Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Summary

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.

Abstract

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.

Introduction

The complete chemical characterization of biomolecules is key to understanding their underlying biological and functional properties. To this end, "omics" 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 establi....

Protocol

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.

1. Preparation of the solvent: 500 µM LiCl in 50:50 H₂O:MeOH (vol./vol.).....

Results

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 m.......

Discussion

The SELECT SERIES Cyclic IMS is a powerful tool that allows selecting a defined ion population—of a given m/z and ion mobility—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.......

Materials

Name	Company	Catalog Number	Comments
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