This article is the final installment in our series on mass spectrometry. In the past two articles, we examined the principles and types of basic units in mass spectrometry. In this article, we will explore tandem mass spectrometry (MS/MS), which enables precise analysis of complex mixtures.
▣ MS/MS
As denoted, MS/MS is an analytical technique that utilizes two stages of mass spectrometry. In the first MS, ions of the desired m/z value are separated. These separated ions, referred to as parent ions or precursor ions, undergo chemical reactions, resulting in changes in their m/z values, and then undergo more precise analysis in the second MS. The ions produced from the decomposition through the chemical reaction are called product ions, which are detected in the second MS. The analytical process of MS/MS can be represented by the following equation. (mp=parent ion, md=product ion, mn=neutral fragment)
The basic concept of MS/MS is similar to using MS in conjunction with chromatography (such as LC/MS, GC/MS, etc.). However, in the case of LC/MS, the analysis depends on the analytes eluted from the chromatography column. This makes the MS act as a dependent variable to the LC in the analysis. In contrast, MS/MS has the advantage of treating each stage as an independent variable that can be manipulated for analysis. There are also analyses that are possible only with MS/MS and not with LC/MS. One such analysis is the ability to quickly screen for specific types of substances in complex mixtures. For instance, MS/MS can determine whether a peptide mixture is glycosylated or phosphorylated.
Both parent ion scans and product ion scans play significant roles in drug development. In cases where substances have similar core structures but various modifications, MS/MS can help identify the most effective therapeutic structure within a homologous series, potentially affecting therapeutic efficacy. It can also screen for unknown metabolites and monitor chemical reactions between substances.
Compared to single MS, MS/MS has the advantage of improved signal-to-noise ratio. Detectors in most single MS instruments are susceptible to interference from individual ions, leading to background noise that is not due to detector noise but actual ions, known as chemical noise. This limits detection accuracy.
In MS/MS, however, MS1 acts as a chemical filter, removing ions of other mass spectra except for the targeted m/z ions. Consequently, the noise observed in MS2 when analyzing product ions can be attributed to detector noise rather than chemical noise, which is generally at a lower level, thereby increasing the reliability of the analysis.
▣ Dissociation methods
Another key point that determines the performance of MS/MS is the reaction that occurs between the two MS stages. The most common reaction is unimolecular dissociation, which is promoted by ion activation. By increasing the internal energy of the parent ion through ion activation, the ion dissociates before entering MS2, generating product ions. Therefore, ion activation can be considered a method of ion separation.
A representative separation method is Collision-Induced Dissociation (CID). In CID, the parent ion collides with a neutral gas, increasing its internal energy and causing it to dissociate, thereby generating product ions. While CID is used in all MS instruments, there are other separation methods specific to certain devices. Other separation methods include Surface-Induced Dissociation (SID) and Photodissociation. Initially, photodissociation used UV or visible light lasers, but to enhance efficiency, Infrared Multiphoton Dissociation (IRMPD) has been developed and is now used.
▣ MS/MS instruments
MS/MS instruments can be classified into two types: tandem-in-space and tandem-in-time. Tandem-in-space requires individual analyzers for each MS stage, and beam-type analyzers are primarily used. In contrast, trap-type analyzers are used in tandem-in-time instruments, where each stage is conducted within the same analyzer but with a time delay. This feature allows tandem-in-time instruments to perform multiple stages of MS analysis with a single analyzer. However, it is challenging to perform parent ion scans or neutral-loss scans with this configuration. Therefore, beam-based instruments are preferable for screening complex mixtures.
The earliest MS/MS instruments used sectors for both stages, but soon the triple quadrupole (QqQ) was developed and became widely used. The QqQ is cheaper and easier to operate compared to sector-based MS/MS, leading to its rapid adoption. In a QqQ instrument, the first and third quadrupoles act as MS analyzers, while the middle quadrupole serves as the collision cell for CID.
Subsequent developments led to hybrid instruments combining different types of analyzers, such as Q-TOF. Currently, the most commonly used tandem-in-space instruments are QqQ and Q-TOF. Although Q-TOF, being a pulsed instrument, cannot perform scanning, it enables fast analysis, making it popular for LC/MS/MS. For scanning, QqQ instruments can be used.
Tandem-in-time instruments, such as the quadrupole ion trap and FTICR, do not require ions to move to the next analyzer stage. Instead, they operate within a time frame, allowing sufficient time for the parent ions to dissociate, thus achieving high MS/MS efficiency. The quadrupole ion trap, in particular, is widely used due to its simplicity and speed in conducting MS/MS experiments, as well as its relatively small size and low cost.
Over the course of these three articles, we have covered the fundamental principles of MS, the roles and types of various analyzers, and the advantages and classifications of MS/MS analysis. Mass spectrometry is expected to remain a crucial tool in drug development, with its broad functionality ranging from the discovery of new drug candidates to quality control.
Reference : Glish, G., Vachet, R. The basics of mass spectrometry in the twenty-first century. Nat Rev Drug Discov 2, 140–150 (2003). https://doi.org/10.1038/nrd1011