Use of periodic ion mobility spectrometry for tandem ion mobility experiments.
The protocol describes how to record the ion mobility profile of fragments produced upon activation of a molecule in a mass spectrometer, and differentiate between isomeric structures. The protocol allows glycans to be classified into 11 groups with respect to some of their main structural features, thus providing information that is otherwise difficult to obtain. It can serve to construct highly general molecular networks suitable for many molecular families, such as metabolites or drugs of interest in many areas of research.
This approach takes full advantage of a new concept in ion mobility spectroscopy and will be useful when mass spectrometry alone fails to distinguish between two structures. The experiment requires good command of mass spectrometry and ion mobility. However, the experimenter should be successful in implementing the method if he carefully follows each of the steps.
The procedure will be demonstrated by Simon Olivier, a doctoral student from my laboratory. To get started, set up a single-pass IMS sequence from the Tune page, put the tool in navigation mode, and open the Cyclic Sequence Control window. Select Advanced mode from the Cyclic Functions tab in this new window, select Add Package, then Single/Multi-Pass.
Wait for a sequence of navigation events to appear on the Sequence tab in the same window. Adapt the sequence so that all titer ions make a single pass around the periodic IMS racetrack. Do not change the injection time or the output and acquisition time, however, reduce the separate time to 1 millisecond
If some of the calibration mixture ions do not fit in the displayed arrival time window, change the IMS synchronization with the TOF analyzer impeller to orthogonal acceleration by increasing the number of impulses per bin in the ADC Settings tab. To record a 2-minute acquisition in the Cyclic Sequence Control window, click Acquire to open the Acquisition Settings pop-up window, enter the file name, description, acquisition length, minutes and click Save. Switch the device to TOF mode from the MS Tune page to check signal stability.
Complete MS acquisition record of the sample for 1 minute, which will be useful for verifying the isotopic pattern and presence of potential contaminants. Put the tool into MSMS mode from the Quad/MS Profile tab on the Tune home page. Specify the target iron mass in the MSMS mass field for isolation in the quadrilateral.
Record a 1-minute acquisition to verify isolation of precursors when processing the data. To perform a navigation-based selection of the isomer of interest, switch the instrument to navigation mode in the Cyclic Sequence Control window, from the Cyclic Functions tab, select Add Band, then Chopping. Wait for a complex sequence of navigation events to appear on the Sequence tab.
Place the Output and Gain event immediately after the first discrete event, and then click Run. Look for preliminary class results to be displayed in real time. Increase the duration of the first discrete event of a multipath separation by changing the time value of this event in the sequence until the resolution of the IMS peaks is satisfactory.
Record a 1-minute acquisition for reference. Click Pause, place the Output and Gain event below Output, Output to Prestorage, and Retain and Output Events. Adjust the duration of events so that the target peak in the output region is to pre-storage, and any other ion is either in the output region or wait and eject
Place the Output and Gain event at the end of the sequence below the re-injection from pre-storage and the second discrete events. Click Play to view the selected demographic group. Check the quality of insulation.
Record a 1-minute acquisition for reference. On the Sequence tab, in the column next to User-defined event times, find the summary times for all events. Note the time found on the Reinject from Pre-Store event line to perform the CCS calibration.
Set the duration of the separate event that directly tracks the output and acquisition to 1 millisecond. In the Re-injection from pre-storage line, check the Enable activation box and optimize fragmentation using the built-in control. If fragmentation is not satisfactory using the built-in control, uncheck the Enable Activation box and proceed to manually optimizing the re-injection voltage, increasing the array pre-gradient, and decreasing the array offset voltage until the results are satisfactory.
To record a 2-minute acquisition in the Acquisition pop-up window, select the Preserve Drift Time option to create a file containing only the arrival times, corresponding to M over Z named _dt.raw. After performing MS analysis of the arabinoxylan pentasaccharide mixture, the spectrum displayed an isotopic pattern with a single peak at M above Z 685.24 indicating that the two compounds are isotropic in nature. The pentamer channels were separated by a periodic IMS cell, and three peaks with different arrival times were separated.
Pure XA3XX shows peaks at 83 and 90 ms, while XA2XX displays a peak at 94 ms. After the first stage of IMS separation, the ions belonging to XA3XX were eluted, and the peak at 94 ms was selected for IMS-IMS analysis. Three-pass separation was performed after ion re-injection without activation, and an XA2XX peak was obtained at 199 ms.
The generated IMS-IMS MS data were decomplexed using access time and M over Z dimensions, generating IMS-IMS spectra. Peaks greater than 0.2% of the relative intensity were exported to the CCS calibration, yielding a central CCS-IMS-calibrated 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 contaminants.
We have previously used IMS-IMS spectra to build networks and classify compounds, but they can also be used to search against databases for compound identification. This technique is very new, but we expect it to be very useful in glycomics as well as in any context in which analysts encounter isomeric molecules.