY UPLC
Source	Michrom	Thermo Advance Source
Differential Analysis Software	Thermo Scientific	SIEVE 2.0
nanoACQUITY C18 BEH130	Waters	186003546	1.7 μm particle size, 150 mm x 100 μm
Acentis Express C8	Sigma-Aldrich	54262	2.7 μm particle size, 15 cm x 200 μm