Anomeric Retention Of Carbohydrates In Multistage Cyclic Ion Mobility (Imsn): De Novo Structural Elucidation Of Enzymatically Produced Mannosides

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 IMSn 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 alpha 1,2- and beta 1,2-mannooligosaccharides, which provided us with reference drift times for fragments of known conformation. Then, we performed IMSn analyses of enzymatically produced mannosides and, by comparison with the references, we succeeded in determining the intrachain anomerism of a alpha 1,2-mannotriose and a mix-linked beta/alpha 1,2-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 IMSn 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 alpha- and beta-glycosidic linkages.