Ionic-liquid-based catch and release mass spectroscopy tags for enzyme monitoring

Abstract

A novel, inexpensive and versatile ionic-liquid-based catch and release mass spectrometry tag (I-Tag) that facilitates substrate purification, fast, robust and sensitive enzymatic reaction monitoring and quantitative kinetic analysis has been developed. The applicability of the system has been demonstrated in an enzymatic assay with β-1,4-galactosyltransferase.

Imidazolium-labeled glycosides as probes to harness glycosyltransferase activity in human breast milk

“…Four different ITagged species were identified in the mixture: starting material 1 ([M + ] 513), LacNAc-ITag 3 ([M + ] 675) and trisaccharides 4 ([M + ] 821) and 9 ([M + ] 966) in a ratio of 12 : 42 : 2 : 44 for 1 : 3 : 4 : 9 as determined by LC-MS. Comparison of retention times and MS data to that of isolated ITag-materials, 13 – 15 confirmed the structure identity of the individual ITag-glycosides.…”

“… 10 Our group 10c , 11 and others 12 have reported the synthesis of inexpensive and versatile imidazolium-based tags (ITags) and their application to chromatography-free chemical oligosaccharide synthesis. Furthermore, in our work, we demonstrated that the cationic labels are also compatible with enzymatic transformations and that ITag-glycoside substrates could be employed for the qualitative and quantitative reaction monitoring, using MS techniques, of bovine milk β-1,4-galactosyltransferase (β-1,4-GalT), 13 human α-1,3-fucosyltransferase (α-1,3-FucT VI) 14 and α-2,3-sialyltransferase (α-2,3-ST) from pasteurella multocida , as well as α-2,6-sialyltransferase (α-2,6-ST) 15 from photobacterium damsela . More recently, ITag-labeled mannosamine was also employed for the non-covalent labeling of live cells, 16 demonstrating the versatility of the probes.…”

Biochemical characterisation of an α1,4 galactosyltransferase from Neisseria weaveri for the synthesis of α1,4-linked galactosides

“…This allows for relative quantitative studies by MALDI analysis, by integrating the isotopic envelope peak area of starting substrate and product. [30][31][32][33] According to the results obtained by MALDI-ToF (Table 1), NwLgtC could perform α1,4-galactosylation on both Lac-ITag and LacNAc-ITag using not only galactose, but also 6deoxygalactose, 6-fluorogalactose and galactosamine (ESI, Figures S13-S20). It is apparent that Lac-ITag acts as a much better acceptor than LacNAc-ITag, giving consistently higher conversion rates (Table 1).…”

In vitro biosynthesis of poly-β-1,4-glucan derivatives using a pro-miscuous glycosyltransferase

“…2A). Our previous work demonstrated that such I-Tags generate strong signals in massspectrometry that dominate the analyte ionization and can be employed for yield estimation in biotransformations 48,55,56 . To assess the scope of the glucose polymerization activity, we chemically synthesized the novel 4-(1-methyl-3-methyleneimidazolium) benzyl β-glucoside ITag-Glc-2 (2) ( Fig.…”