1M Resolution - FAQs

Speed is impacted by the cleanliness and complexity of your samples as well as the processing speed of the computer.

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AcquireX only works for LC experiments because the algorithm specifically looks for things that elute as peaks.

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No, AcquireX is specifically used for small molecule (ions with a charge state of 2 or less) applications.

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AcquireX is available on the Thermo Scientific Orbitrap Fusion Lumos, Orbitrap Fusion, and Orbitrap ID-X Tribrid MS instruments. In regards to the first two instruments, you will need to upgrade to Instrument Control Software Version 3.1 and XCalibur Version 4.2.

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To achieve in-depth MSⁿ analysis, this method exhaustively interrogates a precursor using a combination of fragmentation mechanisms, MSⁿ levels, and m/z analyzers. At high MSⁿ levels, the product ion signals can become quite weak. To address this concern, we developed an “MSⁿ Quality Trigger”, which enables the user to trigger complementary ITMSⁿ scans if the corresponding FTMSⁿ scan S/N drops too low. Mass Frontier 8.0 software can curate “MSⁿ library” data into MSⁿ “tree” libraries, and the software can search unknown MSⁿ data against this local library.

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The assisted collision energy acquisition strategy uses a series of hidden ion trap scans to determine the optimal CE to use in the subsequent analytical scan. These IT spectra only scan a small mass range around the precursor ion (2 Th) to increase the analysis speed. The TIC of these IT scans is compared to the TIC of an IT scan of 0 collision energy to produce a pseudo-breakdown curve of the precursor. Once the amount of unreacted precursor crosses a user-defined threshold (e.g., 20%), the analytical MS/MS scan uses that 'optimal' collision energy. View images and learn more here.

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Assisted collision energy is now an available feature on all Tribrid instruments, software version 3.1. Through the Method Editor GUI, users can select different 'Collision Energy Modes' for both HCD and CID activation types in the 'Scan Properties' panel. View images and learn more here.

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Internal calibrant (reference) ions are used for real-time fine adjustment of the instrument's mass-to-charge ratio calibration, enabling a correction for otherwise uncompensated errors that are due to temperature changes scan-to-scan. The purpose of the internal calibration is to ensure high mass measurement accuracy, regardless of the MSⁿ level. This is especially important for low m/z ions over extended periods of time.

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The three modes offered are:

'Background exclusion workflow'
The background exclusion workflow allows users to generate DDMSⁿ data with automatic application of instrument generated background exclusion list. This allows detection of low level analytes in the presence of a complex background.

'Background exclusion and component inclusion workflow'
The background exclusion and component inclusion workflow allows DDMSⁿ data generation with automatic application of instrument generated inclusion and exclusion list. Multiple samples can be submitted in one sequence for their respective inclusion and exclusion list generation followed by one identification experiment using those lists.

'Deep scan workflow'
The deep scan workflow allows the generation of DDMSⁿ data with automatic application of inclusion and exclusion lists followed by automated re-injection of the sample. The inclusion and exclusion lists are updated prior to re-injection by moving triggered targets from the inclusion list to the exclusion list allowing deeper interrogation of complex samples.

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Inclusion and exclusions lists contain m/z values, retention times and intensity threshold.

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First, make sure your spray is stable while running the eFT check.
If the spray is stable and the check still fails, calibrate the eFT. If the eFT fails due to “peak splitting”, then zoom in on target ions at high resolution to see if the peak is truly splitting. If not, the eFT calibration is probably fine, and the failed calibration could be due to poor cal mix quality, poor spray stability, or interfering ions. It is best to obtain fresh cal mix, replace the infusion lines and ion transfer tube, replace the HESI needle insert, or possibly “bake out” the HESI source. Then, repeat the calibration. Make sure your spray is stable. If it is, perform the Orbitrap transmission, Isotope ratio, and Isotope interactions (positive mode only) diagnostics as shown in this screenshot. Follow up with the eFT and OT mass calibrations.

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Yes. The yellow/orange indicates that the settings are outside the recommended range. For some applications (top-down), AGC and other parameters may be intentionally set outside the recommended ranges.

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You need to select “Show All” (see screenshot here) to see all the settings,

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Double check that your spray is stable and repeat the mass calibration. If it continues to fail, and exhibits large deviations from the expected mass value, then go to diagnostics and run “OT coarse mass calibration”. If the calibrant masses are found, but are continually outside of the 2 ppm specification of any target value, check spray stability. If spray stability is functioning, then run OT Transmission in Diagnostics (see this screenshot. Calibrate the eFT and OT masses.

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The isotope threshold settings are typically the cause of this issue. The typical threshold is selected for larger proteomic samples that have >40% M+1 peaks. Therefore, the system will probably fail for small molecules. If MIPS isn't turned on, the system will use the 40% threshold as described. If an MIPS filter is on, the system will rely on the clustering algorithm of the FTPC data analysis to group the peaks into clusters. Anything that has the same cluster ID is then excluded, regardless of intensity.

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Yes. The calibrations are designed to start with the ion optics and conclude at the ion trap. Subsequent calibrations rely on the previous ones being calibrated correctly. In particular, if anything other than mass is recalibrated, pAGC will have to be recalibrated. If eFT is calibrated, then mass must also be recalibrated.

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Start by checking each group of calibrations, one at a time. If they fail the check repeatedly, perform calibration.
If any coarse calibrations are performed, the main calibrations must be performed as well.
Upon instrument install, the instrument needs to be calibrated.
After venting/replacing hardware/cleaning, the instrument needs to be calibrated.

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Yes. The floranthene radical anion will need the calibrated ion optic and ion trap parameters for effective transmission and trapping for ETD.

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Yes. The ion routing multipole has multiple functions. The IRM not only functions as a collision cell for HCD fragmentation, but also as an ion storage-routing device so ions can be routed to the linear ion trap and back to the Orbitrap via the IRM cell.

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Quadrupole isolation is faster because ions must travel through the quadrupole analyzer regardless of the analysis type chosen. As a result, there is little to no time penalty when using the quadrupole for MS² isolation. In such a case, quadrupole transmission efficiency will depend on isolation width, m/z, and the cleanliness of the analyzer. Ion trap isolation is best suited for MS³ experiments, including those performed using multinotch isolation for TMT analysis; these types of isolation events are not possible with the quadrupole analyzer.

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