The development of mass spectrometry (MS) as we know it today was made possible by the groundbreaking work of three exceptional men: Wilhelm Wien, J.J. Thomson, and Francis Aston. The 1907 paper by Thomson “On rays of positive electricity”1 is frequently cited as the pivotal work that announced the emergence of MS, although there are several other works that bear consideration as well. The tale of MS’s history is fascinating on its own, and readers who are interested in learning more are directed to Griffiths2 and Munzenberg’s 3 writings.
Since then, MS has benefited from important technological advancements. Today, MS affects a wide range of aspects of daily life. The analysis of food products to ensure food safety and that food we consume has not been tainted with chemicals that are harmful . Many of these will be tested by MS when we see a doctor and body fluids are extracted for testing. The use of MS to detect suspected bombs before they board an aeroplane helps ensure our safety in the skies. Additionally, it can help with many elements of the petrochemical industry, including the design and quality control of pharmaceuticals and biopharmaceuticals, as well as the detection of contaminants like PFAS in our food supply, wine, and water. The possibilities are essentially limitless.
How does mass spectrometry operate, and what is it?
MS measures the mass-to-charge ratio (m/z) of atoms and/or molecules in a sample. There are numerous varieties of mass spectrometers, but they all share three characteristics (Figure 1). The first is a method for ionising atoms or molecules from the sample. It is required to make ions because the electric fields employed in mass spectrometers cannot control neutral species. Ion sources, which relate to various methods for achieving this, are used to describe all of them.
Mass spectrometry, ion sources:
- Gas phase techniques
- Desorption techniques
- Spray techniques
Mass analyser types:
- Mass spectrometry in tandem (tandem MS)
- Flight time (ToF)
- The quadrupole
- Magnetic sector
- Ion trap.
- The Orbitrap
Ion detector types:
- Electron Multiplier (EM)
- Faraday Cup (FC)
- Photomultiplier conversion Dynode
- Array detectors
Using other methods in addition to mass spectrometers:
- Mass spectrometry with gas chromatography (GC-MS)
- Mass spectrometry with liquid chromatography (LC-MS)
- Mass spectrometry with crosslinking (XL-MS)
- Mass spectrometry with hydrogen exchange (HX-MS)
- Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI)
How does mass spectrometry operate, and what is it?
There are numerous varieties of mass spectrometers, but they all share three characteristics . The first is a method for ionising atoms or molecules from the sample. It is required to make ions because neutral species cannot be controlled by the electric fields employed in mass spectrometers. Ion sources, which relate to a variety of methods for achieving this, are used to describe all of them.
The mass analyzer itself is the second element in any mass spectrometer. The m/z ratio of ions can be determined using a variety of techniques. The most popular analyzers are tandem quadrupole mass spectrometers, time-of-flight (ToF), and single quadrupole each with their own advantages and disadvantages.
The last element shared by all mass spectrometer systems is a method for counting or detecting the presence of ions with a certain m/z value. The most popular types of these devices, which are sometimes referred to as detectors, include electron multipliers, Faraday cups, channeltrons, and channel plates. Once more, each has a unique strength and weakness.
How to relate the ion source to the sample in order to produce the ions for measurement is the final aspect to take into account, especially given that all mass spectrometers must be operated in vacuum. The sample will occasionally additionally be kept in a vacuum, occasionally at atmospheric pressure (known as ambient MS techniques), and occasionally another type of separation technology will be utilised before the sample is introduced to the ionisation chamber. These three standard parts for mass spectrometers will be covered in more detail in the sections that follow.
Any MS analysis requires ionisation, for which there are numerous techniques appropriate for various sample types and purposes. These can be broadly classified into three categories: gas phase methods, desorption methods, and spray methods. The following provides a summary of each.
Electron ionization (EI): In order to interact effectively with the electrons created in a vacuum by a heated filament, analyte molecules must be in the vapour phase. EI is frequently used when samples are low in molecular weight and relatively volatile. It can be thought of as a pretty severe process of molecule fragmentation and ionisation. 6
Chemical ionisation (CI): A gas is introduced with a concentration greater than the analyte into an EI ionisation chamber. Several molecular ions will be created as a result of the carrier gas’s contact with the electrons. These molecular ions will then react with extra carrier gas to create further molecular ions. The analyte molecules will then interact with these ions in a variety of ways to create analyte molecular ions. CI is a very gentle ionisation method that does not result in significant fragmentation.
– matrix-assisted laser desorption ionisation (MALDI), in which an extra “matrix” that depends on the kind of molecule to be found is added to the sample to be examined. The analyte molecules are then vaporised with little to no fragmentation or disintegration by exposing the sample to laser radiation. Ions can be produced that are both positively and negatively charged. One of the main “soft” ionisation techniques, MALDI, is especially beneficial for the study of large or labile molecules. 9
Liquid metal ion sources (LMIS): These are low-melting-point metals, frequently Ga, that used because they may create ions at a small point source when heat and an electric field are applied to them. The smallest spot sizes and greatest brightness of the ion beams produced by LIMS make them particularly beneficial for MS imaging, which demands excellent spatial resolution.
Electrospray ionisation (ESI): This process involves shrinking a mist of charged droplets through solvent evaporation until gas-phase ions are released. This soft ionisation method can be used to analyse macromolecules and large molecules.
Desorption electrospray ionisation (DESI) is very similar to electrospray ionisation (ESI), with the exception that the charged droplets produced in the ESI source are directed to a material that is kept at room temperature. The desorbed and ionised material is then carried by reflected droplets.
Mass analyzer types
The ions must then be separated after sample ionisation, which takes place in the mass analyzer. Typical mass spectrometers include:
Tandem mass spectrometry, sometimes known as tandem MS or MS/MS, is a hybrid technique that uses more than one type of mass spectrometer to improve selectivity and/or mass resolving capacity.
Time-of-flight (ToF): Ions are separated depending on the time it takes them to pass through a flight tube of a defined length and arrive at a detector in order to determine their m/z ratio.
Quadrupole: As ions enter the quadrupole, electrical potential causes a proportionate m/z-dependent deflection of their trajectories. Only ions with a certain m/z value can enter the chamber and be identified by adjusting the voltage.
Magnetic sector: Similar to how a glass prism divides light into its various wavelengths or hues, magnetic fields scatter ions in trajectories based on their m/z ratios.
Ion traps function similarly to quadrupoles, but instead of detecting ions with stable oscillations, they separate and detect ions by discharging those with unstable oscillations from the system and into the detector.
Orbitrap uses technology that is common to several different kinds of mass analyzers. With a centre electrode that resembles a spindle and two electrically isolated exterior electrodes in the form of cups facing one another, ions with a certain mass-to-charge ratio form orbiting rings. The outer electrodes are employed for current sensing after the conical form of the electrodes forces ions toward the broadest area of the trap. It is the only approach discussed here that detects the ions using an image current rather than a detecting device.
Combining mass analyzers and ionisation processes in different types of mass spectrometers
There are countless permutations and combinations of systems that could be developed with some technical work given the wide variety of ion sources, ionisation mechanisms, and mass analyzers. However, some ionisation sources and mass analyzers are perfect complements to one another, and these make up the majority of commercially available equipment. For instance, a TOF mass analyzer, which needs a pulsed ion source as its foundation for mass discrimination, fits perfectly with the pulsed nature of many laser systems. In more detail, this section will examine some of the typical source and mass spectrometer combinations.
As was already established, this pair of ionisation mechanisms and mass analysis are perfectly matched to one another due to the pulsed nature of many laser systems and the need for ToF analysis. The matrix/sample spot, which is kept in vacuum, receives a laser pulse, which causes the ions to form and accelerate into the ToF flight tube. The measurement of the mass spectrum occurs as the “clock” “starts.”
The approach can also produce images by scanning the stage stepwise, continuously while the laser is being fired repeatedly, or by scanning the laser beam.16 The resulting images can provide a plethora of information about samples such as large tissue sections.
Because MALDI uses a soft ionisation approach rather than a hard one like fluorescence microscopy, molecular information is preserved. As a result, it offers a method for “label-free” imaging.
The bulk of ICP-MS systems currently use TOF mass analyzers, although they were initially used with quadrupole mass analyzers. The main benefit is that, in comparison to systems using quadrupoles, the whole mass spectrum is created faster and with a greater mass resolution. A few specialised systems make use of magnetic sector instruments, frequently in conjunction with multicollector detection systems, which are utilised to make highly accurate measurements of isotope ratios.
The method can also be modified to create images as a result of the mass analysis of the ablated material by combining with a laser beam to create laser ablation (LA)-ICP-MS. The ability to mine and process the ToF data in the past is very advantageous because this is a destructive approach and the material can only be examined once. ToF imaging stores the whole mass spectrum in each (x, y) pixel location of the final image, making it simple to create fresh ion images after analysis.
Ion detector types
The type of detector used to transform a stream of mass-separated ions into a detectable signal is a crucial component of all MS systems. Depending on variables including dynamic range, spatial information retention, noise, and compatibility with the mass analyzer, different types of detectors are utilised.
The following detectors are frequently used:
“electromultiplier” (EM): A serial connection of discrete metal plates that amplify an ionic current by a factor of 108 into a detectable current of electrons.
Faraday cup (FC): ions striking the collector result in a flow of electrons from ground through the resistor, amplifying the potential drop that follows across the resistor.
Photomultiplier conversion dynode: When ions hit a dynode, they first release electrons. When the generated electrons hit a phosphor screen, photons are then released. The photons then enter the multiplier, where amplification takes place in a cascade-like pattern, much like the electromagnetic field (EM).
Array detectors cover a wide range of detector types and systems that may integrate several detection methods, such as detectors for the simultaneous measurement of numerous ions of different m/z and detectors for position-sensitive ion detection.
Using other methods in addition to mass spectrometers
Techniques for gas and liquid separation are frequently used with MS to improve sensitivity and facilitate interpretation. Examples include capillary electrophoresis (CE), gel electrophoresis (GE), liquid chromatography (LC), gas chromatography (GC), and capillary electrophoresis. Particularly frequently used in tandem with ICP-MS and DART-MS is the technique combination.
Mass spectrometry using gas chromatography (GC-MS)
A complicated combination of substances is injected into a column and separated using the analytical/separation process based on their affinity for a chromatographic column and respective boiling points.
High molecular weight molecules, such as proteins, are not appropriate for GC due to the high temperatures utilised since heat denatures them. It is ideal for application in industrial chemicals, environmental monitoring, and remedial areas. Solid, liquid, or gaseous samples are all acceptable.
Following separation, the chemicals can be evaluated for identification using a mass spectrometric method like ICP-MS or ionised using EI or CI and examined in a single or triple quadrupole mass spectrometer. A recent review of GC-MS advancements was published.
Mass spectrometry using liquid chromatography (LC-MS)
Contrary to gas phase chromatography, liquid phase chromatography uses a sample that is in a liquid phase. The sample is dissolved in a solvent before being injected into a chromatographic column that has a stationary phase made of solid materials and a mobile phase made of solubilized chemicals.
As the effluents are in the liquid phase, this separation approach lends itself particularly well to being combined with ICP-MS and ESI-MS procedures, but is also found linked to ion trap and Orbitrap mass spectrometers as well. Pitt (19) and, more recently, Seger (20) have addressed the concepts and applications of clinical biochemistry, while Korfmacher has studied the applications of drug discovery.
Mass spectrometry for crosslinking (XL-MS)
Understanding multiprotein complexes’ structure and organisation is essential to comprehending how cells work. In addition to structural biology techniques like cryogenic electron microscopy (cryo-EM) and X-ray crystallography, chemical crosslinking coupled with mass spectrometry (XL-MS) is a technique that offers lower-resolution structural data.
In XL-MS, a crosslinking agent is used to treat a protein or protein complex, creating covalent bonds between particular functional groups in the protein. The broken-down protein(s) from the crosslinked protein are then digested, and the resulting mixture is examined using LC-MS techniques to identify the crosslinked peptides and ascertain their sequences. Crosslink locations reveal structural details about the system under investigation. Interpretation is nevertheless difficult because samples made this way include a far greater number of unusual chemical species than a digest of the non-crosslinked protein. With increasing sequence length, there are quadratically more possible crosslinked peptides. In spite of this, XL-MS can be a helpful tool for creating structural models of protein-protein interactions.22
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI)
The technique is known as matrix-assisted laser desorption/ionization mass spectrometry imaging and can produce images by scanning the stage in steps, continuously while the laser is being fired repeatedly, or by scanning the laser beam. MALDI-TOF is a fantastic tool for MS analysis (MALDI-MSI). With a spatial resolution ranging from 50 to 200 mm, the generated images can offer a plethora of information on huge tissue sections, for instance. Because MALDI uses a soft ionisation technique rather than a hard one like fluorescence microscopy, molecular information is preserved, negating the need to tag compounds of interest before detecting them. As a result, it offers a method of “label-free” imaging.
What does m/z indicate? What does a molecular ion peak mean?
The chemical formula for pentane is C5H12. Therefore, the molecule’s approximate mass is ((12 x 5) + (1 x 12)), or 72 unified atomic mass units (u), often known as atomic mass units (amu). Be aware that at m/z = 72 u, a peak with a relative intensity of less than 10% is visible on the mass spectrum. The molecular peak is at hand. In the source, the entire molecule was ionised as a single unit without any fragmentation. What about the other, higher peaks? These are the byproducts of pentane’s ionisation, which caused fragmentation. How should the next reported largest mass (at m/z = 57 u with 20% relative intensity) be understood? We can speculate that it might be C4H9 with a little help from mathematics. This would indicate that one of the CH3 groups was broken off during the ionisation process, leaving the C4H9 fragment as a molecular ion. Similar to this, it is possible to interpret the highest signal at m/z = 43 u as C3H7, which indicates that a C2H5 molecule was broken apart. This is comparable to either the CH3 or CH2 groups. Additionally, strong lines can be seen at m/z = 41 and 42. These result from extra Hs being taken during fragmentation away from the C3H7 molecular ion. This serves as the foundation for interpreting mass spectra and necessitates an understanding of both the parent molecule’s structure and chemistry. It is obvious that all of the existing biological materials could find this to be a difficult undertaking. Fortunately, mass spectra may be found in databases for many of these, making interpretation easier.
One more complication is more frequently seen in the mass spectra of elements and tiny 1% 13C.molecules. This is a result of the many isotopes of each element. We assumed that carbon has a mass of 12 u in the pentane case. Since carbon has two stable isotopes, one with a mass of 12 and the other of 13, this is not absolutely true (the atom contains an extra neutron). These two isotopes are naturally abundant in proportions of roughly 99% 12C and 1%13C. Therefore, if one were to look at the mass spectrum in this region using a hard ionisation technique, one would see peaks at m/z = 12 and 13, with the peak at 12 being around 100 times larger than the peak at m/z = 13. It should be noted that the peak at m/z = 13 might just as easily be caused by 12C1H, demonstrating the value of a mass spectrometer’s mass resolution.
However, having several isotopes of a single element can be advantageous. It serves as the foundation for HX-MS, which was previously reported, as well as multi-isotope imaging mass spectrometry,which generates isotope ratio images from samples after purposefully adding stable isotopes to molecules. The portions of the sample where the compound has been integrated are those where the isotope ratio is greater than the natural abundance. The stable isotopes 13C and 15N are frequently employed for this purpose.
Abbreviations for mass spectrometry:
CCD Charge couple device
CE Capillary electrophoresis
CI Chemical Ionization.
DART Direct Analysis is performed in real time
DC direct current
DESI Desorption Electrospray Ionization.
EI electron ionisation
EM Electron multiplier
ESI Electrospray Ionization
FAB Fast atom bombardment
FC Faraday Cup
GC gas chromatography.
GE Gel electrophoresis
HX-MS Hydrogen Exchange Mass Spectrometry
ICP inductively coupled plasma.
LC liquid chromatography.
MALDI Matrix-assisted laser desorption ionisation
MCP Microchannel Plate
MS mass spectrometry
RAE Resistive anode encoder
RF radio frequency
SIMS Secondary Ion Mass Spectrometry
ToF Time of Flight
XL-MS Mass spectrometry using cross-linking
1. Thomson JJ. XLVII. On rays of positive electricity. London, Edinburgh, Dublin Philos Mag J Sci. 1907;13(77):561-575. doi:10.1080/14786440709463633
2. Griffiths J. A Brief History of Mass Spectrometry. Anal Chem. 2008;80(15):5678-5683. doi:10.1021/ac8013065
3. Münzenberg G. Development of mass spectrometers from Thomson and Aston to present. Int J Mass Spectrom. 2013;349-350(1):9-18. doi:10.1016/j.ijms.2013.03.009
4. Koppenaal DW, Barinaga CJ, Denton MB, et al. MS Detectors. Anal Chem. 2005;77(21):418 A-427 A. doi:10.1021/ac053495p
5. John Roboz. A History of Ion Current Detectors for Mass Spectrometry. In: The Encyclopedia of Mass Spectrometry. Elsevier; 2016:183-188. doi:10.1016/B978-0-08-043848-1.00023-7
7. Munson B. Chemical Ionization Mass Spectrometry: Theory and Applications. In: Encyclopedia of Analytical Chemistry. John Wiley & Sons, Ltd; 2000. doi:10.1002/9780470027318.a6004
8. Cody RB, Laramée JA, Durst HD. Versatile New Ion Source for the Analysis of Materials in Open Air under Ambient Conditions. Anal Chem. 2005;77(8):2297-2302. doi:10.1021/ac050162j
9. Karas M, Bachmann D, Hillenkamp F. Influence of the wavelength in high-irradiance ultraviolet laser desorption mass spectrometry of organic molecules. Anal Chem. 1985;57(14):2935-2939. doi:10.1021/ac00291a042
10. Rinehart KL. Fast Atom Bombardment Mass Spectrometry. Science (80- ). 1982;218(4569):254-260. doi:10.1126/science.218.4569.254
11. Barber M, Bordoli RS, Sedgwick RD, Tyler AN. Fast atom bombardment of solids as an ion source in mass spectrometry. Nature. 1981;293(5830):270-275. doi:10.1038/293270a0
12. Ho CS, Lam CWK, Chan MHM, et al. Electrospray ionisation mass spectrometry: principles and clinical applications. Clin Biochem Rev. 2003;24(1):3-12. https://pubmed.ncbi.nlm.nih.gov/18568044
13. Banerjee S, Mazumdar S. Electrospray Ionization Mass Spectrometry: A Technique to Access the Information beyond the Molecular Weight of the Analyte. Int J Anal Chem. 2012;2012:1-40. doi:10.1155/2012/282574
14. Takáts Z, Wiseman JM, Cooks RG. Ambient mass spectrometry using desorption electrospray ionization (DESI): instrumentation, mechanisms and applications in forensics, chemistry, and biology. J Mass Spectrom. 2005;40(10):1261-1275. doi:10.1002/jms.922
15. Glish GL, Burinsky DJ. Hybrid mass spectrometers for tandem mass spectrometry. J Am Soc Mass Spectrom. 2008;19(2):161-172. doi:10.1016/j.jasms.2007.11.013
16. Caprioli RM, Farmer TB, Gile J. Molecular Imaging of Biological Samples: Localization of Peptides and Proteins Using MALDI-TOF MS. Anal Chem. 1997;69(23):4751-4760. doi:10.1021/ac970888i
17. Špánik I, Machyňáková A. Recent applications of gas chromatography with high-resolution mass spectrometry. J Sep Sci. 2018;41(1):163-179. doi:10.1002/jssc.201701016
18. Honour JW. Gas Chromatography-Mass Spectrometry. Methods Mol Biol. 2006:53-74. doi:10.1385/1-59259-986-9:53
19. Pitt JJ. Principles and applications of liquid chromatography-mass spectrometry in clinical biochemistry. Clin Biochem Rev. 2009;30(1):19-34. https://pubmed.ncbi.nlm.nih.gov/19224008
20. Seger C, Salzmann L. After another decade: LC–MS/MS became routine in clinical diagnostics. Clin Biochem. 2020;82:2-11. doi:10.1016/j.clinbiochem.2020.03.004
21. Korfmacher WA. Foundation review: Principles and applications of LC-MS in new drug discovery. Drug Discov Today. 2005;10(20):1357-1367. doi:10.1016/S1359-6446(05)03620-2
22. Merkley ED, Cort JR, Adkins JN. Cross-linking and mass spectrometry methodologies to facilitate structural biology: finding a path through the maze. J Struct Funct Genomics. 2013;14(3):77-90. doi:10.1007/s10969-013-9160-z
23. Marcsisin SR, Engen JR. Hydrogen exchange mass spectrometry: what is it and what can it tell us? Anal Bioanal Chem. 2010;397(3):967-972. doi:10.1007/s00216-010-3556-4
24. Mayne L. Hydrogen Exchange Mass Spectrometry. In: Methods Enzymol.; 2016:335-356. doi:10.1016/bs.mie.2015.06.035
25. Barnes JH, Hieftje GM. Recent advances in detector-array technology for mass spectrometry. Int J Mass Spectrom. 2004;238(1):33-46. doi:10.1016/j.ijms.2004.08.004
26. McMahon G, Glassner BJ, Lechene CP. Quantitative imaging of cells with multi-isotope imaging mass spectrometry (MIMS)-Nanoautography with stable isotope tracers. Appl Surf Sci. 2006;252(19). doi:10.1016/j.apsusc.2006.02.170
27. Lechene C, Hillion F, McMahon G, et al. High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry. J Biol. 2006;5. doi:10.1186/jbiol42