General Label-Free Mass Spectrometry-Based Assay To Identify Glycosidase Substrate Competence

Common glycosidase assays rely on the hydrolysis of non-natural labeled sugar substrates that thereby preclude obtaining information as to the specificity of the leaving group and therefore the most kinetically competent natural substrates. A β-mannosidase could be known to hydrolyze β-mannose, for example, but from what is presently hard to determine by any high-throughput means. Herein, the first chiral dopant-based mass spectrometric assay, with its foundation rooted in the Cooks’ fixed ligand kinetic method, is presented to screen label-free monosaccharide-containing substrates for their kinetic competency with a given glycosidase as a step to name these enzymes not just for the sugar that is removed but also for the leaving group that is produced. This work also presents the first information about the substrate specificity of two specific hyperthermophilic enzymes and the first test of some native, unlabeled substrates (α-1-4 mannobiose and β-1-galactosylphingosine) with mesophilic enzymes.