Mass spectrometry

Mass spectrometry (MS) is an analytical technique useful for measuring the mass-to-charge ratio of ions of one or more molecules present in a sample. These measurements can often be used to calculate the exact molecular weight of the sample components as well. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.

Typically, mass spectrometers can be used to identify unknown compounds via molecular weight determination, to quantify known compounds, and to determine structure and chemical properties of molecules.

A mass spectrum will usually be presented as a vertical bar graph, in which each bar represents an ion having a specific mass-to-charge ratio (m/z) and the length of the bar indicates the relative abundance of the ion. The most intense ion is assigned an abundance of 100, and it is referred to as the base peak. Most of the ions formed in a mass spectrometer have a single charge, so the m/z value is equivalent to mass itself. Modern mass spectrometers easily distinguish (resolve) ions differing by only a single atomic mass unit (amu), and thus provide completely accurate values for the molecular mass of a compound. The highest-mass ion in a spectrum is normally considered to be the molecular ion, and lower-mass ions are fragments from the molecular ion, assuming the sample is a single pure compound.

Mass spectrometer

In order to measure the characteristics of individual molecules, a mass spectrometer converts them to ions so that they can be moved about and manipulated by external electric and magnetic fields. The three essential functions of a mass spectrometer, and the associated components, are:

  1. ionization source
  2. mass analyzer
  3. ion detection system

Because ions are very reactive and short-lived, their formation and manipulation must be conducted in a vacuum. Atmospheric pressure is around 760 torr (mm of mercury). The pressure under which ions may be handled is roughly 10-5 to 10-8 torr (less than a billionth of an atmosphere). Each of the three tasks listed above may be accomplished in different ways. In one common procedure, ionization is effected by a high energy beam of electrons, and ion separation is achieved by accelerating and focusing the ions in a beam, which is then bent by an external magnetic field. The ions are then detected electronically and the resulting information is stored and analyzed in a computer.

Stage 1: the ion source

The heart of the spectrometer is the ion source. Here molecules of the sample (black dots) are bombarded by electrons (light blue lines) issuing from a heated filament. This is called an EI (electron-impact) source. Gases and volatile liquid samples are allowed to leak into the ion source from a reservoir. Non-volatile solids and liquids may be introduced directly. Cations formed by the electron bombardment (red dots) are pushed away by a charged repeller plate (anions are attracted to it), and accelerated toward other electrodes, having slits through which the ions pass as a beam. Some of these ions fragment into smaller cations and neutral fragments. A perpendicular magnetic field deflects the ion beam in an arc whose radius is proportional to the mass of each ion. Lighter ions are deflected more than heavier ions. By varying the strength of the magnetic field, ions of different mass can be focused progressively on a detector fixed at the end of a curved tube (also under a high vacuum).

The role of the ion source is to ionize the molecules of the sample. Molecules are converted to gas-phase ions so that they can be moved about and manipulated by external electric and magnetic fields. Several ionization methods exist, of which the most common is electron impact ionization (EI). Sample molecules are initially vaporized. The gaseous sample is then transferred into a chamber where it is bombarded by an electron beam from a hot cathode. The energy can be varied, but in most cases it is set to around 70 electron volts. Under this bombardment, the beam knocks electrons out of the molecules, creating monovalent, positively charged ions. If more energy is transferred in this ionization phase fragmentation, i.e. the degradation of the molecular ion, occurs. The generated fragments are composed of fragment ions and neutral particles (radicals). In the subsequent steps, the mass spectrometer is able to determine the mass of the fragment ions and display it in a mass spectrum. This creates characteristic ion patterns for each substance.

In addition to EI, other ionization techniques exist, such as chemical ionization (CI), field ionization (FI) or photoionization (PI). Liquids and solids can also be bombarded with fast ions or atoms for ionization. The methods are called fast atom bombardment (FAB) and secondary ion mass spectrometry (SIMS).

Stage 2: the mass analyzer

In the next stage, the ions leave the ion source as a focused beam and fly into the so-called mass analyzer. Its task is to ensure that only ions of identical velocity reach the downstream detector, because only then can the mass of the particles be determined with precision.

There are a number of mass analyzers currently available, each of which has trade-offs relating to speed of operation, resolution of separation, and other operational requirements. The mass analyzer often works in concert with the ion detection system.

Their ion separator is a velocity filter, named Wien filter after its developer Wilhelm Wien. It consists of an upper, positively charged capacitor plate and a negatively charged lower plate. When ions fly through the Wien filter towards a pinhole, the Lorentz force directs them upwards and the electric force downwards. Only ions for which both forces are in balance fly straight through the pinhole, while ions that are too slow or too fast are deflected upwards or downwards and cannot pass. Other ion separation techniques use time-of-flight (TOF), quadrupole or cyclotron resonance analyzers.

Stage 3: the ion detection system

After the sorted ions fly through the pinhole, they enter another magnetic field of the detector. The Lorentz force effective there deflects the ions from their straight path into a curved one. This deviation leads the ions to the detector. This is where Faraday cups, photomultiplier tubes or photographic plates come into play. The greater the mass, the greater the radius of the curved path. When flying through the magnetic field, high-mass fragment ions end up on an outer track, smaller fragments on an inner one.

The extent of deflection is decisive for evaluation. Mass spectrometers do not measure the ions’ mass as such, but the mass-to-charge ratio (m/z), which is plotted on the abscissa of the mass spectrum. In this spectrum, the isotopes of an element appear as separate peaks. For monovalent ions, the mass-to-charge value corresponds to the atomic mass unit u, while for ions with a higher valence, the mass is z times higher. In this way, each molecular compound creates a pattern that is as characteristic as a fingerprint. A comparison with mass spectra stored in databases subsequently lets almost any organic compound be reliably identified.

The separated ions are then measured and sent to a data system where the mass-to-charge ratios are stored together along with their relative abundance. A mass spectrum is simply the mass-to-charge ratios of the ions present in a sample plotted against their intensities. Each peak in a mass spectrum shows a component of unique mass-to-charge in the sample, and heights of the peaks connote the relative abundance of the various components in the sample.

Application areas for mass spectrometry

Mass spectrometry is an established technique in numerous industries. In chemistry the method is used to analyze chemical elements and molecules. It is so sensitive that very small amounts of substances down to one trillionth of a milligram (i.e., a femtogram) can be detected. The method is also popular with toxicologists who can identify poisons and drugs in blood, while environmental scientists find pollutants in soil samples. Biologists, on the other hand, use mass spectrometry for proteomics, i.e. to investigate proteins in living organisms. Physicists determine the mass of atomic nuclei. And archaeologists analyze the ratios of isotopes in bones to draw conclusions about the diet of the human or animal. Meanwhile, the technology is even used at airports to detect residues of explosives or drugs on passengers or luggage.

Research

  • MolDiscovery: learning mass spectrometry fragmentation of small molecules – Identification of small molecules is a critical task in various areas of life science. Recent advances in mass spectrometry have enabled the collection of tandem mass spectra of small molecules from hundreds of thousands of environments. To identify which molecules are present in a sample, one can search mass spectra collected from the sample against millions of molecular structures in small molecule databases. The existing approaches are based on chemistry domain knowledge, and they fail to explain many of the peaks in mass spectra of small molecules. Here, we present molDiscovery, a mass spectral database search method that improves both efficiency and accuracy of small molecule identification by learning a probabilistic model to match small molecules with their mass spectra. A search of over 8 million spectra from the Global Natural Product Social molecular networking infrastructure shows that molDiscovery correctly identify six times more unique small molecules than previous methods. DOI: https://doi.org/10.1038/s41467-021-23986-0 – Cao L., Guler M., Tagirdzhanov A. et al. Nat Commun 12, 3718 (2021).
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