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## University of Birmingham

Skip to 0 minutes and 8 secondsCATHERINE WINDER: In this course, we will focus on one of the analytical techniques we use at the University of Birmingham to study the metabolome. That is mass spectrometry. Other analytical techniques are applied to study the metabolome, and we will provide an overview of these later on this week. But first, let us look at what mass spectrometry is and why it's important to study the metabolome. A mass spectrometer is a scientific instrument applied to separate positively or negatively charged metabolites in time or space based on their mass to charge ratio. Metabolites are constructed of elements like carbon, nitrogen, and hydrogen. Every element has a different mass.

Skip to 0 minutes and 54 secondsMass is normalised under the unified atomic mass scale so that the mass of carbon is 12. For example, hydrogen weighs about 1/12 of that of carbon. Metabolites are constructed with different elements and with different numbers of each element. This means that metabolites typically have different formulae and therefore different masses. However, some metabolites have the same formula and therefore the same mass. We call these isomers, and examples include glucose and fructose or leucine and isoleucine. Mass is the property that we measure using mass spectrometry. Although mass spectrometry measures the mass to charge ratio, many metabolites carry a single positive or negative charge, and therefore the mass is measured directly. Mass can be measured as the nominal mass.

Skip to 1 minute and 49 secondsThis is the mass of the ion or the molecule calculated using the mass of the most abundant isotope of each element rounded up to the nearest integer value. The nominal mass of glucose is 180. The monoisotopic mass is the exact mass of the molecule calculated from the accurate mass of the most abundant isotope of each element. This is normally reported to four decimal places, which is important in untargeted metabolomic studies to differentiate metabolites with the same nominal mass but a differ molecular formula and therefore monoisotopic mass. For example, glucose has a monoisotopic mass of 180.0634, whereas aspirin has a monoisotopic mass of 180.0422. The mass determined in the mass spectrometer measurements depends on the mass resolution of the instrument.

Skip to 2 minutes and 49 secondsIt is important in untargeted studies is to apply high mass resolution instruments to allow for the separated detection of metabolites with a similar but not identical monoisotopic mass, as seen for the example in glucose and aspirin. There are many different types of mass spectrometers, but they all have the same basic components, a sample inlet, an ionisation source, a mass analyzer, a detector, and a data analysis system, which is usually a personal computer. There are two important aspects of mass spectrometers which allow them to operate. Firstly, metabolites have to carry a positive or negative charge so that they can be easily manipulated in a magnetic or electrical field based on their mass to charge ratio.

Skip to 3 minutes and 43 secondsMetabolite ions of different mass to charge ratios are manipulated by the magnetic or electrical forces to traverse different pathways before detection. Secondly, all mass spectrometers work at a low vacuum pressure, at least 10 to the minus 6 or one millionth of an atmosphere. Operating at vacuum pressures prevents many ion-molecular collisions that would divert metabolite ions from their required path and therefore introduce error into the measurement of the mass to charge ratio. The first step in mass spectrometry analysis is the production of metabolite ions in the gas phase during the ionisation process. Electrospray ionisation is the ionisation method applied in many of the mass spectrometry techniques that we will discuss this week in the analysis of the metabolome.

Skip to 4 minutes and 39 secondsIt is suitable for a wide range of molecules and imparts very small amounts of energy to the analytes and results in minimal source fragmentation. Protonation or deprotonation occurs to produce either positive or negatively charged metabolite ions, though the addition of other ions apart from the proton can be observed, and we will call these adduct ions. Importantly, electrospray ionisation allows the high-pressure liquid chromatography method to be coupled to the vacuum region of the mass spectrometer via an atmospheric pressure electrospray ionisation source. This was a hugely important development which allowed us for the first time to analyse biological chemicals in liquid solutions continuously and with high reproducibility.

Skip to 5 minutes and 31 secondsFenn and Tanaka were awarded the Nobel Prize for chemistry in 2002 for the development of electrospray ionisation. In electrospray ionisation, the liquid containing the analytes is dispersed by electrospray. A strong electrical field is applied at the atmospheric pressure to a liquid passing through the capillary tube. Ions are formed in solution, and highly charged liquid droplets are formed in the electrical field at the end of the capillary. The charged droplets are dispersed into a spray by a nebulising gas and are further dried by this gas to remove the solvent molecules and allow ions to be dissolved into the gas phase.

Skip to 6 minutes and 14 secondsThe gas phase ions are focused into the vacuum region of the mass spectrometer via an orifice and separated to their mass to charge ratios in the mass analyzer and the number of ions counted by the detector. The resulting data is presented in a mass spectrum. This is a plot illustrating the abundance of the ion versus the mass to charge ratio. There are many mass analyzers such as the time-of-flight or the Orbitrap. Each has a different mass resolution capability. The performance of a mass analyzer is characterised by the mass range of the analyzer, the analysis speed-- this is the rate at which the analyzer measures each mass spectrum-- the mass accuracy, and the mass resolution.

Skip to 7 minutes and 4 secondsThe mass accuracy refers to the difference of the measured mass to charge ratio compared to the theoretical mass to charge ratio. It is usually expressed in the unit of parts per million. The mass accuracy of the instrument is connected to the mass resolution and calibration stability. The mass resolution is the ability to separate two metabolites with a similar monoisotopic mass, so in the high mass resolution we can detect peaks of very similar mass to charge ratios as discussed for glucose and aspirin. Quadrupole instruments have a low mass resolution. Time-of-flight instruments have medium mass resolution. And Orbitrap instruments have a high mass resolution.

Skip to 7 minutes and 55 secondsTherefore Orbitrap mass analyzers can separate metabolites with a smaller difference in mass to charge ratio than quadrupole mass analyzers. And as an example, the importance of high mass resolution in untargeted studies, we can detect 1.5 to 3 times more mass peaks in direct infusion mass spectrometry experiments when implying a mass resolution of 100,000 compared to 7,500. There have been many advances in the field of mass spectrometry over the past 20 years, including the development of atmospheric pressure ionisation sources, improvements to existing mass analyzers, and the introduction of a new mass analyzer, the Orbitrap, in 2005. And the development of hybrid instruments using a combination of mass analyzers in sequence to increase the versatility of instruments.

Skip to 8 minutes and 52 secondsImprovements in the sensitivity, detection limits, speed, and diversity of the instruments has opened up the application of mass spectrometry to analyse complex biological samples.

# Introduction to mass spectrometry

Dr Catherine Winder provides an introduction to mass spectrometry.