Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

The protocol describes how to record an ion mobility profile for the fragments produced upon activation of a molecule in the mass spectrometer, and to discriminate between isomeric structures. The protocol allows glycans to be classified into 11 groups regarding some of their key structural features, and thus provide an information that is otherwise difficult to obtain. It can serve to build molecular networks which are extremely generic and suited to many molecular families, such as metabolites or drugs of interest in many research areas.

The approach makes the most of a new concept in ion mobility spectrometry, and will be useful where mass spectrometry alone will fail to discriminate between two structures. The experiment requires a good command of mass spectrometry and ion mobility. However, the experimenter should be successful in implementing the method if one follows carefully each of the steps.

Demonstrating the procedure will be Simon Ollivier, a PhD student from my laboratory. To begin, set up a single-pass IMS sequence from the Tune page, put the instrument in Mobility mode, and open the Cyclic Sequence Control window. Select Advanced mode from the Cyclic Functions tab of this new window, select Add Bundle, then Single/Multipass.

Wait for a sequence of mobility events to appear in the Sequence tab of the same window. Adapt the sequence so that all the calibrant ions make a single pass around the cyclic IMS racetrack. Do not change the Inject time or the Eject and Acquire time, however, lower the Separate time to one millisecond.

If some ions of the calibration mixture do not fit in the displayed arrival time window, change the synchronization of the IMS with the pusher of the orthogonal acceleration TOF analyzer by increasing the number of pushes per bin in the ADC Settings tab. To record a two-minute acquisition in the Cyclic Sequence Control window, click on Acquire to open the Acquisition Settings popup window, input the Filename, Description, and Length of Acquisition, minutes, and click Save. Switch the instrument to TOF mode from the MS Tune page to check the stability of the signal.

Record a full MS acquisition of the sample for one minute, which will be useful to check the isotopic pattern and the presence of potential contaminants. Put the instrument in MSMS mode from the Quad/MS profile tab of the main Tune page. Select the mass of the targeted iron in the MSMS Mass field for isolation in the quadrupole.

Record a one-minute acquisition to check the precursor isolation when processing the data. To perform a mobility-based selection of the isomer of interest, switch the instrument to Mobility mode in the Cyclic Sequence Control window, from the Cyclic Functions tab, select Add Bundle, and then Slicing. Wait for a complex sequence of mobility events to appear in the Sequence tab.

Position the Eject and Acquire event right after the first Separate event, and then click Run. Look for the results of the initial separation to be displayed in real-time. Increase the duration of the first Separate event for a multipass separation by changing the time value for this event in the sequence until the resolution of the IMS peaks is satisfactory.

Record a one-minute acquisition for reference. Click Pause, position the Eject and Acquire event below the Eject, Eject to Pre-Store, and Hold and Eject events. Adjust the duration of the events so that the targeted peak is in the Eject to Pre-Store region, and any other ion is either in the Eject or Hold and Eject region.

Position the Eject and Acquire event at the end of the sequence below the Reinject from Pre-Store and the second Separate events. Click Run to display the selected population. Check the quality of the isolation.

Record a one-minute acquisition for reference. In the Sequence tab, in the column next to the user-defined event times, look for the summed-up times of all events. Take note of the Time Abs found on the line of the Reinject from Pre-Store event for performing the CCS calibration.

Set the duration of the Separate event directly proceeding the Eject and Acquire to one millisecond. On the Reinject from Pre-Store line, tick the Enable Activation box and optimize the fragmentation with the built-in control. If the fragmentation is not satisfactory with the built-in control, uncheck the Enable Activation box and proceed to manually optimize the reinjection voltages, increase the Pre-Array Gradient, and lower the Array Offset voltage until the results are satisfactory.

To record a two-minute acquisition in the Acquisition popup window, tick the Retain Drift Time option to generate a file containing only the arrival times, versus M over Z labeled as _dt.raw. After performing the MS analysis of the arabinoxylan pentasaccharide mixture, the spectrum displayed an isotopic pattern with a single peak at M over Z 685.24 suggesting that the two compounds are isomeric in nature. The adducts of the pentasaccharides were separated through the cyclic IMS cell, and three peaks were separated with different arrival times.

The pure XA3XX shows the peaks at 83 and 90 milliseconds, while the XA2XX exhibits a peak at 94 milliseconds. After the first stage of IMS separation, ions belonging to XA3XX were ejected, and the peak at 94 milliseconds was selected for IMS-IMS analysis. A three-pass separation was performed after re-injecting the ion without activation, and a peak for XA2XX was obtained at 199 milliseconds.

The generated IMS-IMS MS data were de-convolved using the arrival time and M over Z dimensions, generating the IMS-IMS spectra. The peaks above 0.2%relative intensity were exported for CCS calibration, resulting in a centroided CCS-calibrated IMS-IMS spectrum. It is always important to check the isolation of the precursor ion, otherwise, the spectrum may come from a mixture of the compound of interest and the contaminants.

We previously used IMS-IMS spectra to build networks and classify compounds, but they could also be used to search against databases for compound identifications. This technique is very recent, but we expect it will be extremely useful in glycomics as well as in any context where analysts are confronted with isomeric molecules.