Tandem mass spectrometry (MS/MS) involves more than one-step of mass selection or analysis, and fragmentation is usually induced between the steps (9). One example of an application of tandem mass spectrometry is protein identification. The first mass analyzer isolates ions of a particular m/z value that represent a single species of peptide among many introduced into and then emerging from the ion source. Those ions are then accelerated into a collision cell containing an inert gas such as argon to induce ion fragmentation. This process is designated collisionally induced dissociation (CID) or collisionally activated dissociation (CAD). The m/z values of fragment ions are then measured in a 2nd mass analyzer to obtain amino acid sequence information.
There are several types of tandem mass spectrometers, including triple stage quadrupoles (TSQ), 3D and linear ion traps, quadrupole/time-of-flight (QTOF) hybrid instruments, quadrupole-linear ion trap hybrid instruments (QTRAP), and time-of-flight-time-of-flight (TOF/TOF) instruments. With 3D or linear quadrupole ion traps, tandem MS can also be performed in a single mass analyzer over time and in these instruments this process may be iterated more than once to yield MSn spectra. These instruments achieve fragmentation by resonance excitation, which induces collisions with the trap bath gas (helium) of sufficiently high energy to induce fragmentation. The other instruments mentioned above employ CID in a collision cell. Other methods that can be used to fragment molecules for tandem MS include electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD) (9).
Triple stage quadrupole (TSQ) tandem mass spectrometers have two mass analyzing quadrupoles separated by a quadrupole operated in the Rf-only mode that serves as a containment device and collision cell (Figure 18).In the first stage quadrupole,ions formed in the ion source and focused into the quadrupole are mass-analyzed, and ions of asingle m/z value are transmitted to the second stage quadrupole, which contains an inert gas such as argon. The potential that accelerates the ions into the second stage can be varied to produce collisions at various energy levels, and these collisions induce dissociation of the precursor (or parent) ion into a set of product (or daughter) fragment ions. Those ions are then mass-analyzed in the third stage quadrupole to yield a product ion spectrum.
Other kinds of experiments can be performed on triple quadrupoles by coordinating the activities of the two mass analyzing quadrupoles, a process sometimes designated “linked scanning”. One such experiment is to identify precursor ions that yield a product ion of a specific m/z value, and this is designated precursor ion scanning or parent ion scanning. This can be used to identify parent ions that contain a specific substituent. An example is negative ion scanning for parent ions of m/z 303 when analyzing phospholipids; this will identify species that contain an arachidonic acid substituent (32). A second linked scan mode identifies precursor ions that undergo a loss of a specific number of m/z units, and that is designated a constant neutral loss scan. Such scans are useful in identifying members of a structural class. An example is that a constant neutral loss of m/z 59 scan identifies Li+ adducts of phospholipid molecules that contain a choline head group because such molecules undergo a characteristic loss of trimethylamine (33). Other examples include selected reaction monitoring (SRM) in which one measures the ion current generated by selecting a parent ion of a specific m/z value in the first quadrupole, subjecting the ion to CAD in the second stage, and then monitoring signal from a product ion of another specific m/z value in the third stage quadrupole. One can also monitor several such reactions, which is designated multiple reaction monitoring (MRM). Examples of application of such scan modes will be provided in subsequent sections on applications of tandem MS.
Quadrupole/time-of-flight (QTOF or QqTOF) tandem mass spectrometers (Figure 19) have a mass-analyzing quadrupole (Q) interfaced with the ion source (typically ESI), an Rf-only hexapole collision cell (q) and a time-of-flight (TOF) second mass analyzer in sequence (34). The configuration can be viewed as re-placing the third quadrupole (Q3) in a triple stage quadrupole by a TOF MS, and this yields high sensitivity, mass resolution, and mass accuracy in both precursor MS and product ion (MS/MS) modes (34). Excellent full-scan sensitivity over a wide mass range is achieved in both modes by the parallel detection feature of TOF/MS. This advantage does not inherently apply to the more specialized modes analogous to the precursor ion, neutral loss, and multiple reaction monitoring scans of triple stage quadrupole systems, and these scan modes could not be performed with any reasonable efficiency on early QTOF instruments. Computational approaches have now been developed to approximate these ‘scan’ modes, although TOF instruments are not strictly speaking scanning devices. For MS/MS, Q1 is operated in the mass filter mode to transmit only the parent ion of interest, which is then accelerated into collision cell Q2, where it undergoes collision induced dissociation. Resultant fragment ions and residual parent ions are then mass analyzed by TOF. An ion mirror compensates for the initial energy and spatial spread of the ions. The detector is made of two microchannel plates. The electrostatic mirror and the ion detector are similar to those used with MALDI ionization sources.
In both 3D and linear ion trap tandem mass spectrometers, fragmentation is induced by resonance excitation that induces collisions of the parent ion of interest with the trap bath gas (helium) of sufficient energy to induce dissociation. The operation of the Finnigan LTQ linear ion trap is illustrated in Figure 20 (35). Ions are introduced into the trap and confined by applying appropriate Rf and DC voltages. To isolate the desired parent ions, the Rf voltage is adjusted, and multi-frequency resonance ejection waveforms are applied to the trap to eliminate all but the desired ion(s) in preparation for subsequent fragmentation and mass analysis. The voltages are then adjusted to stabilize the selected ions and to allow for collisional cooling in preparation for excitation. The energy of the selected ions is then increased by applying a supplemental resonance excitation voltage to the two rods on the X-axis. This increase in energy causes collisions with the trap bath gas that induce dissociation into product ions that are retained in the trapping field. Scanning the contents of the trap to produce a mass spectrum is achieved by linearly increasing the Rf voltage applied to the trap and using a supplemental resonance ejection voltage. This results in sequential movement of ions from stable orbits; they become unstable in the x-direction and are ejected from the trapping field. The ions are then accelerated into high voltage dynode detectors, where they produce secondary electrons. Signal is amplified by electron multipliers, and the analog signals are integrated and digitized.
The QTRAP (Applied Biosystems) is a hybrid tandem mass spectrometer that combines the capabilities of a triple quadrupole and an ion trap (36, 37). The ionpath of the QTRAP (Figure 21) is that of a standard triple quadrupole, but the final quadrupole may be operated either as a conventional transmission Rf/DC quadrupole mass analyzer or as an axial ejection linear ion trap MS. The instrument can operate either as a triple quadrupole or as an ion trap MS and perform novel scan modes not available on other instruments. In the survey MS scan, ions flow through orifice and skimmer, Q0 ion guide, Q1 quadrupole operated as an ion guide, Q2 collision cell at low energy, and into the Q3 linear ion trap. After cooling, trapped ions are scanned out of the Q3 ion trap in an axial direction toward the ion detector to yield a high sensitivity survey MS scan designated an “Enhanced MS”, or EMS scan (36).
Product ion MS/MS scans can be generated on the QTRAP as with 3D ion traps. This “in-trap” fragmentation scan mode first involves allowing most of the ions from the source to enter the Q3 ion trap, followed by an ion isolation step to eject all ions except for the desired precursor ions. Next, the trapping conditions of the ion trap are changed, the fragmentation step performed, the product ions cooled, and a mass analysis scan performed. A shortcoming of this “tandem-in-time” MS/MS process is that the ion trap is normally initially filled with a broadm/zrange of ions from the ion source, and then the precursor ion isolated. This reduces the dynamic range because every ion trap has a finite ion capacity, some of which is used to store ions that will eventually be ejected without interrogation. In addition, MS/MS product ion scans from in-trap fragmentation results in a low mass cutoff, and no product ions are observed with m/z values less than 30% of that of the parent ion (36, 37).
Because of these limitations, a more useful product ion MS/MS scan mode on the QTRAP is the hybrid “Enhanced Product Ion Scan” (EPI) which employs the tandem-in-space capabilities of the ion path with the high sensitivity ion trap mass scan (36, 37). In the EPI scan, the precursor ion is selected in the first quadrupole, Q1. This removes contaminant species from the ion beam before ions enter the ion trap. This allows the full ion trap capacity to be used for each scan. Fragments are generated in EPI MS/MS scansviaion acceleration between Q1 and a pressurized Q2 just as with a triple quadrupole MS, and identical fragmentation patterns are observed. Fragments are produced more quickly than with conventional ion traps, and since the fragmentation step and the ion trapping step are spatially separated, there is no inherent low mass cutoff for the EPI spectrum. Low mass fragments are trapped and analyzed by reducing the ion trap fill mass to the appropriate value. A variety of novel scans are possible with this unique ion path not possible with other mass spectrometers. In addition, the ability to perform both the very selective MS/MS scans of a triple quadrupole MS instrument and the extremely sensitive product ion scans of an ion trap reduces the limits of identification and detection (36, 37).