Bottom-Up Elucidation of Glycosidic Bond Stereochemistry

Abstract

The lack of robust, high-throughput, and sensitive analytical strategies that can conclusively map the structure of glycans has significantly hampered progress in fundamental and applied aspects of glycoscience. Resolution of the anomeric α/β glycan linkage within oligosaccharides remains a particular challenge. Here, we show that "memory" of anomeric configuration is retained following gas-phase glycosidic bond fragmentation during tandem mass spectrometry (MS). These findings allow for integration of MS with ion mobility spectrometry (IM-MS) and lead to a strategy to distinguish α- and β-linkages within natural underivatized carbohydrates. We have applied this fragment-based hyphenated MS technology to oligosaccharide standards and to de novo sequencing of purified plant metabolite glycoconjugates, showing that the anomeric signature is also observable in fragments derived from larger glycans. The discovery of the unexpected anomeric memory effect is further supported by IR-MS action spectroscopy and ab initio calculations. Quantum mechanical calculations provide candidate geometries for the distinct anomeric fragment ions, in turn shedding light on gas-phase dissociation mechanisms of glycosidic linkages.

A Careful Consideration of the Influence of Structure, Partial charges and Basis Sets on Collision Cross Sections of Monosaccharides when Comparing Values from DFT Calculated Conformers to those Obtained Experimentally

“…5 Carbohydrates were diluted to 5 pmol/µL with 1:1 methanol:water prior to analysis; to study lithiated glycans, 10 eq. of N-ethylaniline, colchicine and reserpine; all Sigma), whose CCS have been previously verified.…”

“…2 Both IM-MS techniques have been applied to a broad scope of species to elucidate structural information, including small drug-like molecules, 3,4 carbohydrates, 5,6 DNA and RNA, 7 proteins 8,9 and protein complexes. 9 It has also used to study protein unfolding, 8 refolding, 9,10 proton and electron transfer mechanisms 11,12 and solvent adduct effects on the protein structure.…”

“…13 A great advantage of IM-MS is the ability to elucidate detailed structural information about the studied system by separation of isomers, 3 protomers 14 and conformers. 5 The popularity of IM-MS has grown rapidly since the release of commercial instrumentation from Waters (Synapt and Vion instruments) 15 and Agilent (qToF 6560); historically, IM-MS was carried out on in-house designed instruments with home-built drift tubes commonly filled with helium.…”

“…For instance, Warnke et al 14 demonstrated that combining IM-MS and infrared multiple photon dissociation (IRMPD) spectroscopy reveals two distinct protomers of benzocaine, where depending on the protonation site (N or O), a unique N-H and O-H stretch frequencies appear. Similarly, Voronina et al 28 utilised IM-MS and cold-ion spectroscopy to study the bradykinin fragment (BK [1][2][3][4][5] 2+ ) ions elucidating cis-trans and trans-cis conformations for the more compact conformer and kinetically trapped trans-trans configuration for the larger conformer, highlighting the cis-trans isomerization of the peptide bonds during the desolvation process. In both cases, IM-MS data was supplemented with gas-phase IR experiments which in turn were supported by high-level calculations to assign potential in vacuo species.…”

“…http://dx.doi.org/10.1101/162305 doi: bioRxiv preprint first posted online Jul. 11, 2017; species generated by CID of the α-precursor (magenta) result in two species for both B-and Cfragment ions, whereas β-product ions (blue) consist of single species; Data shown in this figure has been adapted from Figure 2 from Gray et al 5 here, reporting Glcα1-4GlcNAc and Glcβ1-4GlcNAc only.…”