Separation and Identification of Glycan Anomers Using Ultrahigh-Resolution Ion Mobility Spectrometry and Cryogenic Ion Spectroscopy

The analysis of carbohydrates, or glycans, is challenging for established structure-sensitive gas-phase methods. The multitude of possible stereo-, regio- and structural isomers make them substantially more complex to ana-lyze than DNA or proteins, and no one method is currently able to fully resolve them. While the combination of tandem mass spectrometry (MS) and ion mobility spectrometry (IMS) have made important inroads in gly-can analysis, in many cases this approach is still not able to identify the precise isomeric form. To advance the techniques available for glycan analysis we employ two important innovations. First, we perform ultrahigh-resolution mobility separation using structures for lossless ion manipulations (SLIM) for isomer separation and pre-selection. We then complement this IMS-MS stage with a cryogenic IR spectroscopic dimension, since a glycan's vibrational spectrum provides a fingerprint that is extremely sensitive to the precise isomeric form. Using this unique approach in conjunction with oxygen-18 isotopic labelling, we show on a range of disaccha-rides how the two  and  anomers that every reducing glycan adopts in solution can be readily separated by mobility and identified based on their IR spectra. In addition to highlighting the power of our technique to detect minute differences in the structure of isomeric carbohydrates, these results provide the means to determine if and when anomericity is retained during collision-induced dissociation (CID) of larger glycans.

A New Strategy Coupling Ion-Mobility-Selective CID and Cryogenic IR Spectroscopy to Identify Glycan Anomers

Priyanka Bansal, Ahmed Ben Faleh, Eduardo Carrascosa Casado, Robert Paul Pellegrinelli, Thomas Rizzo, Stephan Warnke, Lei Yue

Determining the primary structure of glycans remains challenging due to their isomeric complexity. While high-resolution ion mobility spectrometry (IMS) has recently allowed distinguishing between many glycan isomers, the arrival-time distributions (ATDs) frequently exhibit multiple peaks, which can arise from positional isomers, reducing-end anomers, or different conformations. Here, we present the combination of ultrahigh-resolution ion mobility, collision-induced dissociation (CID), and cryogenic infrared (IR) spectroscopy as a systematic method to identify reducing-end anomers of glycans. Previous studies have suggested that high-resolution ion mobility of sodiated glycans is able to separate the two reducing-end anomers. In this case, Y-fragments generated from mobility-separated precursor species should also contain a single anomer at their reducing end. We confirm that this is the case by comparing the IR spectra of selected Y-fragments to those of anomerically pure mono- and disaccharides, allowing the assignment of the mobility-separated precursor and its IR spectrum to a single reducing-end anomer. The anomerically pure precursor glycans can henceforth be rapidly identified on the basis of their IR spectrum alone, allowing them to be distinguished from other isomeric forms.

2022

Identification of Mobility-Resolved N-Glycan Isomers

Priyanka Bansal, Ahmed Ben Faleh, Robert Paul Pellegrinelli, Thomas Rizzo, Stephan Warnke

ABSTRACT: Glycan analysis has evolved considerably during the last decade. The advent of high-resolution ion-mobility spectrometry has enabled the separation of isomers with only the slightest of structural differences. However, the ability to separate such species raises the problem of identifying all the mobility-resolved peaks that are observed, especially when analytical standards are not available. In this work, we report an approach based on the combination of IMSn with cryogenic vibrational spectroscopy to identify N-glycan reducing-end anomers. By identifying the reducing-end alpha and beta anomers of diacetyl-chitobiose, which is a disaccharide that forms part of the common core of all N-glycans, we are able to assign mobility peaks to reducing anomers of a selection of N-glycans of different sizes, starting from trisaccharides such as Man-1 up to glycans containing nine monosaccharide units, such as G2. By building an infrared fingerprint database of the identified N-glycans, our approach allows unambiguous identification of mobility peaks corresponding to reducing-end anomers and distinguishes them from positional isomers that might be present in a complex mixture.

AMER CHEMICAL SOC2022

Using SLIM-Based IMS-IMS Together with Cryogenic Infrared Spectroscopy for Glycan Analysis

Priyanka Bansal, Ahmed Ben Faleh, Thomas Rizzo, Stephan Warnke, Natalia Yalovenko, Vasyl Yatsyna

The isomeric heterogeneity of glycans poses a great challenge for their analysis. While combining ion mobility spectrometry (IMS) with tandem mass spectrometry is a powerful means for identifying and characterizing glycans, it has difficulty distinguishing the subtlest differences between isomers. Cryogenic infrared spectroscopy provides an additional dimension for glycan identification that is extremely sensitive to their structure. Our approach to glycan analysis combines ultrahigh-resolution IMS-IMS using structures for lossless ion manipulation (SLIM) with cryogenic infrared spectroscopy. We present here the design of a SLIM board containing a series of on-board traps in which we perform collision-induced dissociation (CID) at pressures in the millibar range. We characterize the on-board CID process by comparing the fragments generated from a pentapeptide to those obtained on a commercial tandem mass spectrometer. We then apply our new technique to study the mobility and vibrational spectra of CID fragments from two human milk oligosaccharides. Comparison of both the fragment drift times and IR spectra with those of suitable reference compounds allows us to identify their specific isomeric form, including the anomericity of the glycosidic linkage, demonstrating the power of this tool for glycan analysis.

AMER CHEMICAL SOC2020