Thomas R. Rizzo

While approximately 200 human milk oligosaccharide structures (HMOs) have been completely characterized, the precise isomeric structures of even more HMOs remain ambiguous. To address this analytical challenge, we employ tandem mass spectrometry (i.e., MS/MS) coupled with cryogenic infrared ion spectroscopy (CIRIS) to characterize and identify novel fucosylated HMOs isolated from pooled human milk. In this approach, the precise structure of isomeric precursor molecules purified by liquid chromatography (LC) is identified by IR fingerprinting their fragments. Using this technique, we have identified four novel isomers of trifucosylated oligosaccharides (i.e., trifucosyl-lacto-N-heptaoses and trifucosyl-lacto-N-neoheptaoses), some of which could not have been produced by previously known biosynthetic pathways for HMO synthesis in the mammary gland. Graphical Abstract

The structures of alkali metal ion-crown ether (CE) 1:2 heterocomplexes of benzo-18-crown-6 (B18C6), dibenzo-18-crown-6 (DB18C6), and 18-crown-6 (18C6) were studied under cold (∼10 K) conditions in the gas phase. The structures and intermolecular interactions of M⁺(DB18C6)(18C6) and M⁺(B18C6)(18C6) (M = Rb and Cs) were determined based on their ultraviolet (UV) and infrared (IR) spectra, with the assistance of quantum chemical calculations, and compared with those of K⁺ complexes reported previously. The electronic transitions of the M⁺(DB18C6)(18C6) and M⁺(B18C6)(18C6) complexes occurred in the 35,500-36,000 cm^-1 range, in the vicinity of those of the DB18C6 and B18C6 monomers, and at lower energies than the M⁺(DB18C6) and M⁺(B18C6) complexes. This indicates that the interaction between the M⁺ ion and DB18C6 or B18C6 is substantially weaker in the M⁺(DB18C6)(18C6) and M⁺(B18C6)(18C6) heterocomplexes than in M⁺(DB18C6) and M⁺(B18C6). In the structures determined for M⁺(DB18C6)(18C6) and M⁺(B18C6)(18C6), the M⁺ ion was mainly encapsulated by the 18C6 moiety, and the DB18C6 and B18C6 molecules were weakly bound to the M⁺(18C6) complexes. Based on the structural determination, high-energy conformers were trapped and detected owing to the incomplete structural relaxation in the ion trap of the M⁺(DB18C6)(18C6) and M⁺(B18C6)(18C6) complexes. A previous study reported that UV excitation of K⁺(DB18C6)(18C6) and K⁺(B18C6)(18C6) complexes produces photofragment K⁺(18C6) and K⁺(B18C6) ions, respectively. Upon UV photodissociation (UVPD) of the M⁺(DB18C6)(18C6) and M⁺(B18C6)(18C6) (M = Rb and Cs) complexes, M⁺(18C6) ions were formed as the main fragments, which is reasonable based on the predominant encapsulation by 18C6 in these complexes. Hence, the UVPD patterns of the Rb⁺ and Cs⁺ complexes highlight the peculiarity of the K⁺(B18C6)(18C6) complex, in which K⁺ is effectively transferred from 18C6 to B18C6 upon UV excitation.

We obtained gas-phase UV and IR spectra of 1:2 potassium ion-crown ether (CE) complexes (benzo-15-crown-5 (B15C5), benzo-18-crown-6 (B18C6), dibenzo-18-crown-6 (DB18C6), and 18-crown-6 (18C6)) at approximately 10 K and determined the structure and intermolecular interactions between K⁺ and the two CEs, supported by quantum chemical calculations. The vibronic features of K⁺(B15C5)₂ revealed two conformers in the UV photodissociation spectrum, and the K⁺ ion was held equally by both B15C5 moieties. For K⁺(B18C6)₂ and K⁺(DB18C6)₂, the K⁺ ions were not equally shared between the two CEs. For K⁺(B18C6)(18C6) and K⁺(DB18C6)(18C6), the K⁺ ion mainly interacted with 18C6, and the interaction between K⁺ and B18C6 or DB18C6 was very weak. In contrast, K⁺(B18C6)(18C6) produced photofragment K⁺(B18C6) ion exclusively, whereas K⁺(DB18C6)(18C6) produced K⁺(18C6). The photodissociation of K⁺(B18C6)(18C6) indicates that K⁺ transfer from 18C6 to B18C6 occurs upon UV excitation, probably in the electronically excited states.

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.

The high isomericcomplexity of glycans makes them particularlydifficult 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 approachto solving this problem is to identify mobility-separated isomersby 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 spectroscopictechnique that allows measuring vibrational spectra of all speciesseparated in both IMS and mass spectrometry dimensions in a singlelaser scan. In the current work, we further develop the multiplexingtechnique using ion traps incorporated directly into the IMS devicebased on structures for lossless ion manipulations (SLIM). We alsoshow that multiplexed spectroscopy using perfect sequence matricescan outperform standard multiplexing using Simplex matrices. Lastly,we show that we can increase the measurement speed and throughputfurther by running multiple multiplexing schemes using several SLIMion traps in combination with simultaneous spectroscopic measurementsin the segmented cryogenic ion trap.

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.

Cryogenic infrared spectra of CID fragments separated by IMS are used to identify human milk oligiosaccharide 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 β 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.

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.