Anomeric Retention of Carbohydrates in Multistage Cyclic Ion Mobility (IMSⁿ): De Novo Structural Elucidation of Enzymatically Produced Mannosides

Abstract

Carbohydrates are complex structures that still challenge analysts today because of their different levels of isomerism, notably the anomerism of the glycosidic bond. It has been shown recently that anomerism is preserved upon gas-phase fragmentation and that high-resolution ion mobility (IMS) can distinguish anomers. However, these concepts have yet to be applied to complex biological products. We have used high-resolution IMS on a cyclic device to characterize the reaction products of Uhgb_MS, a novel mannoside synthase of the GH130 family. We designed a so-called IMS n sequence consisting of (i) separating and isolating specific IMS peaks, (ii) ejecting ions to a pre-array store cell depending on their arrival time, (iii) inducing collisional activation upon reinjection, and (iv) performing multistage IMS analysis of the fragments. First, we applied IMS2 sequences to purely linked α1,2- and β1,2-mannooligosaccharides, which provided us with reference drift times for fragments of known conformation. Then, we performed IMS n analyses of enzymatically produced mannosides and, by comparison with the references, we succeeded in determining the intrachain anomerism of a α1,2-mannotriose and a mix-linked β/α1,2-mannotetraosea first for a crude biological medium. Our results show that the anomerism of glycosides is maintained through multiple stages of collisional fragmentation, and that standalone high-resolution IMS and IMS n can be used to characterize the intrachain anomerism in tri- and tetrasaccharides in a biological medium. This is also the first evidence that a single carbohydrate-active enzyme can synthesize both α- and β-glycosidic linkages.

Molecular Networking of High-Resolution Tandem Ion Mobility Spectra: A Structurally Relevant Way of Organizing Data in Glycomics?

“…The similarity arises from the isobaric fragments between cellopentaose and maltopentaose, which differ only in their intrachain anomerism, and because MS is inherently incapable of resolving isobaric fragments. On the other hand, the IMS/IMS spectra reveal clear differences between the two glycans (Figure 5B), thanks to the ability of ion mobility to distinguish between isomeric fragments, including fragments differing by their intrachain anomerism 21 (NB. the ATD of the pentasaccharides before any processing are available in Supplementary Figure 5).…”

“…IMS-MS raised interest from the glycomics community as it was notably able to discriminate intrachain anomers, or match monomeric fragments with the corresponding monosaccharide . IMS has recently gained more momentum in carbohydrate analysis for two main reasons: (i) the demonstration that major structural features of carbohydrates are retained during MS/MS, such as the linkage or the anomericity of the glycosidic bond, − and (ii) the development of ultrahigh-resolution IMS technologies, among which the TWIMS-based Cyclic IMS and the multipass Structures for Lossless Ion Manipulation (SLIM SUPER) . Both possess the ability to increase the IMS resolving power almost infinitely by allowing multiple passes in the ion mobility cell (therefore increasing the separation length), as well as to perform tandem IMS/IMS experiments, pending some modifications for the SLIM .…”

Mass Spectrometry-Based Techniques to Elucidate the Sugar Code

“…Glycosidic cleavage yields information about glycan sequence, whereas cross-ring cleavage is important to deduce information about linkage and branching. Glycosidic B-fragments are also particularly interesting for chemical synthesis of glycans as they are believed to occur as intermediates of S N 1 reactions in solution, and their reactivity can be predicted by gas-phase studies. , Glycosidic B- and C-fragments showed in some cases a memory of the stereoinformation on the glycosidic linkage in unprotected glycosides − but not in protected glycosides …”

Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases

“…Overall, great improvements in these methodologies allow a faster separation and the handling of more samples (with data acquisition per sample within minutes), with reduced compound consumption. These methods, previously used for precise product characterization in final screening steps, now offer real possibilities for screening large libraries, while generating good quality data [ 113 ].…”