S.O. is thankful to the French National Research Agency for funding his Ph.D. (grant ANR-18-CE29-0006).

NOTE: An overview of the protocol is provided in Figure 1. The parameters used for the experiments described in the present protocol can be found in Supplemental Table S1 and Supplemental Table S2.
1. Preparation of the sample solution
NOTE: The protocol is described using an arabinoxylan pentasaccharide (23-&#945;-L-arabinofuranosyl-xylotetraose or XA2XX; see the <s...

The SELECT SERIES Cyclic IMS is a powerful tool that allows selecting a defined ion population&#8212;of a given m/z and ion mobility&#8212;without the need for upstream chromatographic separation. The instrument affords the possibility of generating a bidimensional fragmentation map of this ion population, from which both MS/MS and IMS/IMS spectra can be extracted. However, the user must note several critical points that require attention during the experimental process.
First, the us...

The complete chemical characterization of biomolecules is key to understanding their underlying biological and functional properties. To this end, &#34;omics&#34; sciences have developed in recent years, aiming for the large-scale characterization of chemical structures at biological concentrations. In proteomics and metabolomics, MS has become a core tool to unravel the structural heterogeneity found in biological media-notably thanks to its sensitivity and ability to provide structural information through tandem MS (MS/MS). In MS/MS strategies, an ion is selected according to its mass, then fragmented, and finally, the masses of its fragments are acquired to establish a fingerprint of the molecule. MS/MS spectra can, in particular, be used to match spectral databases1,2, or tentatively reconstruct the parent structures3,4. Under the assumption that similar spectra belong to similar compounds, MS/MS data can also be used to build molecular networks (MNs) connecting related species through a similarity score5,6.
However, because of the inherent property of MS to detect the mass-to-charge ratio (m/z) of ions, the technique is blind to a number of structural features that fall within the range of (stereo)isomerism. For example, carbohydrates are made of several monosaccharide subunits, many of which are stereoisomers or even epimers (e.g., Glc vs. Gal or Glc vs. Man). These subunits are linked by glycosidic bonds, which can differ by the position of the linkage (regioisomerism) and the steric configuration of the anomeric carbon (anomerism). These characteristics make it difficult for standalone MS to distinguish between carbohydrate isomers7, and only regioisomerism can be addressed using high-energy activation methods8,9,10. Although derivatization is an option to disrupt the equivalence of stereoisomeric groups11, it requires extensive sample preparation. Another, more straightforward option is to couple MS with an analytical dimension sensitive to isomerism, such as IMS.
Because this protocol is designed for users who are already familiar with the basic concepts of IMS, and because detailed reviews are available elsewhere12,13, only a brief overview of the principles of IMS is given here. IMS is a gas-phase separation method that relies on the interaction of ions with a buffer gas and an electric field, ultimately separating ions according to their gas-phase conformations. Different principles of IMS coupled to MS can be found on commercial instruments: some operate at alternating high and low electric fields (field asymmetric IMS, FAIMS), while most operate within the low field limit&#8212;notably drift tube IMS (DTIMS, linearly decreasing electric field), traveling wave IMS (TWIMS, symmetric potential waves), and trapped IMS (TIMS, high flow of buffer gas trapping ions against electric fields)13. The low-field methods allow access to a so-called CCS, a property of the ion-gas pair that represents the surface (in &#197;2 or nm2) of the ion that interacts with the buffer gas during the separation. CCS is theoretically instrument-independent and is thus useful to generate data that can be reproduced between different laboratories14. Ion mobility separations can be impacted by various parameters and, notably, by fluctuations of the gas pressure and gas temperature in the mobility cell. The CCS calibration is a way to remedy this, as both the calibrant and the species of interest will be similarly affected13. However, it is mandatory to install the instrument in a temperature-controlled room and to have a reliable gas pressure control system.
An interesting evolution of IMS is IMS/IMS, which was first introduced in 2006 by Clemmer&#39;s group as an analog of MS/MS15,16. In IMS/IMS, an ion of interest is selectively isolated based on its ion mobility; it is then activated (until possible fragmentation), and a new IMS analysis of the activated ion or fragments is performed. In the first instrumental design, two IMS cells were put in series, separated by an ion funnel where the activation stood. Since then, although a number of IMS/IMS setups were proposed (for a review, see Eldrid and Thalassinos17), the first commercial mass spectrometer with IMS/IMS capability only became available in 201918. This instrument substantially improved the initial concept by combining it with another technological breakthrough: a cyclic design of the IMS cell.
The cyclic IMS cell theoretically allows increasing near-infinitely the drift path length and, thus, the resolving power of the instrument19. This was achieved by means of a particular instrument geometry, where the cyclic TWIMS cell is placed orthogonally to the main ion optical axis. A multifunction array region at the entrance of the IMS cell allows controlling the direction of the ion path: (i) sending ions sideways for IMS separation, (ii) forward for MS detection, or (iii) backward from the IMS cell to be stored in a prearray cell. From this prearray store cell, the ions can be activated and the fragments reinjected in the IMS cell for ion mobility measurement, an approach that has been successfully used to characterize stereoisomers20. Ultimately, the collected data contain ion mobility and m/z information for the precursor and its fragments.
In a recent publication that used this cyclic design for glycan analyses (Ollivier et al.21), we showed that the mobility profile of the fragments contained in such IMS/IMS data acts as a fingerprint of a biomolecule that can be used in a molecular networking strategy. The resulting network, called IM-MN, led to the organization of glycomics datasets in a structurally relevant way, whereas the network built solely from MS/MS data (MS-MN) revealed little information. To complement this publication and help Cyclic IMS users implement this workflow, this protocol provides a complete description of the protocol used to collect the data. This protocol focuses only on the generation of the IMS/IMS data that users can then use to build IM-MN networks (see21)&#8212;or for any other application of their choice. Building of IM-MN will not be considered herein, as protocols for molecular networking are already available22. The crucial points that must be followed to generate valuable and reproducible IMS/IMS acquisitions are highlighted. Taking the example of one of the oligosaccharides studied by Ollivier et al.21, the following steps are detailed: (i) sample preparation, (ii) tuning of the Cyclic IMS instrument, (iii) automated peak-picking of the data, and (iv) CCS calibration.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>33-&#945;-L- plus 23-&#945;-L-Arabinofuranosyl-xylotetraose (XA3XX/XA2XX) mixture</td>
			<td>Megazyme Ltd., Wicklow, Ireland</td>
			<td>O-XAXXMIX</td>
			<td>XA2XX + XA3XX mixture</td>
		</tr>
		<tr>
			<td>33-&#945;-L-Arabinofuranosyl-xylotetraose (XA3XX)</td>
			<td>Megazyme Ltd., Wicklow, Ireland</td>
			<td>O-XA3XX</td>
			<td>Pure XA3XX standard</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 1.5 mL, Eppendorf Quality, colorless, 1,000 tubes</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030120086</td>
			<td>Used to prepare the carbohydrate stock solution and dilution</td>
		</tr>
		<tr>
			<td>FALCON 50 mL Polypropylene Conical Tube 30 x 115 mm</td>
			<td>Corning Science M&#233;xico S.A. de C.V., Reynosa, Tamaulipas, Mexico</td>
			<td>352070</td>
			<td>Used to prepare the aqueous stock solution of 100 mM LiCl</td>
		</tr>
		<tr>
			<td>Lithium Chloride (ACS reagent, &#8805;99 %)</td>
			<td>Sigma-Aldrich Inc., Saint Quentin Fallavier, France</td>
			<td>310468</td>
			<td>Used to dope the sample with lithium</td>
		</tr>
		<tr>
			<td>Major Mix IMS/Tof Calibration Kit</td>
			<td>Waters Corp., Wilmslow, UK</td>
			<td>186008113</td>
			<td>Calibration solution for MS and IMS</td>
		</tr>
		<tr>
			<td>MassLynx 4.2 SCN1016 Release 6 (Waters Embedded Analyser Platform for Cyclic IMS 2.9.1 Release 9)</td>
			<td>Waters Corp., Wilmslow, UK</td>
			<td>721022377</td>
			<td>Cyclic IMS vendor software for instrument control and data processing</td>
		</tr>
		<tr>
			<td>Methanol for HPLC PLUS Gradient grade</td>
			<td>Carlo-Erba Reagents, Val de Reuil, France</td>
			<td>412383</td>
			<td>High-purity solvent</td>
		</tr>
		<tr>
			<td>MS Leucine Enkephaline Kit</td>
			<td>Waters Corp., Wilmslow, UK</td>
			<td>700002456</td>
			<td>Reference compound used for tuning of the mass spectrometer</td>
		</tr>
		<tr>
			<td>SCHOTT DURAN 100 mL borosilicate glass bottle</td>
			<td>VWR INTERNATIONAL, Radnor, Pennsylvania, US</td>
			<td>218012458</td>
			<td>Used to prepare the solution of 500 &#181;M LiCl in 50:50 MeOH/Water</td>
		</tr>
		<tr>
			<td>SELECT SERIES Cyclic IMS</td>
			<td>Waters Corp., Wilmslow, UK</td>
			<td>186009432</td>
			<td>Ion mobility-mass spectrometer equipped with a cylic IMS cell</td>
		</tr>
		<tr>
			<td>Website: http://mzmine.github.io/</td>
			<td>MZmine Development Team</td>
			<td>-</td>
			<td>Link to download the MZmine software</td>
		</tr>
		<tr>
			<td>Website: https://github.com/siollivier/IM-MN</td>
			<td>INRAE, UR BIA, BIBS Facility, Nantes, France</td>
			<td>-</td>
			<td>Link to an in-house R script containing a CCS calibration function</td>
		</tr>
	</tbody>
</table>

using a cyclic ion mobility spectrometer for tandem ion mobility experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

An arabinoxylan pentasaccharide, XA2XX, was chosen as an example to illustrate this protocol. This compound is commercially available, but only as a mixture with another arabinoxylan pentasaccharide, XA3XX (pure XA3XX is also commercially available). The structures of XA2XX and XA3XX are given in Supplemental&#160;Figure S1. As the ratio of XA2XX and XA3XX in the commercial mixture is ~50:50, a solution at 20 &#181;g/mL of the m...

Watch this Scientific Journal Video about Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments at JoVE.com

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass ...

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.

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3.9K Views.  INRAE, UR BIA, F-44316 Nantes, France. 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 metaboli...