September 29, 2014

Tutorial: Mass spectrometry in plant science, part 1 – how a quadrupole produces a mass spectrum




            Mass spectrometry (MS) is one of the most widely used analytical techniques in chemistry, and it has wide applications in plant science. A mass spectrum can be used to identify an analyte (an object of analysis, such as a metabolite) by matching its pattern of mass fragments to the entries in a library of reference spectra. Understanding the fundamental principles behind mass spectrometry enables the proper interpretation of spectra and library search results and may assist in the identification of unknown plant metabolites. What follows is an introduction to the principles behind the experimental acquisition of a mass spectrum using an analyzer commonly found in most plant research institutes.

            MS is usually coupled to a chromatographic technique such as liquid or gas chromatography so that it can be used to resolve multiple spectra in a complex biological sample in series, in which each spectrum is acquired independently and reflects a single chemical species. Otherwise, spectra obtained from a mixture of compounds analyzed at once can rarely be interpreted. MS involves the ionization and fragmentation of compounds in a sealed chamber followed by their migration to a detector in a way that indicates the molecular masses of the resulting pieces. Ionization allows their movement to be controlled in an electric or magnetic field. The most common analyzer, the transmission quadrupole, uses a combination of voltage and radio waves to manipulate the motion of ionized species. Neutral fragments cannot be detected by MS directly, but their elimination from larger fragments can often be inferred from the mass spectrum. In the most common type of ionization, electron impact ionization (EI), a beam of electrons is used to strip off an electron from the target molecule as it leaves the chromatograph and enters the ionization chamber, leaving it with a positive charge. For the aromatic ring benzene, this reaction looks like this:



If only a single electron is lost, then the resulting mass-to-charge ratio (m/z) will be essentially the same as the molecular weight of that compound, usually denoted M+·. This molecular ion can undergo a variety of reactions, bond cleavages, and rearrangements to produce smaller fragments which appear as lighter mass peaks in the spectrum. Their m/z ratios reflect their particular chemical composition and can be used as clues to solve unknown chemical structures. Consider the isoprene mass spectrum below, which features a prominent peak at m/z 68 which reflects the intact molecular ion, the base peak at m/z 67 which indicates the loss of H·, and another prominent peak at m/z 53. The last peak results from the loss of a methyl group (CH3; 15 amu). Notice too that the molecular ion has an even mass but an odd number of electrons, whereas the fragment at m/z 53 (and most other fragments in the spectrum) has an odd mass and even number of electrons. These details will be covered in a subsequent post. Here, I want to focus on how this data is obtained.






The transmission quadrupole is composed of four parallel metal rods that oscillate through a range of voltage and radio frequency (RF) values. The ionization chamber sits at one end of the poles and the detector at the other.

To be detected, an ion must leave the ionization chamber and travel in a straight line to the detector and enter a small opening without veering off to the side and colliding with a rod or the wall of the chamber. For each voltage and RF combination, there is a corresponding fragment size that has the right m/z to maintain a straight path and enter the detector where a signal is registered. All other masses will follow curved paths under the influence of the poles and fail to reach the detector. As the analyzer scans through the range of m/z values, it registers the abundance of fragments in each mass window. A quadrupole can complete many scan cycles and produce multiple spectra during the elution of a single chromatographic peak, but the bias in the abundance of individual mass peaks caused by the slope of the chromatographic peak results in a minor experimental artefact known as spectral skewing. This problem is particular to quadrupoles but can be compensated for. Other types of analyzer, like time-of-flight and ion trap systems, avoid this problem by using a pulse ionization method. Because of the regularity of the ionization-induced decomposition into component fragments, mass spectra of the same compound acquired on different instruments will usually have a high degree of resemblance to each other if the conditions are similar. This makes it possible to search a library of standard spectra to identify an unknown. For an excellent in-depth explanation of the principles of MS, see “Understanding Mass Spectra: A Basic Approach” by R. Martin Smith (Wiley Interscience, 2nd edition). 

A plant extract may contain tens of thousands of distinct chemical species, and only a small portion will be detectable on any analytical platform. MS is the most broadly useful detector system and is often coupled to gas and liquid chromatography and electrophoretic separations. Compared to other chemical detection techniques (absorbance, NMR, fluorescence, flame ionization, IR spectroscopy, etc.), MS provides a practical combination of detailed structural information, sensitivity and wide applicability to different metabolites that makes it an essential technique in plant research. This brief introduction to the technique covered how a common analyzer like a quadrupole obtains the data in the mass spectrum. The next post in this series will explain how this mass spectrum can reveal useful information in an isotopic labeling experiments.

1 comment:

  1. I found this post very useful and interesting since definitely a deep knowledge of the analytical techniques can help on data interpretation and understand limitations of each technique. Looking forward for the next post.

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