Combining Liquid Chromatography and Cryogenic IR Spectroscopy in Real Time for the Analysis of Oligosaccharides.

While the combination of liquid chromatography (LC) and mass spectrometry (MS) serves as a robust approach for oligosaccharide analysis, it has difficulty distinguishing the smallest differences between isomers. The integration of infrared (IR) spectroscopy within a mass spectrometer as an additional analytical dimension can effectively address this limitation by providing a molecular fingerprint that is unique to each isomer. However, the direct interfacing of LC-MS with IR spectroscopy presents a technical challenge arising from the mismatch in the operational time scale of each method. In previous studies, this temporal incompatibility was mitigated by employing strategies designed to slow down or broaden the LC elution peaks of interest, but this workaround is applicable only for a few species at a time, necessitating multiple LC runs for comprehensive analysis. In the current work, we directly couple LC with cryogenic IR spectroscopy by acquiring a spectrum in as little as 10 s. This allows us to generate an orthogonal data dimension for molecular identification in the same amount of time that it normally takes for LC analysis. We successfully demonstrate this approach on a commercially available human milk oligosaccharide product, acquiring spectral information on the eluting peaks in real time and using it to identify both the specified constituents and nonspecified product impurities.

Using Hadamard Transform Multiplexed IR Spectroscopy Together with a Segmented Ion Trap for the Identification of Mobility-Selected Isomers.

The high isomeric complexity of glycans makes them particularly difficult to analyze. While ultra-high-resolution ion mobility spectrometry (IMS) can offer rapid baseline separation of many glycan isomers, their unambiguous identification remains a challenging task. One approach to solving this problem is to identify mobility-separated isomers by measuring their highly resolved cryogenic vibrational spectra. To be able to apply this approach to complex mixtures at high throughput, we have recently developed a Hadamard transform multiplexed spectroscopic technique that allows measuring vibrational spectra of all species separated in both IMS and mass spectrometry dimensions in a single laser scan. In the current work, we further develop the multiplexing technique using ion traps incorporated directly into the IMS device based on structures for lossless ion manipulations (SLIM). We also show that multiplexed spectroscopy using perfect sequence matrices can outperform standard multiplexing using Simplex matrices. Lastly, we show that we can increase the measurement speed and throughput further by running multiple multiplexing schemes using several SLIM ion traps in combination with simultaneous spectroscopic measurements in the segmented cryogenic ion trap.

New Approach for the Identification of Isobaric and Isomeric Metabolites.

The structural elucidation of metabolite molecules is important in many branches of the life sciences. However, the isomeric and isobaric complexity of metabolites makes their identification extremely challenging, and analytical standards are often required to confirm the presence of a particular compound in a sample. We present here an approach to overcome these challenges using high-resolution ion mobility spectrometry in combination with cryogenic vibrational spectroscopy for the rapid separation and identification of metabolite isomers and isobars. Ion mobility can separate isomeric metabolites in tens of milliseconds, and cryogenic IR spectroscopy provides highly structured IR fingerprints for unambiguous molecular identification. Moreover, our approach allows one to identify metabolite isomers automatically by comparing their IR fingerprints with those previously recorded in a database, obviating the need for a recurrent introduction of analytical standards. We demonstrate the principle of this approach by constructing a database composed of IR fingerprints of eight isomeric/isobaric metabolites and use it for the identification of these isomers present in mixtures. Moreover, we show how our fast IR fingerprinting technology allows to probe the IR fingerprints of molecules within just a few seconds as they elute from an LC column. This approach has the potential to greatly improve metabolomics workflows in terms of accuracy, speed, and cost.

Identification of human milk oligosaccharide positional isomers by combining IMS-CID-IMS and cryogenic IR spectroscopy.

High-resolution ion mobility spectrometry (IMS) coupled with cryogenic infrared spectroscopy has proven to be a powerful technique for the identification of oligosaccharides. However, the need for an extensive database, combined with the scarcity of pure standards, remains a significant barrier to the broad application of this approach. To solve this issue, we demonstrate a method in which ion fragments produced by collision-induced dissociation (CID) are separated using IMS and identified using the vibrational fingerprints of only a few standards. Identification of the fragments allows us to determine the structure of the precursor molecule, the vibrational fingerprint of which is then added to our database. We then show how we can use this approach to identify the structure of mobility separated isomers found in pooled human milk.

Multistage Ion Mobility Spectrometry Combined with Infrared Spectroscopy for Glycan Analysis.

The structural complexity of glycans makes their characterization challenging, not only because of the presence of various isomeric forms of the precursor molecule but also because the fragments can themselves be isomeric. We have recently developed an IMS-CID-IMS approach using structures for lossless ion manipulations (SLIM) combined with cryogenic infrared (IR) spectroscopy for glycan analysis. It allows mobility separation and collision-induced dissociation of a precursor glycan followed by mobility separation and IR spectroscopy of the fragments. While this approach holds great promise for glycan analysis, we often encounter fragments for which we have no standards to identify their spectroscopic fingerprint. In this work, we perform proof-of-principle experiments employing a multistage SLIM-based IMS-CID technique to generate second-generation fragments, followed by their mobility separation and spectroscopic interrogation. This approach provides detailed structural information about the first-generation fragments, including their anomeric form, which in turn can be used to identify the precursor glycan.

Identification of Mobility-Resolved N-Glycan Isomers.

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 α and ß 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.

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

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.

High-Throughput Multiplexed Infrared Spectroscopy of Ion Mobility-Separated Species Using Hadamard Transform.

Coupling vibrational ion spectroscopy with high-resolution ion mobility separation offers a promising approach for detailed analysis of biomolecules in the gas phase. Improvements in the ion mobility technology have made it possible to separate isomers with minor structural differences, and their interrogation with a tunable infrared laser provides vibrational fingerprints for unambiguous database-enabled identification. Nevertheless, wide analytical application of this technique requires high-throughput approaches for acquisition of vibrational spectra of all species present in complex mixtures. In this work, we present a novel multiplexed approach and demonstrate its utility for cryogenic ion spectroscopy of peptides and glycans in mixtures. Since the method is based on Hadamard transform multiplexing, it yields infrared spectra with an increased signal-to-noise ratio compared to a conventional signal averaging approach.

Identification of N-glycan positional isomers by combining IMS and vibrational fingerprinting of structurally determinant CID fragments.

While glycans are present on the surface of cells in all living organisms and play key roles in most biological processes, their isomeric complexity makes their structural characterization challenging. Of particular importance are positional isomers, for which analytical standards are difficult to obtain. We combine ultrahigh-resolution ion-mobility spectrometry with collision-induced dissociation and cryogenic infrared spectroscopy to determine the structure of N-glycan positional isomers. This approach is based on first separating the parent molecules by SLIM-based IMS, producing diagnostic fragments specific to each positional isomer, separating the fragments by IMS, and identifying them by comparing their IR fingerprints to a previously recorded spectral database. We demonstrate this strategy using a bottom-up scheme to identify the positional isomers of the N-linked glycan G0-N, in which a terminal N-acetylglucosamine (GlcNAc) is attached to either the α-3 or α-6 branch of the common N-glycan pentasaccharide core. We then use IR fingerprints of these newly identified isomers to identify the positional isomers of G1 and G1F, which are biantennary complex-type N-glycans with a terminal galactose attached to either the α-3 or α-6 branch, and in the case of G1F a fucose attached to the reducing-end GlcNAc. Starting with just a few analytical standards, this fragment-based spectroscopy method allows us to develop a database which we can use to identify positional isomers. The generalization of this approach would greatly facilitate glycan analysis.

Toward High-Throughput Cryogenic IR Fingerprinting of Mobility-Separated Glycan Isomers.

Infrared (IR) spectroscopy is a powerful tool used to infer detailed structural information on molecules, often in conjunction with quantum-chemical calculations. When applied to cryogenically cooled ions, IR spectra provide unique fingerprints that can be used for biomolecular identification. This is particularly important in the analysis of isomeric biopolymers, which are difficult to distinguish using mass spectrometry. However, IR spectroscopy typically requires laser systems that need substantial user attention and measurement times of tens of minutes, which limits its analytical utility. We report here the development of a new high-throughput instrument that combines ultrahigh-resolution ion-mobility spectrometry with cryogenic IR spectroscopy and mass spectrometry, and we apply it to the analysis of isomeric glycans. The ion mobility step, which is based on structures for lossless ion manipulations (SLIM), separates glycan isomers, and an IR fingerprint spectrum identifies them. An innovative cryogenic ion trap allows multiplexing the acquisition of analyte IR fingerprints following mobility separation, and using a turn-key IR laser, we can obtain spectra and identify isomeric species in less than a minute. This work demonstrates the potential of IR fingerprinting methods to impact the analysis of isomeric biomolecules and more specifically glycans.

Identifying Mixtures of Isomeric Human Milk Oligosaccharides by the Decomposition of IR Spectral Fingerprints.

The analysis of glycans presents a significant challenge that arises from their isomeric heterogeneity. While high-resolution ion mobility spectrometry (IMS) has shown the ability to resolve subtly different glycan isomers, their unambiguous assignment remains difficult. Here, we demonstrate an infrared (IR) spectroscopic approach for identifying isomers in a glycan mixture. To display the feasibility of this approach, we have constructed a small database of cryogenic spectra of five lacto-N-fucopentaose (LNFP) and six disaccharide isomers and demonstrated that in the cases where they cannot be separated by IMS, we can use a cryogenic IR spectrum to identify the isomeric components of a mixture.

A new approach for identifying positional isomers of glycans cleaved from monoclonal antibodies.

Glycosylation patterns in monoclonal antibodies (mAbs) can vary significantly between different host cell types, and these differences may affect mAbs safety, efficacy, and immunogenicity. Recent studies have demonstrated that glycan isomers with the terminal galactose position on either the Man α1-3 arm or the Man α1-6 arm have an impact on the effector functions and dynamic structure of mAbs. The development of a robust method to distinguish positional isomers of glycans is thus critical to guarantee mAb quality. In this work, we apply high-resolution ion mobility combined with cryogenic infrared spectroscopy to distinguish isomeric glycans with different terminal galactose positions, using G1F as an example. Selective enzymatic synthesis of the G1(α1-6)F isomer allows us to assign the peaks in the arrival-time distributions and the infrared spectra to their respective isomeric forms. Moreover, we demonstrate the impact of the host cell line (CHO and HEK-293) on the IgG G1F gycan profile at the isomer level. This work illustrates the potential of our approach for glycan analysis of mAbs.

Unravelling the structures of sodiated ß-cyclodextrin and its fragments.

We present cryogenic infrared spectra of sodiated ß-cyclodextrin [ß-CD + Na]+, a common cyclic oligosaccharide, and its main dissociation products upon collision-induced dissociation (CID). We characterize the parent ions using high-resolution ion mobility spectrometry and cryogenic infrared action spectroscopy, while the fragments are characterized by their mass and cryogenic infrared spectra. We observe sodium-cationized fragments that differ in mass by 162 u, corresponding to Bn/Zm ions. For the m/z 347 product ion, electronic structure calculations are consistent with formation of the lowest energy 2-ketone B2 ion structure. For the m/z 509 product ion, both the calculated 2-ketone B3 and the Z3 structures show similarities with the experimental spectrum. The theoretical structure most consistent with the spectrum of the m/z 671 ions is a slightly higher energy 2-ketone B4 structure. Overall, the data suggest a consistent formation mechanism for all the observed fragments.

Structural Insights from Tandem Mass Spectrometry, Ion Mobility-Mass Spectrometry, and Infrared/Ultraviolet Spectroscopy on Sphingonodin I: Lasso vs Branched-Cyclic Topoisomers.

Lasso peptides form a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked topology, where the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Sphingonodin I is a lasso peptide that has not yet been structurally characterized using the traditional structural biology tools (e.g., NMR and X-ray crystallography), and its biological function has not yet been elucidated. In the present work, we describe structural signatures characteristic of the class II lasso peptide sphingonodin I and its branched-cyclic analogue using a combination of gas-phase ion tools (e.g., tandem mass spectrometry, MS/MS, trapped ion mobility spectrometry, TIMS, and infrared, IR, and ultraviolet, UV, spectroscopies). Tandem MS/MS CID experiments on sphingonodin I yielded mechanically interlocked species with associated bi and yj fragments demonstrating the presence of a lasso topology, while tandem MS/MS ECD experiments on sphingonodin I showed a significant increase in hydrogen migration in the loop region when compared to the branched-cyclic analogue. The high-mobility resolving power of TIMS permitted the separation of both topoisomers, where sphingonodin I adopted a more compact structure than its branched-cyclic analogue. Cryogenic and room-temperature IR spectroscopy experiments evidenced a different hydrogen bond network between the two topologies, while cryogenic UV spectroscopy experiments clearly demonstrated a distinct phenylalanine environment for the lasso peptide.

Cryogenic Infrared Action Spectroscopy Fingerprints the Hydrogen Bonding Network in Gas-Phase Coumarin Cations.

We report cryogenic vibrational spectra of gas-phase cations of two common hydroxycoumarins, scopoletin and esculetin, as well as their glycosidic derivatives, scopolin and esculin. The study allows direct observation of the intramolecular interactions between the hydroxyl groups of these molecules. We use cryogenic messenger-tagging IR action spectroscopy to detect vibrational bands in the 3100-3800 cm-1 spectral range and discuss the corresponding structural characteristics and hydrogen bonding networks that they imply. The experimental data are supported by a thorough computational evaluation, including investigation of the conformational space. Through comparison of the calculated conformers with the experimental results, we identify the main types of OH oscillators and infer how protonation and sodiation affect the structural arrangement of these molecules. The results presented here provide direct evidence of how slight structural differences sensitively affect the hydrogen bonding network in coumarin derivatives.

Analyzing glycans cleaved from a biotherapeutic protein using ultrahigh-resolution ion mobility spectrometry together with cryogenic ion spectroscopy.

Glycans covalently attached to protein biotherapeutics have a significant impact on their biological activity, clearance, and safety. As a result, glycosylation is categorized as a critical quality attribute that needs an adequate analytical approach to guarantee product quality. However, the isomeric complexity and branched structure of glycans makes their analysis a significant challenge. In this work, we propose a multidimensional approach for monitoring released glycans that combines ultrahigh-resolution ion mobility spectrometry (IMS) and cryogenic vibrational spectroscopy, and we demonstrate this technique by characterizing four N-glycans cleaved from the therapeutic fusion protein etanercept that range in abundance from 1% to 22% of the total N-glycan content. The recorded vibrational spectra exhibit well-resolved transitions that can be used as a fingerprint to identify a particular glycan. This work represents an important advance in the analysis of N-linked glycans cleaved from biopharmaceutical proteins that could eventually be used as tool for monitoring biopharmaceutical glycoforms.

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

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.

How General Is Anomeric Retention during Collision-Induced Dissociation of Glycans?

Despite the essential role that glycans play in many biological processes, their isomeric complexity makes their structural determination particularly challenging. Tandem mass spectrometry has played a central role in glycan analysis, and recent work has shown that fragments generated by collision-induced dissociation (CID) of disaccharides can retain the anomeric configuration of the glycosidic bond. If this result proves to be general, it would provide a powerful new tool for glycan sequencing. In this work, we use messenger-tagging infrared (IR) spectroscopy to investigate the generality of anomer retention in CID by exploring different fragmentation channels in glycans of increasing complexity. Our results demonstrate that anomericity seems to be retained irrespective of fragment size and branching.

Combining Cryogenic Infrared Spectroscopy with Selective Enzymatic Cleavage for Determining Glycan Primary Structure.

Given the biological relevance and intrinsic structural complexity of glycans, increasing efforts are being directed toward developing a general glycan database that includes information from different analytical methods. As recently demonstrated, cryogenic infrared (IR) spectroscopy is a promising technique for glycan analysis, as it provides unique vibrational fingerprints of specific glycan isomer ions. One of the main goals of a glycan database is the identification and detailed characterization of unknown species. In this work, we combine enzymatic digestion with cryogenic IR-spectroscopy and demonstrate how it can be used for glycan identification. We measured the IR-spectra of a series of cationic glycan standards of increasing complexity and compared them with spectra of the same species after enzymatic cleavage of larger glycans. We show that the cryogenic IR spectra of the cleaved glycans are highly structured and virtually identical to those of standards after both single and multiple cleavages. Our results suggest that the combination of these methods represents a potentially powerful and specific approach for the characterization of unknown glycans.

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 makes 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 labeling, we show on a range of disaccharides how the two α and ß 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.