Accurate Collisional Cross Section Measurement by Multipass Cyclic Ion Mobility Spectrometry.

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.

Direct Oligosaccharide Profiling Using Thin-Layer Chromatography Coupled with Ionic Liquid-Stabilized Nanomatrix-Assisted Laser Desorption-Ionization Mass Spectrometry.

The in-depth characterization of glycan structures is crucial to understanding their structure-function relationships and their effects on health and various diseases. Despite advances in rapid analysis, the utility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is limited for complex mixtures of carbohydrates due to their low ionization efficiency and the difficulty in separating oligosaccharides because of their high structural similarity. In this study, we developed an ionic liquid (IL)-stabilized, nanomatrix-decorated, thin-layer chromatography (TLC)-MALDI MS method for simultaneous and rapid separation, detection, and identification of oligosaccharides to achieve efficient profiling. The IL demonstrated good dispersion and stabilization for the spin coating of dihydroxybenzoic acid-functionalized magnetic nanoparticles (DHB@MNPs) on the TLC plate with spot homogeneity, which contributed to the observed high reproducibility (<20% CV) and 12- and 28-fold signal enhancement. Although the TLC was not able to separate isomeric glycans, the DHB@MNPs generate diagnostic glycosidic and cross-ring cleavage ions, enabling on-spot structural elucidation of composition, sequence, branching, and linkage of glycans in each separated spot. Without chemical derivatization of glycan samples, glycan visualization by TLC and tandem MS, our integrated platform, allowed the identification of 25 oligosaccharides from human milk, and heatmap analysis revealed the variability in the oligosaccharide abundance in samples from individual donors at different lactation times, which may provide insight into the microbiota and immunity of infants. With the demonstrated simplicity of our sample preparation method along with the achieved separation and in-depth structural characterization, our approach can be used for the rapid screening of other oligosaccharide-rich samples.

ZIC-cHILIC Functionalized Magnetic Nanoparticle for Rapid and Sensitive Glycopeptide Enrichment from <1 µL Serum.

Due to their unique glycan composition and linkage, protein glycosylation plays significant roles in cellular function and is associated with various diseases. For comprehensive characterization of their extreme structural complexity occurring in >50% of human proteins, time-consuming multi-step enrichment of glycopeptides is required. Here we report zwitterionic n-dodecylphosphocholine-functionalized magnetic nanoparticles (ZIC-cHILIC@MNPs) as a highly efficient affinity nanoprobe for large-scale enrichment of glycopeptides. We demonstrate that ZIC-cHILIC@MNPs possess excellent affinity, with 80-91% specificity for glycopeptide enrichment, especially for sialylated glycopeptide (90%) from biofluid specimens. This strategy provides rapidity (~10 min) and high sensitivity (<1 µL serum) for the whole enrichment process in patient serum, likely due to the rapid separation using magnetic nanoparticles, fast reaction, and high performance of the affinity nanoprobe at nanoscale. Using this strategy, we achieved personalized profiles of patients with hepatitis B virus (HBV, n = 3) and hepatocellular carcinoma (HCC, n = 3) at the depth of >3000 glycopeptides, especially for the large-scale identification of under-explored sialylated glycopeptides. The glycoproteomics atlas also revealed the differential pattern of sialylated glycopeptides between HBV and HCC groups. The ZIC-cHILIC@MNPs could be a generic tool for advancing the glycoproteome analysis, and contribute to the screening of glycoprotein biomarkers.