Understanding the modes of operation on the SELECT SERIES™ MRT
INTRODUCTION
The SELECT SERIES MRT is a hybrid quadrupole Multi-Reflecting Time-of-Flight (Q-MRT) mass spectrometer capable of ultra-high mass resolution and mass accuracy in the parts-per-billion range1. There are three modes of analyzer operation on the SELECT SERIES MRT, namely Multi Reflecting Tof (MRT) mode, Resolution Enhancement Mode (REM) mode and Diamond mode.
The MRT Tof analyzer is comprised of two opposing gridless ion mirrors with periodic focusing lenses located midway between the mirrors. Ions enter the Tof via a double orthogonal accelerator and are reflected in a zig zag motion away from the orthogonal accelerator, the periodic lens limit divergence of the beam minimizing ion losses within the analyzer. The distal periodic lens also acts to deflect the ions back towards the detector which is situated above the orthogonal accelerator.
Multi Reflecting Tof (MRT) mode
Figure 1 shows the ion path of the MRT operating in a standard single pass mode, the ions enter the analyzer through an orthogonal accelerator and follow a trajectory between the periodic lenses P1 and P23 (flightpath shown in blue), once the ions reach lens P23 they are deflected back in a trajectory towards P1 and the detector (flightpath shown in red), resulting in a pathlength of ~48m and a resolving power of >200,000 FWHM.
Figure 1 - Flight path of MRT operating in a standard single pass (known as MRT) mode.
The time of flight for an ion of 1000 m/z corresponds to approximately 1.4ms for the 47m pathlength of the MRT, if a traditional Tof ‘push and wait’ approach was adopted this would result in a poor duty cycle resulting in low sensitivity. To overcome the low duty cycle the MRT is operated in an overpushing mode, where multiple pushes are performed within a defined time interval, the time difference between pushes are encoded such that ions of the same m/z will have different arrival times at the detector, the resulting data are subsequently decoded into a coherent spectrum, where all the individual pushes are aligned. Using this approach up to two orders of magnitude of signal recovery per scan can be achieved over a conventional ‘push and wait’ approach. This mode of operation is called Multi Reflecting Tof (MRT) mode with a resolving power of >200,000 FWHM and should be considered as the general mode of operation for most applications (the exception being studies of intact proteins that are >50kDa in mass).
Resolution Enhancement Mode (REM)
In the MRT mass analyzer ions travel a fixed pathlength from the orthogonal accelerator to the detector, with the observed resolving power, R (being defined as R = time of flight/2x time spread), increases with an increased pathlength, this can be achieved by making the analyzer physically longer or by deflecting the ions within the analyzer and enabling them to make multiple passes of the device. The former has practical physical limitations whereas the later can be achieved by applying a timed pulse to an electrode deflecting the ions within the device.
To accomplish an increased pathlength and hence increased resolving power in the MRT a timed pulse can be applied to lens P1, deflecting a restricted mass range so as to undergo two passes, increasing the pathlength and hence observed resolving power to >300,000 FWHM. This is approach is shown in Figure 2, the ions enter the analyzer through a orthogonal accelerator and follow a trajectory between lenses P1 and P23 (flightpath shown in blue in Figure 2A), once the ions reach lens P23 they are deflected back in a trajectory towards P1 (flightpath shown in red in Figure 2B), a timed pulse is then applied to lens P1 (shown in orange) deflecting the ions back towards P23 (flight path shown in green in Figure 2C), once the ions reach lens P23 they are once more deflected back in a trajectory towards P1 and finally on towards the detector (flight path shown in yellow in Figure 2D), resulting in a pathlength of ~92m and a resolving power of >300,000 FWHM.
Figure 2 - Flight path of the MRT analyzer operating in a Resolution Enhancement Mode (REM) a double pass mode.
 |
A): ions pass (shown in blue) from orthogonal accelerator into analyzer and are focussed and reflected towards lens P23. B): ions are deflected by lens P23 back towards lens P1, shown in red. C): a timed pulse applied to P1 (shown highlighted in orange) deflects the ions back towards lens P23, shown in green. D): ions are deflected a second time by lens P23 back towards P1 (pulse off) and onto the detector (shown in yellow). |
The transmittable mass range is a function of the selected low m/z, with the high m/z limit being approximately four times that of the lowest m/z, so for a start mass of m/z 300 the upper m/z would be ~1100 m/z.
The time of flight for an ion of 1000 m/z corresponds to approximately 2.8ms for the 92m pathlength of the MRT when two passes of the analyzer are undertaken, further reducing duty cycle compared to a single pass of the analyzer. An overpushing approach, as used for a single pass of the analyzer in MRT mode, cannot be employed as ions of differing m/z would arrive at lens P1 at differing times resulting in significant problems in decoding the multiplexed data.
An alternative approach to enhancing duty cycle would be through a synchronization of the time-of-flight of an ion packet release between the upstream gas cell and the orthogonal accelerator for the m/z range of interest. Ions are accumulated in the collision cell and are released with a delay to the orthogonal accelerator such that the m/z range of interest arrives in the orthogonal accelerator to coincide with the extraction voltages being applied. The bunching effect of this accumulation approach can result in up to two orders of magnitude of signal recovery per scan over a conventional ‘push and wait’ approach.
The combination of the two passes through the analyzer and the accumulation duty cycle enhancement mode of operation is called Resolution Enhancement Mode (REM) with a resolving power of >300,000 FWHM and should be considered as a targeted mode of operation where a higher resolving power is required for specific (targeted) analytes.
Diamond Mode
The third mode of operation of the analyzer on the MRT is Diamond mode, this is shown in Figure 3. The ions enter the analyzer through an orthogonal accelerator and follow a trajectory towards lens P1 (flightpath shown in purple) and are deflected towards the detector, resulting in a pathlength of ~1.9m and a resolving power of >10,000 FWHM and should be considered as the mode of operation for the analysis of intact proteins that are >50kDa in mass (especially in conjunction with Protein Mode).
Figure 3, Flight path of MRT operating in Diamond mode.
Standard and Protein Modes
Within MRT and Diamond there are two sub-modes of operation, Standard (previously described) and Protein modes. Protein mode uses different voltages in the mass analyzer that are optimized to provide better transmission of high molecular weight ions, with improved transmission observed for analytes >5kDa. MRT Protein mode offers high resolution (>200,000 FWHM) of analytes in the ~5 to 50kDa range, whereas Diamond Protein mode offers superior transmission of analytes >50kDa where high mass resolving power has limited benefit owing to the isotope limit.
1. Novel Hybrid Quadrupole-Multireflecting Time-of-Flight Mass Spectrometry System, DA Cooper-Shepherd et al J.Am. Soc. Mass Spectrometry 2023 34 (2), 264-272. DOI: 10.1021/jasms.2c00281