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

The authors have no conflict of interest to disclose.

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 user should carefully check the MS isolation window for the presence of possible isobaric contaminants. Indeed, the isolation window of a quadrupole is relatively wide, and knowing ions of a slightly different m/z that may be coselected in the quadrupole will help the user properly assign the peak of interest in ion mobility.
Second, when performing the initial separation, the user must ensure that all ions undergo the same number of passes around the Cyclic ion mobility cell. This is an important and tricky aspect of ion mobility separation in a cyclic device. An erroneous evaluation of the number of passes for a given ion may lead to an improper identification and interpretation of the peaks. Controlling the number of passes of different ion populations can be tricky owing to the relatively short single-pass length (~1 m), and species with very different mobilities can quickly overlap.
In particular, a peak can split between two different passes if the array switches direction when this ion population passes through (this is relatively easy to identify: the split peak will appear sharper with a population right at the beginning of the Eject and Acquire event). To properly set the number of passes, the user should start with a short separation time (1-5 ms) that will give the 1-pass profile. Then, the user should gradually increase the separation time until the entire population has moved to higher arrival times, which will give the 2-pass profile. The 2-pass profile should look similar to the 1-pass profile but with better-resolved peaks. The time a given ion population takes to make one pass around the cyclic cell is a constant that the user can use to calculate the number of passes as a function of the separation time. For instance, if there is a 10 ms difference between the first and second pass, there will also be a 10 ms difference between the second and the third.
Third, during the IMS selection stage, the user should carefully check the quality of the isolation, as demonstrated in Figure 4. It is especially important to check the reinjected profile because if the TW height and velocity settings are too low, the ejection of the other populations might not be complete. Advanced users can correct this by adjusting the Driftcell RF radiofrequency voltage in the RF tab of the Tune page.
Fourth, the user should be careful in generating the fragmentation spectrum and, notably, in selecting the appropriate collision energy, especially if the voltages are tuned manually. Indeed, excessively lowering the Array Offset voltage can negatively impact the overall ion intensity by hindering the reinjection. In addition, the precursor and fragments might span over a wide range of mobilities. Thus, they will rapidly undergo a different number of passes if the final separation time is high, so it is important to keep a 1-ms Separate event as explained in protocol step 2.3.3. This is a major limitation since the single-pass length is relatively short, limiting the single-pass resolving power to ~100 for oligosaccharides27. In this respect, an increased path length in a single pass would be beneficial (i.e., the TWIMS-based Structures for Lossless Ion Manipulation or SLIM, with a path length of 13 m28). The SLIM setup was launched commercially very recently29.
Finally, the user should be careful in defining the final acquisition mobility range using the Pushes per bin command, particularly if working on multiply charged ions. Ion mobility is indeed a function of the charge12, and, for example, singly-charged fragments generated from a doubly-charged precursor are likely to be slower than the precursor (although they are smaller compounds).
A major limitation of using only MS and IMS separations to select the precursor (and not, for instance, an upstream step of chromatography) is that a given m/z can yield multiple peaks in IMS and that multiple peaks can come from the same compound. This is illustrated by the distributions of m/z 685.2, for both the XA2XX+XA3XX mixture and pure XA3XX, in Figure 4A. Multimodal IMS distributions of a single m/z result from different gas-phase conformations. For species analyzed as cation adducts (in positive mode), the different conformations possibly arise from differences in coordination with the counter-ion30,31,32.
For oligosaccharides, they can also arise from the separation of reducing-end anomers, although separating reducing-end anomers typically requires higher IMS resolving power than what is used here33,34,35. In the present case, the multimodal IMS distribution in Figure 4A results partly from the individual contributions of XA2XX and XA3XX. It is, however, notable that XA3XX yields two peaks, which are likely cation-coordination conformers. It was easy to identify which peak corresponded to XA2XX (i.e., the species of interest) because pure XA3XX is available commercially, and its mobility profile could be recorded separately. To work on complex mixtures such as biological media, it may be important to consider adding a chromatographic separation stage.
Two points must be noted regarding the processing workflow used to obtain mass-deconvolved IMS/IMS spectra. First, in this protocol, it is proposed to use MZmine 224 to deconvolve the IMS/IMS spectrum using the MS dimension and, notably, use the ADAP algorithm36 to split the EIMs into different peaks. Although it gives fairly good results, as illustrated in Figure 6, the ADAP algorithm was designed for chromatographic analyses and thus accounts for asymmetry factors inherent to liquid phase chromatography, such as peak tailing. Therefore, the ADAP algorithm might result in not identifying some of the features when applied to IMS peaks (e.g., shoulders). In essence, IMS data are simpler than chromatographic data: because there is no chemical interaction of the compounds with a column, IMS data acquired under appropriate conditions (i.e., without saturating the IMS cell) are expected to follow gaussian distributions37,38. Ideally, the ADAP deconvolution step would be best replaced by a gaussian fitting function, such as that used by software destined for IMS like CUISuite 239. However, as it stands, gaussian deconvolution was not directly adapted to the complete chain of treatment described in this protocol. Therefore, using the free, open-source software MZmine appeared to be a good compromise for end-users.
The second part of the processing that warrants discussion is the CCS calibration. This protocol proposes using a logarithmic fit calibration25,26 and a commercial calibrant mixture from the same provider as that of the spectrometer (see the Table of Materials). This procedure is the most straightforward to implement in the lab. Regarding the choice of the calibrant mixture, the user should consider that, as mentioned in several studies, the accuracy of the CCS calibration is improved when using calibrants of the same molecular class and charge state as the analyte26,40,41. The error introduced when calibrating with relatively similar types of compounds (e.g., carbohydrates vs. peptides) is moderate. However, it is recommended not to use very different ions such as, for example, using salt clusters as calibrants when measuring carbohydrates41. Regarding the choice of the calibration method, Richardson et al.42 recently reported a new calibration method that takes into account the physics of TWIMS to improve the accuracy of the calibration (with a provided software). However, the approach requires the evaluation of highly specific parameters through the analysis of various types of compounds&#8212;ranging from metabolites to native proteins. Because no mixture of such a variety of compounds can be found commercially, this method was not implemented in the present protocol.
Finally, to evaluate the reproducibility of the method, we evaluated the interday reproducibility by repeating the IMS/IMS-MS acquisition at day 1 and day 95 (Supplemental Figure S2). The experiment showed that IMS/IMS-MS data are highly reproducible, with no IMS peak shifting by more than 0.2 ms over this extended period. The IMS/IMS spectrum generated in this work was further compared to another spectrum of XA2XX acquired under different conditions for previous work on ion mobility-molecular networking21. Some instrumental parameters that can impact the ion structure and ion mobility profile13 were deliberately changed&#8212;the source parameters and the activation voltage gradient (a comparison of the varying instrumental conditions is given in Supplemental Table S2). Then, the two spectra were compared using the cosine similarity score&#8212;which is popular for the comparison of MS/MS spectra in metabolomics&#8212;on the GNPS platform5 (CCS tolerance for matching fragments = 0.015 nm2). 
The comparison showed a cosine similarity score of 0.87 (Figure 7), which can be considered high with regard to the important instrumental variations applied. This leads to the idea that IMS/IMS spectral libraries could be used to dereplicate glycans in complex mixtures with a high level of confidence, which would not be the case with MS/MS spectra. Note that although the current approach only uses the CCS dimension of the fragmentation spectrum, the IMS/IMS-MS data also contains MS information, which is not redundant with the CCS. To optimize the dereplicative power of IMS/IMS, a bidimensional scoring system must be developed.

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><tbody><tr><td>33-&amp;alpha;-L- plus 23-&amp;alpha;-L-Arabinofuranosyl-xylotetraose (XA3XX/XA2XX) mixture</td><td>Megazyme Ltd., Wicklow, Ireland</td><td>O-XAXXMIX</td><td>XA&lt;sup&gt;2&lt;/sup&gt;XX + XA&lt;sup&gt;3&lt;/sup&gt;XX mixture</td></tr><tr><td>33-&amp;alpha;-L-Arabinofuranosyl-xylotetraose (XA3XX)</td><td>Megazyme Ltd., Wicklow, Ireland</td><td>O-XA3XX</td><td>Pure XA&lt;sup&gt;3&lt;/sup&gt;XX 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&amp;eacute;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, &amp;ge;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 &amp;micro;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 mixture was prepared to reach an XA2XX concentration of ~10 &#181;g/mL in 50:50 MeOH/H2O + 500 &#181;M LiCl.
First, an MS analysis of the XA2XX + XA3XX mixture was performed using high-resolution MS. As the two compounds are isomers, a single peak was observed at [M+Li]+m/z 685.24. This MS peak was selected with the quadrupole and the selection window adjusted to remove the -1 Da lithium isotope, which could be mistaken as the monoisotopic peak by processing algorithms (Figure 2).
The [M+Li]+ adducts of the pentasaccharides were then submitted to the first stage of IMS separation: after 3 passes around the cyclic IMS cell, 3 peaks were separated with arrival times of 83, 90, and 94 ms. This profile was compared to that of pure XA3XX (infused at 10 &#181;g/mL), showing that the peaks at 83 and 90 ms corresponded to XA3XX, while the peak at 94 ms corresponded to XA2XX (Figure 4A). The peak at 94 ms was selected for IMS/IMS analysis: the ions belonging to XA3XX were ejected (Figure 4B), and the peak of interest was sent to the prearray store cell. A 3-pass separation was performed after reinjecting the ion without activation to ensure that only the XA2XX peak remained after the selection (arriving at 199 ms in Figure 4C).
Then, the ion was fragmented upon reinjection from the prestore area, and a single-pass IMS separation was performed on all the fragments. Two different activations were tried: the maximum setting of the built-in prestore activation function was first tried (Figure 5B,C); however, the precursor remained the base peak of the spectrum. This is not desired because, for reference spectra, fragments below a certain intensity threshold would typically be removed. Thus, a manually defined prearray gradient &#8594; pre-array bias &#8594; array offset voltage gradient was chosen (Figure 5D,E).
The generated IMS/IMS-MS data were deconvolved with MZmine 2.51, using the arrival time and m/z dimensions (Figure 6A), to give IMS/IMS spectra containing only the mobility information of the fragments. The peaks above 0.2% relative intensity were exported for CCS calibration (the detailed MZmine parameters are given in Supplemental&#160;Table S1). The CCS calibration was performed using the calibration solution (R2 = 0.995, mean absolute deviation of control = 1.63%, see Supplemental&#160;Table S3). This processing finally afforded a centroided, CCS-calibrated, IMS/IMS spectrum (Figure 6B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63451/63451fig01.jpg" /> 
Figure 1: Overview of the IMS/IMS data generation process. Abbreviations: IMS = ion mobility spectrometry; IMS/IMS = tandem IMS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63451/63451fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63451/63451fig02.jpg" /> 
Figure 2: Isotopic pattern of an XA3XX + XA2XX arabinoxylan pentasaccharide mixture. (A) Saturated signal without DRE; (B) signal corrected using DRE with a 5% ion transmission (i.e., 95% attenuation); and (C) profile after quadrupole selection to remove the -1 Da peak corresponding to a lithium isotope. In purple: the region where artifact peaks can appear due to saturation is magnified 6 times. Abbreviations: DRE = dynamic range enhancement; MS = mass spectrometry; MSMS = tandem MS; LM Res = low-mass resolution; HM Res = high-mass resolution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63451/63451fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63451/63451fig03.jpg" /> 
Figure 3: Overview of the Cyclic IMS control window, in which the user defines the IMS/IMS sequence. The sequence displayed shows how to check the quality of the isolation in IMS/IMS, with a selection of XA2XX after 3 passes (the spectrum displayed corresponds to the setting of the selection events after the first-stage separation). The sequence consists in running a first 3-pass IMS separation over 58 ms, then ejecting the two faster isoforms from the IMS cell (segment 3), ejecting the slower isoform (ATD between 92 and 96 ms) in the prestore (segment 4), reinjecting it in the IMS cell without activation (segment 6), allowing the ions to undergo a further 3-pass (58-ms) separation (segment 7), then ejecting ions from the IMS cell, and acquiring data (segment 8). Abbreviations: IMS = ion mobility spectrometry; cIMS = cyclic IMS; IMS/IMS = tandem IMS; ADC = analog-to-digital converter; TW = traveling wave; PE = potential energy; ATD = arrival time distribution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63451/63451fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63451/63451fig04.jpg" /> 
Figure 4: Selection of XA2XX from the mixture of XA2XX and XA3XX. (A) Separation of the arabinoxylan pentasaccharides, XA3XX and XA2XX, after 3 passes (corresponding to a separation time set at 58 ms) around the Cyclic IMS cell. (B) Fraction ejected directly after the first stage of IMS separation. (C) Fraction selected for IMS/IMS on which another 3-pass separation was performed after reinjection. The XA2XX peak of interest is highlighted in gray. The ion mobility spectra are shown in data bins and annotated with their arrival time (ms). Abbreviations: IMS = ion mobility spectrometry; IMS/IMS = tandem IMS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63451/63451fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63451/63451fig05.jpg" /> 
Figure 5: Principles of collision-induced dissociation using the prearray store area. (A) Schematics of the multifunction array region detailing key voltages (in red) used for the selection, the reinjection, and the activation during IMS/IMS experiments. The blue arrows show the direction of the traveling wave in the multifunction array. (B, C) IMS/IMS and MS/MS spectra obtained for XA2XX using the built-in prestore activation function (+150 V). The color bar represents the ion intensity scale (blue = low; red = high). (D, E) IMS/IMS and MS/MS spectra obtained for XA2XX with manual optimization of the voltages (prearray gradient 195 V, prearray bias 180 V, array offset -10 V). Precursor ions are indicated by asterisks on the spectra. The ion mobility spectra are shown in data bins and annotated with their arrival time (ms). Abbreviations: IMS = ion mobility spectrometry; IMS/IMS = tandem IMS; TOF = time-of-flight. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63451/63451fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63451/63451fig06.jpg" /> 
Figure 6: Illustration of the processing steps. Results of (A) the MZmine peak picking and (B) the CCS calibration of arabinoxylan pentasaccharide XA2XX. A shows the mass deconvolution of the IMS/IMS spectrum through a color code. B shows the final IMS/IMS spectrum after centroiding and CCS calibration. Abbreviations: IMS = ion mobility spectrometry; IMS/IMS = tandem IMS; CCS = collision cross section. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63451/63451fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63451/63451fig07.jpg" /> 
Figure 7: A comparison of two IMS/IMS spectra of XA2XX illustrates the reproducibility of the method. The final calibrated spectrum from this paper (top) is compared to the spectrum from the work by Ollivier et al.21 (bottom, flipped). Abbreviations: IMS = ion mobility spectrometry; IMS/IMS = tandem IMS; CCS = collision cross section. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63451/63451fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure S1: Structures of the XA2XX and XA3XX arabinoxylan pentasaccharides. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63451/JoVE_FigS1.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure S2: Evaluation of the interday repeatability using XA2XX. IMS/IMS acquisitions were repeated at Day 1 (top) and Day 95 (bottom). Abbreviations: IMS = ion mobility spectrometry; IMS/IMS = tandem IMS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63451/JoVE_FigS2.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Table S1: Detailed MZmine parameters. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63451/Table S1.docx" target="_blank">Please click here to download this Table.</a>
Supplemental Table S2: Instrument parameters changed to evaluate the reproducibility. Abbreviation: ESI = electrospray ionization. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63451/Table S2.xlsx" target="_blank">Please click here to download this Table.</a>
Supplemental Table S3: Control of the CCS calibration using a second acquisition of calibration solution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63451/Table S3.xlsx" target="_blank">Please click here to download this Table.</a>

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 ...

using-cyclic-ion-mobility-spectrometer-for-tandem-ion-mobility

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4.2K Views.  INRAE, UR BIA, F-44316 Nantes, France. Het protocol beschrijft hoe een ionenmobiliteitsprofiel kan worden vastgelegd voor de fragmenten die worden geproduceerd bij activering van een molecuul in de massaspectrometer en hoe onderscheid kan worden gemaakt tussen isomere structuren. Het protocol maakt het mogelijk om glycanen in 11 groepen te classificeren met betrekking tot enkele van hun belangrijkste structurele kenmerken, en zo een informatie te verstrekken die anders moeilijk te verkrijgen is. Het kan dienen om moleculaire netwe...

Video: Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Biochemistry

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, penicillin, paclitaxel, rapamycin, or artemisinin, commonly used in medical practice, have been first identified and isolated from natural products. However, the identification and isolation of biologically active specialized metabolites from natural sources is a challenging and time-consuming process. Traditionally, individual metabolites are isolated and purified from complex mixtures, following the extraction of biomass. Subsequently, the isolated molecules are tested in functional assays to verify their biological activity. Here we present the use of cellular membrane affinity chromatography (CMAC) columns to identify biologically active compounds directly from complex mixtures. CMAC columns allow for the identification of compounds interacting with immobilized functional transmembrane proteins (TMPs) embedded in their native phospholipid bilayer environment. This is a targeted approach, which requires knowing the TMP whose activity one intends to modulate with the newly identified small molecule drug candidate. In this protocol, we present an approach to prepare CMAC columns with immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders. In this article, we provide a detailed protocol to assemble the CMAC column with immobilized TrkB receptors using neuroblastoma cell lines overexpressing TrkB receptors. We further present the approach to investigate the functionality of the column and its use in the identification of specialized plant metabolites interacting with TrkB receptors.

The protocol describes the preparation of cell membrane affinity chromatography (CMAC) columns with immobilized cell membrane fragments containing functional transmembrane tropomyosin kinase receptor B proteins. The use of CMAC columns in the identification of specialized plant metabolites interacting with these receptors and present in complex natural mixtures is also explained.

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, ...

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold collision-induced dissociation (TCID) to measure the thermochemistry of the dissociation of negatively-charged [amb+M(II)+NTA]- ternary complexes into two product channels: [amb+M(II)] + NTA or [NTA+M(II)]-&#160;+ amb, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contain one of the alternative metal binding (amb) heptapeptides with the primary structures acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7 or acetyl-Asp1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, where the amino acids&#39; Aa1,2,6,7 positions are the potential metal-binding sites. Geometry-optimized stationary states of the ternary complexes and their products were selected from quantum chemistry calculations (presently the PM6 semi-empirical Hamiltonian) by comparing their electronic energies and their collision cross-sections (CCS) to those measured by ES-IM-MS. From the PM6 frequency calculations, the molecular parameters of the ternary complex and its products model the energy-dependent intensities of the two product channels using a competitive TCID method to determine the threshold energies of the reactions that relate to the 0 K enthalpies of dissociation (&#916;H0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (&#916;H298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.

Thermochemical Studies of NiII and ZnII Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry, semi-empirical quantum calculations, and energy-resolved threshold collision-induced dissociation to measure the relative thermochemistry of the dissociation of related ternary metal complexes.

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Biology

Histone Proteomic Analysis and Post-Translational Modification Characterization

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

Enhanced Isomeric Separation of Ergot Alkaloids Using LC-IMS-MS

Detection of Regulated Ergot Alkaloids in Food Matrices by Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometry

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) workflows. LC-IMS-MS methods provide higher peak resolution, enhanced separation of isobaric and isomeric compounds, and improved signal-to-noise ratio (S/N) compared to traditional LC-MS methods. IMS provides another molecular characteristic for the identification of analytes, namely the collision cross section (CCS) parameter, reducing false positive results. Therefore, LC-IMS-MS methods address important analytical challenges in the field of food safety (i.e., detection of compounds at trace levels in complex food matrices and unambiguous identification of isobaric and isomeric molecules).
Ergot alkaloids (EAs) are a family of mycotoxins produced by fungi that attack a wide variety of grass species, including small grains such as rye, triticale, wheat, barley, millet, and oats. Maximum levels (MLs) of these mycotoxins have been established in several foodstuffs, as detailed in the Commission Regulation EC/2023/915. This new legislation includes six main EAs and their corresponding epimers, so an efficient methodology is required to properly distinguish these isomeric molecules considering their co-occurrence.
Therefore, the goal of this protocol is to show how the integration of IMS in LC-MS workflows contributes to the separation of isomeric EAs, enhancing the selectivity of the analytical method. Additionally, it illustrates how the generation of CCS libraries through the characterization of analytical standards provides higher confidence for the identification of mycotoxins. This protocol is designed to clearly explain the benefits of implementing IMS in food safety, taking as an example the determination of EAs in cereals. A QuEChERS-based extraction followed by an LC-trapped ion mobility spectrometry (TIMS)-MS analysis provided limits of quantification ranging from 0.65 to 2.6 ng/g with acceptable accuracy (although low recovery for ergotaminine) at 1.5x, 1x, and 0.5x the ML and exhibited a negligible matrix effect.

This protocol presents a validated liquid chromatography-ion mobility-high resolution mass spectrometry method to determine the presence of ergot alkaloids in food in compliance with the recently released Commission Regulation (EU) 2023/915.

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) ...

Tandem Mass Spectrometry

Tandem mass spectrometry is a technique that uses multiple mass analyzers in series to obtain a higher selectivity and signal-to-noise ratio for the analyte. Instruments with multiple analyzers separated by an interaction cell enable secondary fragmentation and selected study of the fragment ions.
Secondary fragmentations occur in the interaction cell and can be induced by various factors. Fragmentation induced by collision with inert gases, such as N2, Ar, He, etc., is called collision-induced dissociation (CID). Other techniques involve interaction with the surface through surface-induced dissociation (SID); interaction with low-energy electrons, called electron-capture dissociation (ECD); or through an intense laser beam, called photo-induced dissociation (PID).
Tandem mass spectrometry can be used for various experiments by varying the mode of operation of the two mass analyzers. When the first mass analyzer selects a particular precursor ion and the second mass analyzer scans all the fragmented product ions, it is called a product-ion scan, and the spectra obtained by this experiment are called product-ion spectra. Alternately, when the first mass analyzer scans all the precursor ions and the second mass analyzer selects a particular product ion, it is called &#39;precursor-ion mode&#39;, and the spectra are termed precursor-ion spectra. Another experiment can be performed by scanning both the analyzers simultaneously with an offset of a mass equal to a neutral fragment lost by the ion to be monitored. Such a mode is called &#39;neutral-loss scanning mode&#39;, and the corresponding spectra are called neutral-loss spectra.
The instrumentation of tandem mass spectrometry can be varied. Tandem-in-space spectrometers use two independent mass analyzers in two different regions in space, for example, a triple quadrupole mass spectrometer and TOF&#8211;TOF spectrometer. On the other hand, a tandem-in-time spectrometer, such as a quadrupole ion-trap instrument, uses the same spatial region to fragment the ions and dissociate and analyze a selected ion of interest, but at different times by expelling the unwanted ions. Tandem-in-time spectrometers can be used to perform MSn experiments. &#160;&#160;

Tandem mass spectrometry, or MS/MS, combines two mass analyzers in sequence, connected by a collision or interaction cell.
First, the samples are ionized ...

Education

JoVE Core

Analytical Chemistry

Unit 1

Principles of Mass Spectrometry

KEY TERMS AND CONCEPTS

Mobility Shift Affinity Capillary Electrophoresis: A Method to Analyze Sample-Ligand Interactions Depending on Differential Migration of Protein-Ligand Complexes

After preparing the method for the measurements without ligands, prepare the method for the measurements with ligands, by first using 0.1 molar EDTA solution to rinse the capillary at 2.5 bar for 1 minute. Then, use deionized water to rinse the capillary. Next, equilibrate the capillary by using ligand solution to rinse it at 2.5 bar for 1.5 minutes.
Then, inject the acetanilide solution at 0.05 bar for 6 seconds, and change the inlet and outlet vials to the ligand-containing buffer vials. Apply 0.05 bar for 2.4 seconds, in order to push the acetanilide solution further in from the tip of the capillary. Apply 10.0 kilovolts for 6 minutes, and detect the acetanilide peak at a wavelength of 200 nanometers.
After rinsing the capillary with EDTA, deionized water, and the ligand solution as before, inject the protein sample, and change the inlet and outlet vials to fresh ligand-containing buffer vials. Ultimately, repeat taking measurements with and without ligands.

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

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