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Direct infusion mass spectrometry

The application of mass spectrometry to analyse metabolome samples
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MARK VIANT: Direct infusion mass spectrometry, otherwise known as DIMS, is the most simple application of electrospray ionisation mass spectrometry in metabolomics. In this approach, the sample is infused directly into the ion source of the mass spectrometer without introducing any bias caused by chromatographic separation. Since no chromatography is used, the sample analysis time can be much shorter. And therefore, the sample throughput is higher compared to either liquid or gas chromatography mass spectrometry approaches. For example, in Birmingham we can analyse more than 1,000 samples each week using our direct infusion mass spec approaches. DIMS can be applied in scientific research for both targeted and untargeted studies.
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To maximise the potential of this technique for untargeted metabolomics, it’s essential to use a mass spectrometer with both high-mass resolution and high-mass accuracy. Fortunately, advances in the scientific instrumentation over the last 20 years have provided us with the capability to do this by trapping ions in a mass analyser, and then measuring the orbital frequency of ions of different mass to charge ratios. Such instruments include Fourier transform ion cyclotorn resonance mass spectrometry, also known as FTICR, and the Orbitrap mass spectrometer. In theory, by using approaches that acquire data at very high-mass resolution, it’s possible to detect and identify a greater number of metabolites with very similar masses.
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If data acquisition is performed at low-mass resolution and metabolites with similar masses are detected as a single peak in the mass spectrum, then it wouldn’t be possible to measure those metabolites uniquely.
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At Birmingham, one of the instruments we use in our DIMS pipeline is an FTICR mass spectrometer. This instrument operates under an ultra high vacuum, typically a billionth of atmospheric pressure, and with a very strong, seven Tesla magnetic field. The mass to charge ratio of each ion is determined by measuring the orbital frequency of that ion in the magnetic field. Ions typically travel many kilometres during the mass measurement process. Metabolites are present across a wide range of concentrations in biological samples. For example, some are present at high concentrations, such as amino acids, and others occur at very low trace concentrations, such as hormones.
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In mass spectrometers that operate by trapping ions and measuring orbital frequencies, the mass accuracy can be adversely reduced if there are too many ions trapped at any one time. This is because when too many ions are forced into the same space in the mass analyzer they repel each other, causing unwanted space charge effects. And this can change the orbital motion of those ions, and it leads to degraded or reduced mass accuracy. To simply infuse a biological sample and collect data across the complete range of mass to charge ratios from low to high mass metabolites would result in a low-quality mass spectrum with poor mass accuracy of the data.
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So to overcome this problem and utilise these mass spectrometers to their full potential, we developed a method called mass spectral stitching. Using this novel approach, the metabolomics data is collected in about a dozen mass spectral windows, where each window is just a small mass to charge range. This ensures that we only track a relatively small number of ions, and therefore this method doesn’t lead to the adverse space charge effects. By limiting the mass to charge range of each window, the data can be collected across a wider dynamic range, meaning we can measure metabolites both at low and at high concentrations without compromising the mass accuracy.
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To acquire this type of data requires a constant infusion of the liquid sample for up to about five minutes. To minimise the volume of sample required, we use a nano-electrospray ion source instead of an electrospray ion source. In the nano-electrospray source the sample enters the mass spectrometer at a flow rate of about 300 nanolitres per minute, whereas for electrospray sources it typically flows at around 500 microlitres per minute. The nano-electrospray direct infusion mass spectrometry approach allows the analysis of very tiny samples. For example, we routinely use it to measure the metabolism of single water fleas that are only a couple of millimetres long and have a mass of about one milligramme.
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An additional advantage of nano-electrospray is that ion suppression is reduced compared to in conventional electrospray ion sources. After collection of this DIMS data using our optimised approach, the spectral windows are stitched together using computational algorithms to construct a single mass spectrum. The steps involved in the processing and analysis of this type of DIMS data will be discussed later in the course. The resulting stitched mass spectrum highlights the advantages of the DIMS approach. The spectrum contains a broader range of metabolites that are detected with a higher mass accuracy than would be obtained using conventional mass spectrometry approaches.
Professor Mark Viant describes the application of direct infusion mass spectrometry to analyse the metabolome of biological samples and explains how scientists at the University of Birmingham are optimising the approach to increase the number of metabolites detected.
For further information relating to the techniques covered in the video see the links below
Daphnia magna – Birth of the Next Generation
TriVersa NanoMate®: Chip-based Infusion
Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
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Metabolomics: Understanding Metabolism in the 21st Century

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