The tremendous isomeric diversity of carbohydrates enables awide range of their biological functions but makes the identification and study of these molecules difficult. We investigated the ability of intermolecular interactions to communicate structural specificity of carbohydrates to protonated aromatic molecules in non-covalent complexes,i solated and cooled in the gas phase.Our study revealed that small structural differences between carbohydrate isomers of any type,i ncluding enantiomers,a re accurately communicated by these interactions to aromatic molecules as detectable changes in their electronic excitation spectra. The specific response of the aromatics to the isomers of carbohydrates is fine-tuned by the interplay of the various involved non-covalent bonds.These findings enable the gas-phase identification and relative quantification of any isomers of oligosaccharides in their solution mixtures using the 2D UV-MS fingerprinting technique.Carbohydrates are by far the most abundant organic molecules present in living organisms. [1] Existing in myriads of structural forms,m any of which are isomeric,c arbohydrates may function as messenger and recognition molecules in signaling the type and the state of living cells in complex chains of biological processes. [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. [5] Them onomeric structural units of carbohydrates exist in av ariety of stereoisomeric forms,s uch as d/l-enantiomers, epimers,a nd a/b-anomers.F or instance,acyclic aldohexo-pyranose has 5stereogenic centers,which implies an existence of 2 5 = 32 stereoisomers (for example, a-d-glucose, b-lgalactose,e tc.), 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. [6] Theenormous isomeric diversity of carbohydrates makes their identification and study exceptionally challenging.Most common analytical methods of isomeric identifications of carbohydrates,such as chromatography [7] and, more recently, ion mobility, [8] demonstrate ah igh capability in separating small is...

Biological functionality of isomeric carbohydrates may differ drastically, making their identifications indispensible in many applications of life science. Due to the large number of isoforms, structural assignment of saccharides is challenging and often requires a use of different orthogonal analytical techniques. We demonstrate that isomeric carbohydrates of any isoforms can be distinguished and quantified using solely the library-based method of 2D UV-MS photofragmentation of cold ions. The two-dimensional "fingerprint" identities of UV transparent saccharides were revealed by photofragmentation of their non-covalent complexes with aromatic molecules. We assess the accuracy of the method by comparing the known relative concentrations of isomeric carbohydrates mixed in solution with the concentrations that were mathematically determined from the measured in the gas phase fingerprints of the complexes. For the tested sets with up to five isomers of di-to heptasaccharides, the root-mean-square deviation of 3-5% was typically achieved. This indicates the expected level of accuracy in analysis of unknown mixtures for isomeric carbohydrates of similar complexity.

The native-like structures of protonated glycine and peptide Gly3H+ were elucidated using cold ion IR spectroscopy of these biomolecules hydrated by a controlled number of water molecules. The complexes were generated directly from an aqueous solution using gentle electrospray ionization. Already with a single retained water molecule, GlyH+ exhibits the native-like structure characterized by a lack of intramolecular hydrogen bonds. We use our spectra to calibrate the available data for the same complexes, which are produced by cryogenic condensation of water onto the gas-phase glycine. In some conformers of these complexes, GlyH+ adopts the native-like structure, while in the others, it remains “kinetically” trapped in the intrinsic state. Upon condensation of 4–5 water molecules, the embedded amino acid fully adopts its native-like structure. Similarly, condensation of one water molecule onto the tripeptide is insufficient to fully eliminate its kinetically trapped intrinsic states.

The tremendous structural and isomeric diversity of lipids enables a wide range of their functions in nature but makes the identification of these biomolecules challenging. We distinguish and quantify isomeric lipids using cold ion UV fragmentation spectroscopy of their noncovalent complexes with aromatic amino acids and dipeptides. On the basis of structural simulations, specific isomer-sensitive aromatic "sensors" have been preselected for lipids of each studied class. Tyrosine appeared to be a good "sensor" to distinguish steroids and prostaglandins, which are rich in functional groups, while diphenylalanine is a better choice for sensing largely hydrophobic phospholipids. With this sensor, the relative concentrations of two isomeric glycerophospholipids mixed in solution have been determined with 3.3% accuracy, which should degrade only to 3.7% for a 14 s express measurement.

Non-covalent binding of proteins to glycans is amazingly selective to the isoforms of carbohydrates, including α/β anomers that co-exist in solution. We isolate in the gas phase and study at the atomic level the simplest model system: non-covalent complexes of monosaccharide α/β-GalNAc and protonated aromatic molecule tyramine. IR/UV cold ion spectroscopy and quantum chemistry calculations jointly solve the structures of the two complexes. Although the onsets of the measured UV absorptions of the complexes differ significantly, the networks of Hbonds in both complexes appear identical and do not include the anomeric hydroxyl. The detailed analysis reveals that, through inductive polarization, the α-to β-re-orientation of this group nevertheless reduces the length of one remote short intermolecular H-bond by 0.03 Å.Although small, this change substantially strengthens the bond, thus contributing to the anomeric selectivity of the binding.Protein-carbohydrate non-covalent interactions are ubiquitous in nature. The flexibility and the diversity of these weak interactions make proteins sensitive to finer structural details of the binding partners, greatly increasing their biological functionality. Despite the tremendous isomeric diversity of carbohydrates, the glycan binding proteins (GBPs), for instance, lectins, are capable of finding specific membrane carbohydrates to adhere viruses and bacteria to appropriate cells for invasion. 1 Hydrogen bonds between the hydroxyl groups of carbohydrates and the polar groups of amino acid residues play a crucial role in binding to lectins and other GBPs. Difference in the relative orientation of the hydroxyl groups in glycans has a pivotal impact on the ability of GBPs to distinguish different isoforms of carbohydrates, in particular their epimers (e.g., monosaccharaides Gal, Glu, Man, etc.) 2-3 Regardless of its epimeric isoform, a glycan in solution exists as two interconverting isomers: α-and β-anomers, which differ only by the orientation of the first OH group (1-OH) in the reducing-end monosaccharide. In contrast to epimers, which are usually recognized through binding to the 3-OH and 4-OH groups, the anomeric 1-OH often is not involved in the binding to GBPs. 2,4 Nevertheless, similar to epimers, the two anomers often exhibit very different affinities to, for instance, lectins and to some artificial receptors, which allows them a selective recognition of anomeric carbohydrates. [3][4] Despite many excellent studies in this field, the detailed mechanism of the anomeric recognition remains obscure, in particular, due to the large size of the interacting partners and the presence of solvent molecules. [5][6][7][8] Here we use cold-ion UV/IR spectroscopy and structural quantum chemistry computations to investigate at the atomic level the simplest anomeric system that models a local protein-glycan interaction: monosaccharaide anomers, α/β-GalNAc, non-covalently bound to protonated tyramine (TrmH +