Ion/Neutral, Ion/Electron, Ion/Photon, and Ion/Ion Interactions in Tandem Mass Spectrometry: Do We Need Them All? Are They Enough?
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- McLuckey, S.A. & Mentinova, M. J. Am. Soc. Mass Spectrom. (2011) 22: 3. doi:10.1007/s13361-010-0004-9
A range of strategies and tools have been developed to facilitate the determination of primary structures of analyte molecules of interest via tandem mass spectrometry (MS/MS). The two main factors that determine the primary structural information present in an MS/MS spectrum are the type of ion generated from the analyte molecule and the dissociation method. The ion type subjected to dissociation is determined by the ionization method/conditions and ion transformation processes that might take place after initial gas-phase ion formation. Furthermore, the range of analyte-related ion types can be expanded via derivatization reactions prior to mass spectrometry. Dissociation methods include those that simply alter the population of internal states of the mass-selected ion (i.e., activation methods like collision-induced dissociation) as well as processes that rely on the transformation of the ion type prior to dissociation (e.g., electron capture dissociation). A variety of ion interactions have been studied for the purpose of ion dissociation and ion transformation, including ion/neutral, ion/photon, ion/electron, and ion/ion interactions. A wide range of phenomena have been observed, many of which have been explored/developed as means for structural analysis. The techniques arising from these phenomena are discussed within the context of the elements of structural determination in tandem mass spectrometry: ion-type definition and dissociation. Unique aspects of the various ion interactions are emphasized along with any barriers to widespread implementation.
Key wordsTandem mass spectrometry Dissociation methods Activation methods
Gas-phase reactions have long played important roles in mass spectrometry, both for the purpose of ionization and—in the case of molecular mass spectrometry—structural characterization. Emphasis is placed here on the primary structural characterization of molecules of interest (i.e., bond connectivity) via their gas-phase ionic surrogates. When reactions take place within the context of tandem mass spectrometry (i.e., between stages of mass analysis), precursor–product relationships can be clearly determined, and a significant reduction in chemical noise can be achieved . The value of a tandem mass spectrometry experiment, however, is also clearly linked to the quality and quantity of information that is forthcoming from the reaction. For this reason, novel reactions have been explored and developed since the earliest days of analytical tandem mass spectrometry. We have seen reactions in tandem mass spectrometry expand from the unimolecular dissociation of metastable ions and ions activated by keV energy collisions to processes that involve the interactions of ions with surfaces, photons, electrons, ions of opposite polarity, and with inert and reactive neutral species over a wide range of collision energies and time-scales .
Given the wide range of interactions that can now be effected in tandem mass spectrometry, it is useful to place them all into the broad context of structural characterization in mass spectrometry. The discussion begins with a few general considerations for structural characterization by tandem mass spectrometry that include options before mass-selected ions are subjected to a reaction as well as options for processes that take place between the stages of MS/MS. With these in mind, it is then possible to discuss each type of interaction from a common standpoint. Collision-induced dissociation (CID), which falls into the ion/neutral interaction category, is regarded as the “default” process in MS/MS. It is the technique against which all others must be compared for reasons discussed below. For any other approach to find broad use, it must offer a compelling advantage over CID in a widely performed measurement. Each interaction is therefore discussed very briefly, with emphasis placed on general considerations and features that might make it attractive relative to CID, while current barriers to widespread implementation are also mentioned.
2 Structural Characterization via MS/MS
A summary of the steps that can be involved in ion-type generation and dissociation in tandem mass spectrometry. Ion-type generation involves the possible use of wet chemistry to derivatize the analyte, the selection of an ionization method, and possible gas-phase ion transformation. Ion dissociation in MS/MS generally follows the formation of an initial ion type. It may involve the application of an activation method, such as CID or IRMPD, to the initially formed ion type, or it may involve an ion transformation step that leads to the dissociation of a different ion type, as occurs with ECD or EDD. The latter are ion dissociation methods but are not strictly ion activation methods, as defined here, because they involve an ion transformation that fundamentally alters the ion chemistry
Summary of ion activation, ion dissociation, and ion transformation approaches according to ion interaction type
No ∆ ion type
No ∆ ion type
Ion/neutral (ion/gas or surface)
CID, SID, MAD
I/M H+, e− transfer, H/D exchange
Collisional ionization, etc.
ECD, HECD, Al-ECD, EDD, El + dissociation
e− detachment (anions)
e− ionization (cations)
I/I H+, e− transfer
Heavy ion transfer
Covalent modification, etc.
The structural information that can ultimately be obtained from a tandem mass spectrum is the primary criterion in assessing an approach. However, there are also important practical considerations for analysis, such as efficiency and speed. Efficiency can be defined here as the fraction of mass-selected precursor ions that are converted to informative products. Clearly, a process of low efficiency will be limited in applicability to highly abundant precursor ions. The time-scale over which the process takes place is important in determining its compatibility with respect to implementation with on-line separations. Other practical considerations include the ease and cost of implementation of a method. The instrument platform usually places constraints on the range of processes and conditions that can be accessed. For example, ions typically travel at keV translational energies between stages of MS in sector and tandem TOF instruments. Therefore, processes that involve low translational energy ions, such as ion/molecule reactions, are difficult to implement between stages of MS in such instruments. Furthermore, the choice of ionization method is generally limited for any particular instrument.
The remaining discussion provides brief commentary on the various methods that fall into each of the interaction type categories within the context provided above. No attempt is made to review the various interactions and methods. Rather, emphasis is placed on unique features of the various interaction types.
2.1 Ion/Neutral Interactions
This broad category includes several types of ion/neutral collisions. Those relevant to tandem mass spectrometry are energetic collisions between ions and ground-state neutral species (e.g., ion/neutral collision-induced dissociation and collisional electron detachment reactions), low-energy reactive collisions (i.e., ion/molecule reactions), collisions of ions with neutral species in excited electronic states (e.g., metastable atoms), and collisions of ions with a surface (e.g., surface-induced dissociation). (Note that elastic ion/neutral collisions also play a major role in ion mobility measurements, but this topic falls outside the present scope.)
2.1.1 Collision-Induced Dissociation
Much has already been written about CID (reaction 2) and the wide range of conditions under which it can be implemented [7, 8, 9]. It is a true activation method in that it transfers energy into the precursor ion without changing its ion-type. While no tandem mass spectrometer can readily access the entire range of conditions that can give rise to CID, essentially all tandem mass spectrometers are capable of implementing CID under some conditions. If a structural characterization problem can be addressed via CID, this will generally be the preferred approach, because virtually all tandem mass spectrometers are designed around the implementation of ion/neutral gas collisions. Important characteristics of CID are its universal nature, its ease of implementation, its high efficiency (at least under some conditions), and its speed, particularly in beam-type instruments.
2.1.2 Ion/Neutral Collisional Ionization (Charge Permutation)
Reactions 3–7) are ion transformation processes that may or may not result in fragmentation. Such processes are not important at low collision energies. Even at high collision energies for polyatomic ions, collision-induced dissociation often dominates. Collisional ionization processes have not found much use in biological tandem mass spectrometry, in part due to the paucity of instruments that employ keV collision energies and the relatively low velocities of high mass ions, which result in small cross-sections for collisional electron detachment.
2.1.3 Ion/Molecule Reactions
Thermal energy ion/molecule reactions involving ground-state reactants are not used as ion activation or ion dissociation methods. Rather, they play an important role in ion transformation applications (e.g., charge state manipulation), and are therefore sometimes used in conjunction with an activation method (e.g., CID). Ion/molecule reactions can be quite efficient (e.g., 100% of the analyte ions can undergo reaction in favorable cases), depending upon the magnitude of the rate constant, the number density of the reagent that can be generated, and the time available for reaction. The range of reactions that can be accessed is constrained by the ability to generate an adequate number density of the neutral reagent. For this reason, neutral reagents are typically limited to relatively small species with relatively high vapor pressures, although desorption methods, such as laser-induced acoustic desorption (LIAD) , can expand the range of neutral analytes available for consideration.
2.1.4 Ion/Metastable Atom Interactions
When the metastable atoms have relatively high translational energies, CID can contribute. The electronic excitation of an analyte ion (without electron transfer or detachment) with the concomitant relaxation of the atom—which would qualify as an activation process—is also possible, but the extent to which such a process contributes is unclear.
MAD/MIDI is relatively new and has been applied to a very limited number of analyte classes, making its ultimate potential as a dissociation method difficult to assess. Reported efficiencies (<5%) probably do not reflect those from a highly optimized system with a bright metastable atom source. Some capabilities that complement CID, such as preservation of post-translational modifications, overlap with those of other methods (e.g., ETD, ECD) but the applicability of MAD to singly charged cations is potentially important. The requirement of a metastable atom source represents a barrier to widespread implementation.
2.1.5 Ion/Surface Interactions
The surface provides for high center-of-mass collision energies and relatively short activation times. The energy transfer distribution has been shown to generally be narrower than those provided under most CID conditions and more precisely variable. The relatively fast activation has been shown to give rise to symmetrical charge state distributions from the dissociation of dimeric and multimeric complexes, whereas most CID studies have shown asymmetric partitioning of charge . SID, therefore, appears to have advantages over CID for the examination of large biomolecule complex ions. SID has largely been implemented with specially constructed instrumentation, and has not seen widespread adoption. If SID could be implemented in commercial instrumentation without compromising CID or adding substantially to the cost, this approach could see wider use. Developments in this direction are underway .
2.2 Ion/Photon Interactions
Ion spectroscopy has been actively pursued for many years, with significant advances made in bio-ion spectroscopy in recent years . Ion/photon interactions are particularly attractive from the standpoint of the potential to obtain wavelength-dependent information and the practical advantage of requiring no gases to be admitted into the vacuum system. While CID is largely “universal” or “nonspecific,” the use of photons as a means for ion excitation promises to add levels of specificity via the dependence of absorbance upon wavelength, which cannot be provided by the other types of interactions. Given the impracticality of measuring the absorbance spectrum of an ion directly, fragmentation is used as a signature of photon absorption. The ability to generate overall dissociation yields and fragmentation patterns as a function of wavelength is very attractive. Furthermore, selective activation via chromophore-specific irradiation is also attractive. However, issues remain with regard to efficiency, speed, ease of implementation, and cost, which must be addressed before the full potential of ion/photon interactions can be realized for ion structure characterization.
IRMPD, usually implemented with a continuous-wave fixed-wavelength IR laser (10.6 μm), shares commonalities with some forms of CID , as it is a slow heating method. It has proven to be particularly useful when the presence of a collision-gas is deleterious to MS performance, as in FT-ICR, and has recently been coupled with derivatization approaches to provide a degree of selectivity in ion activation . Black-body infrared dissociation (BIRD)  is another IR multiphoton approach, typically used in FT-ICR instruments, that relies on the absorption of black-body radiation from the environment. Rather than being used as a structural tool, this broadband activation method is primarily used as a tool to generate information about dissociation dynamics via the measurement of fragmentation kinetics at well-defined temperatures. UV-PD techniques at several fixed wavelengths have been examined. Of particular note for peptides are the unique fragmentation reactions observed for singly protonated peptides using 157 nm photons  that enable de novo sequencing. Single-photon UV-PD is particularly well suited to tandem time-of-flight instruments, for which reaction conditions between mass analysis stages are often limited to relatively short interaction times.
Ion/light interactions are well established in tandem mass spectrometry, and their importance will likely grow as technology advances. The need for a light source (with BIRD constituting an exception) introduces a cost/implementation barrier. However, the variable wavelength provides degrees of specificity and selectivity that are unique among the interaction types. For this reason, we can expect to see further exploration of wavelength dependence and combinations of ion transformation with photoactivation techniques.
2.3 Ion/Electron Interactions
Relatively little extension of the use of electrons beyond EIEIO in MS/MS took place until the discovery of ECD of multiply charged ions  (reaction 1). ECD proved, in many cases, to provide more extensive primary sequence information from multiply protonated peptides and proteins than CID, because CID typically occurs by charge-directed pathways that are sequence and charge-state dependent, and, importantly, preserved labile post-translational modifications so that they could be localized. This addressed a serious shortcoming of CID for a widely shared analytical problem. Subsequent work demonstrated variations of the ECD experiment in which collisional or IR activation of ions prior to (i.e., activated ion ECD, AI-ECD ) or after electron capture was carried out, and in which electron energies were increased (i.e., hot ECD or HECD ), resulting in greater dissociation yields.
The net result of the electron detachment/electron capture process is electronic excitation of the (M+H)+ species, which has been termed electronic excitation dissociation (EED) . Very recently, it was observed that electron capture by an anion can occur via the irradiation of anions with electrons of 4–5 eV . These relatively new processes and the radical ions that are produced via electron detachment from even-electron multiply charged ions have not yet been studied extensively. However, the technologies developed to allow for the generation of high fluxes of electrons for ECD studies have enabled the study of these lower cross-section processes. Furthermore, interest in ion–electron interactions has stimulated the development of electrodynamic ion trap-based technology for their implementation with hybrid tandem mass spectrometers .
2.4 Ion/Ion Reactions
Ion/ion electron transfer to multiply charged cations can give rise to dissociation (i.e., electron transfer dissociation (ETD) , see reaction 20) that is generally analogous to that noted for ECD. Similarly, dissociation from ion/ion electron transfer from multiply charged anions , referred to as negative electron transfer dissociation  (nETD, see reaction 21), is similar to that noted in EDD. Hence, ETD and nETD constitute dissociation methods. Ion/ion reactions are commonly carried out in electrodynamic ion traps due to their ability to store ions simultaneously and in overlapping regions of space. Ion traps are common standalone instruments as well as components in many hybrid instruments. Hence, ETD and nETD can be implemented in many popular tandem MS platforms, provided the means for generating and delivering the reagent ions are in place. Commercially available instruments from several manufacturers support ion/ion electron transfer and proton transfer reactions, and these instruments are seeing growing use in proteomics applications. While there are many useful analytical applications for single-electron transfer and single-proton transfer, the range of chemistries potentially accessible via ion/ion reactions extends much further, making them perhaps the most flexible of the ion interactions for ion transformation.
The preceding descriptions make clear that each of the ion interaction types has unique characteristics, as well as characteristics that overlap with other interaction types either for ion transformation or structural determination. Ion/molecule, ion/electron, and ion/ion reactions are primarily methods for ion transformation that can take place in conjunction with fragmentation, as in ECD and ETD, or can be used in series with an activation method. Electron energy provides a useful variable in electron/ion interactions, but the processes are restricted to electron capture, electron detachment, or electronic excitation. Ion/molecule and ion/ion reactions provide for a much more diverse array of chemical phenomenology, but each has unique challenges in generating and/or delivering the reagent species. Aside from differences in implementation, ion/molecule and ion/ion reactions show overlap in some reaction types but not in others, such as charge inversion, which is restricted to ion/ion reactions, and metastable atom dissociation, which is an ion/neutral interaction process. As for pure activation methods, inelastic collision techniques (i.e., CID and SID) offer “universal” approaches, while the photon-based approaches offer the potential for greater selectivity than collision-based techniques.
Answers to the questions posed in the title can be addressed with analogy to a toolbox. As far as structural characterization is concerned, some of the interactions discussed here serve as tools for ion activation, some serve as tools for ion-type transformation, and some combine ion transformation with dissociation. Some tools are used often, while others are used only in very specialized circumstances. Some approaches have been or are being evaluated for their potential and may find no particular niche. Each of the general reaction types discussed here has unique characteristics and may therefore be uniquely well suited for a particular task. In this sense, the answer to the first question is “it would be nice if our toolbox was equipped with all of these techniques, but some tools are far more useful than others and we might not use some even if we could.” It is impractical to design an instrument that allows for all of the interaction types. Hence, some of the techniques may never find extensive use in solving structural problems. Furthermore, while some may make important contributions in specific areas, others may never find an application for which its characteristics are sufficiently compelling to be adopted by anyone. Such is the way of technology. Hence, based on our current state of knowledge, it would be difficult to argue that we really do need all of the techniques that have been bestowed with acronyms.
As for the second question—whether current tools are enough—there will always be a need for new and improved tools if there are unmet needs. Even with all of the currently available options in derivatization, ionization, and dissociation, there are many unmet needs, as well as room for improvement in measurements that already provide useful information. These structural characterization needs are apparent at the boundaries of all of the application areas in modern molecular mass spectrometry (e.g., carbohydrates, lipids, high-mass biomolecules, large complexes, metabolomics, proteomics, etc.). Clearly, the answer to this question is “no.” For this reason, it is important to continue to explore new reaction conditions and new reaction types, as well as to further develop some types of interactions that provide useful capabilities but may be lacking in, for example, efficiency, speed, or ease of implementation. Novel derivatization approaches, novel ionization methods or conditions, novel ion transformation, and new or improved ion activation approaches will be needed to address current and future challenges. While some of what has already been explored may not prove to be “needed” in the long run, we are certainly far from having everything we need.
The authors’ work in the general area of structural characterization via mass spectrometry has been supported by the Office of Basic Energy Sciences, Division of Chemical Sciences under award no. DE-FG02-00ER15105. Instrument development and the application of ion/ion reactions to peptide and protein ions have been supported by the National Institutes of Health under grant GM 45372.