Glycan Fingerprinting via Cold‐Ion Infrared Spectroscopy

Abstract

The diversity of stereochemical isomers present in glycans and glycoconjugates poses af ormidable challenge for comprehensive structural analysis.T ypically,s ophisticated mass spectrometry (MS)-based techniques are used in combination with chromatography or ion-mobility separation. However,c oexisting structurally similar isomers often render an unambiguous identification impossible.O ther powerful techniques such as gas-phase infrared (IR) spectroscopyh ave been limited to smaller glycans,since conformational flexibility and thermal activation during the measurement result in poor spectral resolution. This limitation can be overcome by using cold-ion spectroscopy. The vibrational fingerprints of cold oligosaccharide ions exhibit aw ealth of well-resolved absorption features that are diagnostic for minute structural variations.T he unprecedented resolution of cold-ion spectroscopy coupled with tandem MS may render this the key technology to unravel complex glycomes.Carbohydrates are ubiquitous in nature,and are historically associated with their prominent roles as structural scaffolds and energy sources within the cell. However,s horter chains, often referred to as oligosaccharides or glycans,a re also essential in numerous biological signaling processes.[1] The field of glycomics,w hich aims to comprehensively elucidate the structure and functions of glycans,i sc urrently profiting from technical breakthroughs in automated chemical synthesis and analysis but remains largely underexplored when compared to genomics and proteomics.T he inherent structural diversity of oligosaccharides creates major challenges for progress in glycobiology.Whereas DNAand proteins are exclusively assembled in alinear and template-driven fashion, glycans are non-template derived, branched, and exhibit complex stereo-and regiochemistry.Exploring the structure, shape,and resulting functions of oligosaccharides is difficult.Detailed structural analyses of glycans typically involve liquid chromatography (LC) and/or mass spectrometry (MS)-based techniques that require only small amounts of sample. [2] In addition to the molecular composition, sophisticated MS techniques such as sequential mass spectrometry (MS n )o r chemical derivatization can yield information about the sequence and connectivity of the constituting monosaccharide building blocks.[3] Ion-mobility mass spectrometry (IM-MS) has proven ap owerful tool to rapidly separate and identify the connectivity and configurational isomers of carbohydrates. [4] Combining MS and infrared (IR) spectroscopy provides av ery sensitive alternative by interrogating the vibrational modes of isolated molecules in the controlled environment of the gas phase.E xploring the characteristic absorption bands of molecules is ar outine means to deduce structural information concerning functional groups,hydrogen-bonding patterns,a nd preferred molecular conformations.A ss uch, gas-phase IR spectroscopy is aw ell-established tool for the structural analysis of peptides,proteins,and small mol...

Cryogenic Vibrational Spectroscopy Provides Unique Fingerprints for Glycan Identification

“…Compagnion and co-workers [26, 27] have recently implemented a similar approach. In a very recent paper, Mucha et al [28] used helium droplets to obtain spectroscopic fingerprints of oligosaccharides, showing that sufficiently resolved spectra could indeed distinguish the various types of isomerism, albeit with an admittedly complex experimental set-up. In the present work, we use messenger-tagging infrared spectroscopy in a cryogenic ion trap in combination with IMS-MS to identify and characterize glycans.…”

Unraveling the isomeric heterogeneity of glycans: ion mobility separations in structures for lossless ion manipulations

“…Even with the growing suite of MS n methods, the deconvolution and identification of co-eluting species remains a challenge since isomers exhibit very similar fragmentation pathways 4 , 5 . Additionally, while spectroscopic approaches have shown promise for glycan fingerprinting, these methods remain of limited utility for accurately characterizing isomeric mixtures 6 .…”

“…Ion mobility-mass spectrometry (IM-MS)-based approaches are quickly becoming an attractive, and rapid, alternative to 1-D chromatographic separations 2 , and even have shown utility as a front-end separation prior to spectroscopic methods 6 . In IM-MS ions are separated in the gas-phase based on their mobilities (size/shape) as well as mass-to-charge ( m/z ).…”

“…HMOs have received widespread attention for their roles in potentially protecting the gut microbiome, promoting growth and immunological development in infants, and their anti-adhesive and probiotic properties 6 , 11 . Lacto-N-tetraose and lacto-N-neotetraose (LNT and LNnT, respectively), which only differ in their linkage position (1–3 versus 1–4) between the D-galactose and N-acetyl D-glucosamine residues, were able to be baseline resolved after 31.5 meters of separation as their protonated adducts (Figure 4).…”

Interplay of H‐Bonds with Aromatics in Isolated Complexes Identifies Isomeric Carbohydrates

“…[2] Based on this,cancer cells can be identified, for instance,b yd etecting their specific membrane glycoproteins, [3] which may differ from those of the healthy cells by the isoform of the glycan only,w hile viruses and bacteria adhere to appropriate cells for invasion by selective binding to specific membrane glycans, [4] distinguishing them from many other structurally similar (for example, isomeric) saccharides.T hese and many other examples illustrate the importance of comprehensive structural studies of carbohydrates,i ncluding the identification of their isoforms. Vibrational spectroscopy in the gas phase (for example,infrared multiple photon dissociation [10] or IR-induced fragmentation of tagged saccharides [11] )w as able to distinguish, though without quantification, many stereoisomers of small oligosaccharides. ), although not all of them are essential in nature.I nc ontrast to amino acids and nucleotides,t hese isomeric units can interconnect through different OH groups, forming regioisomers,but also at multiple points,assembling into linear and branched structural isomers.N atural modifications (for example, N-acetylation) of different units further multiply the number of possible isoforms.Human milk alone, for instance,c ontains at least 200 different oligosaccharides, many of which are essential not only for supplying energy to newborns,but also for their antibacterial defense.…”