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Detailed glycan structural characterization is frequently achieved by collisionally activated dissociation (CAD) based sequential tandem mass spectrometry (MSn) analysis of permethylated glycans. However, it is challenging to implement MSn(n> 2) during online glycan separation, and this has limited its application to analysis of complex glycan mixtures from biological samples. Further, permethylation can reduce liquid chromatographic (LC) resolution of isomeric glycans. Here, we studied the electronic excitation dissociation (EED) fragmentation behavior of native glycans with a reducing-end fixed charge tag and identified key spectral features that are useful for topology and linkage determination. We also developed a de novo glycan sequencing software that showed remarkable accuracy in glycan topology elucidation based on the EED spectra of fixed charge-derivatized glycans. The ability to obtain glycan structural details at the MS2level, without permethylation, via a combination of fixed charge derivatization, EED, and de novo spectral interpretation, makes the present approach a promising tool for comprehensive and rapid characterization of glycan mixtures.

Ion mobility-mass spectrometry (IM-MS) is a powerful analytical tool for structural characterization. IM measurement provides collision cross section (CCS) values that facilitate analyte identification. While CCS values can be directly calculated from mobility measurements obtained using drift tube ion mobility spectrometry (DT-IMS), this method has limited mobility resolution due to the practical constraints on the length of the ion drift path. Consequently, DT-IMS cannot differentiate analytes with similar mobilities or resolve fine mobility features of individual ions. Cyclic IMS (cIMS) instruments leverage a cyclic path enabled by traveling wave ion mobility (TWIM) technology and offer increased mobility solution to address this challenge. While TWIM devices must first be calibrated to enable CCS measurements, current calibration strategies are primarily tailored for single-pass analyses. This preference is partly attributed to the challenges associated with multipass calibration methods, which require both calibrants and analytes to experience the same number of passes. Achieving this consistency can be complicated due to factors like peak splitting and diffusion, and may not be feasible for online IM-MS analyses. A recent report employed average ion velocities obtained from multiple measurements under different separation pathlengths as a path length-independent metric for CCS calibration. However, the ability to exploit this averaging approach is limited by observed variation in ion drift time/velocity in these measurements. In this study, we introduce a novel calibration strategy designed for multipass cIMS analyses, directly targeting the root cause for the path length- and mobility-dependent variations in ion drift time. With this method, we demonstrate that CCS values derived from multipass measurements closely align with those obtained from single-pass analyses, with an average deviation of 0.1%. We apply this method to characterize four isomeric trisaccharides. Our approach not only results in excellent agreement between our measured cIMSCCS values and the reported DTCCS values, with an average difference of only 0.5%, but also allows us to effectively identify subtle mobility characteristics of each compound and determine their respective CCS values. This level of detail and accuracy was previously unattainable using DT-IMS or single-pass cIMS measurements. We developed an algorithm for reconstructing arrival time distribution in cases where wrap-around has resulted in peak splitting. Collectively, the new calibration strategy and the reconstruction procedure maintain reproducibility and precision in CCS measurements while largely eliminating the need for meticulous selection of separation times. We expect that our method will empower researchers to harness the high mobility resolution offered by multipass cIMS analyses without compromising the accuracy of CCS measurement, making it appropriate for straightforward use across a wide range of applications.