Mass spectrometry (MS) is an analytical technique that ionizes chemical species and sorts the ions based on their mass-to-charge ratio. In simpler terms, a mass spectrum measures the masses within a sample. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.
A mass spectrum is a plot of the ion signal as a function of the mass-to-charge ratio. These spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical structures of molecules, such as peptides and other chemical compounds.
In a typical MS procedure, a sample, which may be solid, liquid, or gas, is ionized, for example by bombarding it with electrons. This may cause some of the sample’s molecules to break into charged fragments. These ions are then separated according to their mass-to-charge ratio, typically by accelerating them and subjecting them to an electric or magnetic field: ions of the same mass-to-charge ratio will undergo the same amount of deflection.
The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier. Results are displayed as spectra of the relative abundance of detected ions as a function of the mass-to-charge ratio. The atoms or molecules in the sample can be identified by correlating known masses to the identified masses or through a characteristic fragmentation pattern.
Mass Spectrometry Applications
Mass spectrometry has both qualitative and quantitative uses. These include identifying unknown compounds, determining the isotopic composition of elements in a molecule, and determining the structure of a compound by observing its fragmentation.
Other uses include quantifying the amount of a compound in a sample or studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in a vacuum). MS is now in very common use in analytical laboratories that study physical, chemical, or biological properties of a great variety of compounds.
As an analytical technique it possesses distinct advantages such as:
- Increased sensitivity over most other analytical techniques because the analyzer, as a mass-charge filter, reduces background interference
- Excellent specificity from characteristic fragmentation patterns to identify unknowns or confirm the presence of suspected compounds
- Information about molecular weight
- Information about the isotopic abundance of elements
- Temporally resolved chemical data.
A few of the disadvantages of the method is that often fails to distinguish between optical and geometrical isomers and the positions of substituent in o-, m- and p- positions in an aromatic ring. Also, its scope is limited in identifying hydrocarbons that produce similar fragmented ions.
Pharmacokinetics is often studied using mass spectrometry because of the complex nature of the matrix (often bloodv or urine) and the need for high sensitivity to observe low dose and long time point data. The most common instrumentation used in this application is LC-MS with a triple quadrupole mass spectrometer.
Tandem mass spectrometry is usually employed for added specificity. Standard curves and internal standards are used for quantitation of usually a single pharmaceutical in the samples. The samples represent different time points as a pharmaceutical is administered and then metabolized or cleared from the body.
Blank or t=0 samples taken before administration are important in determining background and ensuring data integrity with such complex sample matrices. Much attention is paid to the linearity of the standard curve; however it is not uncommon to use curve fitting with more complex functions such as quadratics since the response of most mass spectrometers is less than linear across large concentration ranges.
There is currently considerable interest in the use of very high sensitivity mass spectrometry for microdosing studies, which are seen as a promising alternative to animal experimentation.
Mass spectrometry is an important method for the characterization and sequencing of proteins. The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).
In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins.
In the first, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyzer. This approach is referred to as “top-down” strategy of protein analysis. The top-down approach however is largely limited to low-throughput single-protein studies.
In the second, proteins are enzymatically digested into smaller peptides using proteases such as trypsin or pepsin, either in solution or in gel after electrophoretic separation. Other proteolytic agents are also used. The collection of peptide products are then introduced to the mass analyzer.
When the characteristic pattern of peptides is used for the identification of the protein the method is called peptide mass fingerprinting (PMF), if the identification is performed using the sequence data determined in tandem MS analysis it is called de novo peptide sequencing.
These procedures of protein analysis are also referred to as the “bottom-up” approach. A third approach however is beginning to be used, this intermediate middle-down approach involves analyzing proteolytic peptide larger than the typical tryptic peptide.
Mass spectrometry (MS), with its low sample requirement and high sensitivity, has been predominantly used in glycobiology for characterization and elucidation of glycan structures. Mass spectrometry provides a complementary method to HPLC for the analysis of glycans.
Intact glycans may be detected directly as singly charged ions by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or, following permethylation or peracetylation, by fast atom bombardment mass spectrometry (FAB-MS). Electrospray ionization mass spectrometry (ESI-MS) also gives good signals for the smaller glycans. Various free and commercial software are now available which interpret MS data and aid in Glycan structure characterization.
Respired Gas Monitor
Mass spectrometers were used in hospitals for respiratory gas analysis beginning around 1975 through the end of the century. Some are probably still in use but none are currently being manufactured.
Found mostly in the operating room, they were a part of a complex system, in which respired gas samples from patients undergoing anesthesia were drawn into the instrument through a valve mechanism designed to sequentially connect up to 32 rooms to the mass spectrometer. A computer directed all operations of the system. The data collected from the mass spectrometer was delivered to the individual rooms for the anesthesiologist to use.
The uniqueness of this magnetic sector mass spectrometer may have been the fact that a plane of detectors, each purposely positioned to collect all of the ion species expected to be in the samples, allowed the instrument to simultaneously report all of the gases respired by the patient. Although the mass range was limited to slightly over 120 u, fragmentation of some of the heavier molecules negated the need for a higher detection limit.
Isotope Ratio MS: Isotope Dating And Tracing
Mass spectrometry is used to determine the isotopic composition of elements within a sample. Differences in mass among isotopes of an element are very small, and the less abundant isotopes of an element are typically very rare, so a very sensitive instrument is required.
These instruments, sometimes referred to as isotope ratio mass spectrometers (IR-MS), usually use a single magnet to bend a beam of ionized particles towards a series of Faraday cups which convert particle impacts to electric current. A fast on-line analysis of deuterium content of water can be done using flowing afterglow mass spectrometry, FA-MS.
Probably the most sensitive and accurate mass spectrometer for this purpose is the accelerator mass spectrometer (AMS). This is because it provides ultimate sensitivity, capable of measuring individual atoms and measuring nuclides with a dynamic range of ~1015 relative to the major stable isotope.
Isotope ratios are important markers of a variety of processes. Some isotope ratios are used to determine the age of materials for example as in carbon dating. Labeling with stable isotopes is also used for protein quantification.
Tandem Mass Spectrometry
A tandem mass spectrometer is one capable of multiple rounds of mass spectrometry, usually separated by some form of molecule fragmentation. For example, one mass analyzer can isolate one peptide from many entering a mass spectrometer. A second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID). A third mass analyzer then sorts the fragments produced from the peptides.
Tandem MS can also be done in a single mass analyzer over time, as in a quadrupole ion trap. There are various methods for fragmenting molecules for tandem MS, including collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD), blackbody infrared radiative dissociation (BIRD), electron-detachment dissociation (EDD) and surface-induced dissociation (SID). An important application using tandem mass spectrometry is in protein identification.
Tandem mass spectrometry enables a variety of experimental sequences. Many commercial mass spectrometers are designed to expedite the execution of such routine sequences as selected reaction monitoring (SRM) and precursor ion scanning.
In SRM, the first analyzer allows only a single mass through and the second analyzer monitors for multiple user-defined fragment ions. SRM is most often used with scanning instruments where the second mass analysis event is duty cycle limited. These experiments are used to increase specificity of detection of known molecules, notably in pharmacokinetic studies.
Precursor ion scanning refers to monitoring for a specific loss from the precursor ion. The first and second mass analyzers scan across the spectrum as partitioned by a user-defined m/z value. This experiment is used to detect specific motifs within unknown molecules.
Another type of tandem mass spectrometry used for radiocarbon dating is accelerator mass spectrometry (AMS), which uses very high voltages, usually in the mega-volt range, to accelerate negative ions into a type of tandem mass spectrometer.
Sparkman, O. David (2000)
Mass spectrometry desk reference
Pittsburgh: Global View Pub. ISBN 0-9660813-2-3
Downard, Kevin (2004)
Mass Spectrometry – A Foundation Course
Royal Society of Chemistry. doi:10.1039/9781847551306. ISBN 978-0-85404-609-6
Top Image: Dean Calma / IAEA. Technician in Clean Laboratory mounting filaments on a turret for thermal ionization mass spectrometry.