ABSTRACT
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.
Subject(s)
Oligosaccharides , Humans , Chromatography, Liquid , Mass Spectrometry/methods , Isomerism , Spectrophotometry, Infrared , Oligosaccharides/chemistryABSTRACT
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.
ABSTRACT
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.
Subject(s)
Ion Mobility Spectrometry , Milk, Human , Humans , Ion Mobility Spectrometry/methods , Milk, Human/chemistry , Oligosaccharides/analysis , Isomerism , Spectrophotometry, InfraredABSTRACT
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.
Subject(s)
Ion Mobility Spectrometry , Metabolomics , Metabolomics/methods , Isomerism , Databases, FactualABSTRACT
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.
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 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.
Subject(s)
Ion Mobility Spectrometry , Polysaccharides , Humans , Ion Mobility Spectrometry/methods , Isomerism , Polysaccharides/chemistry , Spectrum AnalysisABSTRACT
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.
Subject(s)
Ion Mobility Spectrometry , Polysaccharides , Disaccharides , Ion Mobility Spectrometry/methods , Isomerism , Polysaccharides/analysis , Spectrophotometry, InfraredABSTRACT
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.
Subject(s)
Polysaccharides , Isomerism , Signal-To-Noise Ratio , Spectrophotometry, InfraredABSTRACT
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.
Subject(s)
Ion Mobility Spectrometry , Polysaccharides , Isomerism , Oligosaccharides , Spectrophotometry, InfraredABSTRACT
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.
ABSTRACT
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.
Subject(s)
Milk, Human , Oligosaccharides , Humans , Ion Mobility Spectrometry , Isomerism , PolysaccharidesABSTRACT
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.
Subject(s)
Antibodies, Monoclonal , Polysaccharides , Glycosylation , HEK293 Cells , Humans , IsomerismABSTRACT
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.
Subject(s)
Ion Mobility Spectrometry/methods , Polysaccharides/analysis , Humans , Ion Mobility Spectrometry/standards , Isomerism , Milk, Human/metabolism , Oligosaccharides/analysis , Oligosaccharides/standards , Polysaccharides/standards , Reference Standards , Spectrophotometry, Infrared/standards , Tandem Mass SpectrometryABSTRACT
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.
Subject(s)
Polysaccharides/chemistry , Carbohydrate Conformation , Disaccharides/chemistry , Spectrophotometry, Infrared , Tandem Mass SpectrometryABSTRACT
Mass spectrometry enables the in-depth structural elucidation of membrane protein complexes, which is of great interest in structural biology and drug discovery. Recent breakthroughs in this field revealed the need for design rules that allow fine-tuning the properties of detergents in solution and gas phase. Desirable features include protein charge reduction, because it helps to preserve native features of protein complexes during transfer from solution into the vacuum of a mass spectrometer. Addressing this challenge, we here present the first systematic gas-phase study of azobenzene detergents. The utility of gas-phase techniques for monitoring light-driven changes of isomer ratios and molecular properties are investigated in detail. This leads to the first azobenzene detergent that enables the native mass spectrometry analysis of membrane proteins and whose charge-reducing properties can be tuned by irradiation with light. More broadly, the presented work outlines new avenues for the high-throughput characterization of supramolecular systems and opens a new design strategy for detergents in membrane protein research.
ABSTRACT
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.
ABSTRACT
Double-resonance spectroscopic schemes in combination with cryogenic ion traps are the go-to techniques when isomer-specific high-resolution spectra are required for analysis of molecular ions. Their limitation lies in the requirement for well-resolved, isomer-specific absorption bands as well as in the potentially time-consuming steps to identify each isomer. We present an alternative approach where isomeric species are readily separated using ion mobility spectrometry (IMS) and selected prior to cryogenic spectroscopic analysis. To date, most IMS approaches suffer from relatively low resolution, however, recent technological developments in the field of travelling-wave ion mobility using structures for lossless ion manipulation (SLIM) permit the use of extremely long drift paths, which greatly enhances the resolution. We demonstrate the power of combining this type of ultra-high resolution IMS with cryogenic vibrational spectroscopy by comparing mobility-resolved IR spectra of a disaccharide to those acquired using IR-IR double resonance. This new approach is especially promising for the investigation of larger molecules where spectral congestion interferes with double resonance techniques.
Subject(s)
Disaccharides/analysis , Ion Mobility Spectrometry , Infrared Rays , Ions/analysis , Spectrophotometry, InfraredABSTRACT
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.
Subject(s)
Polysaccharides/analysis , Carbohydrate Conformation , Ion Mobility Spectrometry , Spectrophotometry, InfraredABSTRACT
The amino acid serine has long been known to form a protonated "magic-number" cluster containing eight monomer units that shows an unusually high abundance in mass spectra and has a remarkable homochiral preference. Despite many experimental and theoretical studies, there is no consensus on a Ser8H+ structure that is in agreement with all experimental observations. Here, we present the structure of Ser8H+ determined by a combination of infrared spectroscopy and ab initio molecular dynamics simulations. The three-dimensional structure that we determine is â¼25 kcal mol-1 more stable than the previous most stable published structure and explains both the homochiral preference and the experimentally observed facile replacement of two serine units.
ABSTRACT
The amino acid serine is known to form a very stable octamer that has properties that set it apart from serine complexes of different sizes or from complexes composed of other amino acids. For example, both singly protonated serine octamers and anionic octamers complexed with two halogen ions strongly prefer homochirality, even when assembled from racemic D,L mixtures. Consequently, the structures of these complexes are of great interest, but no acceptable candidates have so far been identified. Here, we investigate anionic serine octamers coordinated with two chloride ions using a novel technique coupling ion mobility spectrometry-mass spectrometry with infrared spectroscopy, in combination with theoretical calculations. The results allow the identification of a unique structure for (Ser8Cl2)2- that is highly symmetric, very stable and homochiral and whose calculated properties match those observed in experiments.