The isomeric complexity of glycans make their analysis by traditional techniques particularly challenging. While the recent combination of ion mobility spectrometry (IMS) with cryogenic IR spectroscopy has demonstrated promise as a new technique for glycan analysis, this approach has been limited by the modest resolution of the ion mobility stage. In this work we report results from a newly developed instrument that combines ultrahigh-resolution IMS with cryogenic IR spectroscopy for glycan analysis. This apparatus makes use of the recent development in traveling-wave IMS called structures for lossless ion manipulation. The IMS stage allows the selection of glycan isomers that differ in collisional cross section by as little as 0.2% before injecting them into a cryogenic ion trap for IR spectral analysis. We compare our results to those using drift-tube IMS and highlight the advantages of the substantial increase in resolution. Application of this approach to glycan mixtures demonstrates our ability to isolate individual components, measure a cryogenic IR spectrum, and identify them using a spectroscopic database.

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 analyze 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 glycan 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 disaccharides how the two a and b 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.

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.

We explore the capability of SLIM-based IMS for isomer selectivity in combination with cryogenic, messenger-tagging IR spectroscopy.