Metabolomics Basics: What is Metabolomics?

• Experimental Design

Because biological and systematic noise is significant in many metabolomics experiments, one cannot underestimate the importance of experimental design. Some important factors to consider are the number of samples required for statistical significance and confidence, number of biological and analytical replicates and the use of paired samples (untreated sample or time zero is its own control for treated or later time point).

• Sample preparation

Internal standards
Before extraction, samples are typically spiked with one or more internal standards to assess recovery during extraction and chromatography and artifacts such as ion suppression. Standards can also be used as a normalization method and to help with chromatographic alignment if chromatography is used. For metabolic fingerprinting, internal standards may be used that represent all major classes of compounds (e.g. carbohydrates, organic acids, steroids, amines, etc.) and for metabolic profiling, use of compounds that represent the metabolites of interest may be of interest.

Metabolite extraction
The overall goal of extraction is to dissolve metabolites of interest and inactivate enzymes that metabolize them. Therefore, it is important that samples are extracted soon after collection and that they are stored at -80^o C before and after extraction because of both biological and chemical instability.

For metabolic fingerprinting, the goal is to develop a reproducible method to extract the most comprehensive set of metabolites possible while removing the highest amount of proteins. This can be challenging because of the physio-chemical diversity and range of abundances of cellular, tissue and biofluidic metabolites.

For metabolic profiling, the goal is to develop a method to extract the set of metabolites of interest while removing the largest amount of protein. Considerations for metabolite extraction include the type of sample, metabolites of interest (targeted or comprehensive) and degree of protein removal.

Direct infusion mass spectrometry (DIMS) – NO SAMPLE PREPARATION NECESSARY
In direct infusion analysis, the analyte solution is in a syringe and flows to the ion source of the mass spectrometer using a syringe pump. Both APCI and ESI have been used to detect metabolites in complex biological samples. This type of analysis can be helpful with metabolites that are challenging to measure by GC-MS and can increase the throughput of the analysis.

Derivatization
Derivatization is most commonly used for GC-MS analysis. Derivatization is the process of chemically modifying a compound to produce a new compound which has properties that are suitable for analysis using a GC (or in some cases LC). Biological samples contain a variety of metabolites such as organic acids, sugars, sugar alcohols, amino acids and steroids some of which are not volatile. Derivatization will reduce their polarity to increase their stability and detectability and/or improve their volatility both of which contribute to their separation by GC and later detection by MS.

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• Separation Methods Coupled to Mass Spectrometry

Gas Chromatography (GC)
The oldest form of automated separation is gas chromatography. Gas chromatography uses a carrier gas as a mobile gas phase and a stationary liquid or polymer phase within a column. This method requires the analyte to be volatile in order to migrate through the capillary. The analyte can be naturally volatile or be rendered volatile via chemical derivatization. The advantages of using GC is that electron ionization (EI) can be used to ionize the molecules before they enter the mass spectrometer.

 

High Performance Liquid Chromatography (HPLC)
Liquid chromatography can reduce ion suppression by decreasing the number of competing analytes entering the mass spectrometer ion source at a given time. Liquid chromatography can separate metabolites that are not volatile, so a more diverse set of metabolites can be analyzed than with GC-MS. An overview of liquid chromatography (LC) columns offered by Thermo Fisher Scientific, with some recommendations based on the known physical and chemical properties of the analyte(s) and the mode of analysis, is available here.

 

 

Capillary electrophoresis (CE)
In capillary electrophoresis, metabolites are separated based on charge and size. Advantages include easily optimized resolution, small dead volume and minimal sample effluent consumption. CE has been shown to work better for analysis of polar and low abundance compounds in complex mixtures. Some researchers have trouble in robustly coupling CE to MS.

 

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• Analyte Detection

Ionization methods for Mass Spectrometry

In order to obtain a comprehensive LC-MS analysis for discovery metabolomics, positive and negative ion
modes are used either by switching between the two modes in the same or different runs. Thermo
Scientific ion traps offer fast polarity switching for this purpose.

 

Ionization methods that do not require sample preparation or upfront separation
The methods described below are advantageous because they do not require sample preparation or
separation before mass spectrometric analysis. Additionally, the methods are used at atmospheric
pressure. Consequently they increase the throughput of metabolomic analyses. Disadvantages of these
methods are due to the lack of upfront separation and include (1) inability to distinguish chemical isomers that have the same mass and (2) signal suppression or enhancement is more likely due to simultaneous introduction of all sample analytes and subsequent ion suppression.

Laser diode thermal desorption (LDTD)
The LDTD source contains an infrared diode laser with an X-Y moveable stage capable of holding one 96-well plate. Upon operation, the plunger powered by nitrogen inserts into a sample well, the programmable laser thermally desorbs the dried sample (2 μL spotted) by heating the back of the plate, and the desorbed neutral sample is passed by the carrier gas (air) along the transfer tube to the corona discharge needle to be ionized and then transmitted into the MS.

Desorption electrospray ionization (DESI)
DESI directs pneumatically assisted electrosprayed droplets onto a sample surface to produce ions. It is a gentle ionization method that produces electrospray-like spectra. This technique has been proven to work for polar and non-polar organic and biological compounds and enables in situ analysis of biological tissues. DESI is compatible with all Thermo Scientific mass spectrometers.

Direct analysis in real time (DART)
This technique uses an atmospheric-pressure ion source that permits analysis of gases, liquids, solids, or materials on surfaces in open air at ground potential under ambient conditions. Samples are exposed to the DART gas stream which will rapidly generate ions that are carried by the gas stream into the sampling orifice of the mass spectrometer atmospheric pressure interface. The gas stream can graze a solid sample surface or be reflected off the sample surface. Liquids can be sampled by dipping an object (such as a glass rod) into the liquid sample to be measured. Vapors are introduced directly into the DART gas stream. DART has been used with Thermo Scientific ion traps and LTQ Orbitrap.

Extractive Electrospray Ionization (EESI)
EESI is a method for the ionization of analytes in complex matrices. EESI does not suffer from ion suppression effects which is common for traditional electrospray ionization (ESI). The technique was established for the direct analysis of vaporized samples such as breath for clinical diagnosis. Desorption of analytes from surfaces with a gentle gas stream allows the fast and efficient investigation of biological samples, or the detection of explosives from surfaces in real time. For EESI, a solvent is sprayed by electrospray while the sample is introduced as vapor. Charge is transferred from the electrospray plume to the analytes which leads to their ionization.

GC-MS ionization methods

Electron impact (EI)
The advantages of EI are that there are extensive spectral libraries that exist for metabolite identification (see section Y), the technique has good sensitivity and unique fragmentation patterns. However, the molecular ion is commonly not detectable because the high energy fragmentation completely fragments the parent ion, which can make identification of unknown metabolites challenging. Also, the application of EI is restricted to thermally stable samples.

Chemical ionization (CI)
This method is less energetic than EI so it tends to produce more stable ions for easier determination of molecular weight of the metabolite. Quantification is almost impossible without internal standards.

LC-MS ionization methods

These methods are soft ionization, atmospheric pressure methods (unlike EI & CI) so that no fragmentation of the molecular ion occurs. For more comprehensive coverage of metabolites, ionization should be performed in both positive and negative ion modes. Thermo Scientific ion traps are known for their fast polarity switching.

Electrospray ionization (ESI)/Nano-ESI
ESI is the most common ionization method used with LC-MS. This method is best used for analyzing polar and ionic metabolites. It provides excellent quantitative analysis and high sensitivity. Nano-ESI is performed at lower flow rates such as 200nL/min which improves sensitivity and dynamic range.

Atmospheric pressure chemical ionization (APCI)
This technique is advantageous for analysis of nonpolar and neutral molecules that are thermally stable such as lipids and is tolerant of higher buffer concentrations. It can also be used with polar compounds so that a wide range of metabolites can be analyzed.

Atmospheric pressure photoionization (APPI)
APPI directly photoionizes metabolites and therefore can ionize compounds that cannot be ionized as effectively using other atmospheric pressure techniques such as less polar metabolites. This ion source uses low ionization energy so it causes minimal fragmentation and relatively few adducts form, which makes it easier to identify the metabolite. This technique has a large linear dynamic range and minimal ionization of air and solvents. This ion source was developed and is sold by Syagen Technology, Inc.and compatible with Thermo Scientific mass spectrometers.

Multimode ESI/APPI (ESPI)
This ion source was developed and is sold by Syagen Technology, Inc. and compatible with Thermo Scientific mass spectrometers.

Interchangeable ESI/APCI
The universal Thermo Scientific Ion Max source features rapidly interchangeable ESI and APCI probes.

Combination APCI/APPI
The PhotoMate combination APCI/APPI ion source from Thermo Scientific allows for effective ionization of non-polar compounds. This unique ion source provides greater flexibility and the capability to analyze a larger range of compounds without changing hardware.