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-α-L-arabinofuranosyl-xylotetraose or XA2XX; see the Table of Materials) as an example.
2. Tuning of the Cyclic IMS mass spectrometer
NOTE: Software-related instructions (windows, menus, and commands) are highlighted in bold.
3. IMS/IMS-MS acquisition
4. IMS/IMS-MS processing with MZmine 224
NOTE: MZmine 2 is available from the URL given in the Table of Materials. The use of MZmine 2.51 is recommended. At the time of preparation of this manuscript, the later versions cannot open RAW files from Cyclic IMS instruments because of a change in the import function.
5. TWCCSN2 of the centroided IMS/IMS spectra
NOTE: In this protocol, a logarithmic fit calibration25,26 will be used, which tends to give better results than linear calibration and is easy to implement in a spreadsheet or an in-house processing script. An in-house script (written in R) is available at the URL given in the Table of Materials.
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 Figure S1. As the ratio of XA2XX and XA3XX in the commercial mixture is ~50:50, a solution at 20 µg/mL of the mixture was prepared to reach an XA2XX concentration of ~10 µg/mL in 50:50 MeOH/H2O + 500 µM 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 µg/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 → pre-array bias → 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 Table S1). The CCS calibration was performed using the calibration solution (R2 = 0.995, mean absolute deviation of control = 1.63%, see Supplemental Table S3). This processing finally afforded a centroided, CCS-calibrated, IMS/IMS spectrum (Figure 6B).
Figure 1: Overview of the IMS/IMS data generation process. Abbreviations: IMS = ion mobility spectrometry; IMS/IMS = tandem IMS. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Supplemental Figure S1: Structures of the XA2XX and XA3XX arabinoxylan pentasaccharides. Please click here to download this File.
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. Please click here to download this File.
Supplemental Table S1: Detailed MZmine parameters. Please click here to download this Table.
Supplemental Table S2: Instrument parameters changed to evaluate the reproducibility. Abbreviation: ESI = electrospray ionization. Please click here to download this Table.
Supplemental Table S3: Control of the CCS calibration using a second acquisition of calibration solution. Please click here to download this Table.
33-α-L- plus 23-α-L-Arabinofuranosyl-xylotetraose (XA3XX/XA2XX) mixture | Megazyme Ltd., Wicklow, Ireland | O-XAXXMIX | XA2XX + XA3XX mixture |
33-α-L-Arabinofuranosyl-xylotetraose (XA3XX) | Megazyme Ltd., Wicklow, Ireland | O-XA3XX | Pure XA3XX standard |
Eppendorf Safe-Lock Tubes, 1.5 mL, Eppendorf Quality, colorless, 1,000 tubes | Eppendorf, Hamburg, Germany | 0030120086 | Used to prepare the carbohydrate stock solution and dilution |
FALCON 50 mL Polypropylene Conical Tube 30 x 115 mm | Corning Science México S.A. de C.V., Reynosa, Tamaulipas, Mexico | 352070 | Used to prepare the aqueous stock solution of 100 mM LiCl |
Lithium Chloride (ACS reagent, ≥99 %) | Sigma-Aldrich Inc., Saint Quentin Fallavier, France | 310468 | Used to dope the sample with lithium |
Major Mix IMS/Tof Calibration Kit | Waters Corp., Wilmslow, UK | 186008113 | Calibration solution for MS and IMS |
MassLynx 4.2 SCN1016 Release 6 (Waters Embedded Analyser Platform for Cyclic IMS 2.9.1 Release 9) | Waters Corp., Wilmslow, UK | 721022377 | Cyclic IMS vendor software for instrument control and data processing |
Methanol for HPLC PLUS Gradient grade | Carlo-Erba Reagents, Val de Reuil, France | 412383 | High-purity solvent |
MS Leucine Enkephaline Kit | Waters Corp., Wilmslow, UK | 700002456 | Reference compound used for tuning of the mass spectrometer |
SCHOTT DURAN 100 mL borosilicate glass bottle | VWR INTERNATIONAL, Radnor, Pennsylvania, US | 218012458 | Used to prepare the solution of 500 µM LiCl in 50:50 MeOH/Water |
SELECT SERIES Cyclic IMS | Waters Corp., Wilmslow, UK | 186009432 | Ion mobility-mass spectrometer equipped with a cylic IMS cell |
Website: http://mzmine.github.io/ | MZmine Development Team | – | Link to download the MZmine software |
Website: https://github.com/siollivier/IM-MN | INRAE, UR BIA, BIBS Facility, Nantes, France | – | Link to an in-house R script containing a CCS calibration function |
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.
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.
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.