Enzymatic conversion continuously monitored with a robotic nanoESI-MS tool: experimental status

Abstract

The presented study describes an adaptation of enzymatic assays to miniaturized and automated nanoelectrospray ionization (nanoESI)-mass spectrometry (MS). The system consists of a liquid handling robot including an ESI chip source and offers several advantages for the investigation of enzymatic reactions. Therewith different parameters can be tested rapidly and automatically. Thus the technique provides a basis for the efficient development of enzymatic assays using MS detection. In the present project the miniaturized setup was applied to become appropriate for the investigation of various enzymatic assays since real-time measurements using automated nanospray robots are not reported in detail so far. The reaction of hen egg white lysozyme (HEWL) and the saccharide hexa-N-acetylchitohexaose (GlcNAc)₆ was used as an exemplary model system. The generation of a stable nanoelectrospray using aqueous solution with enzymes/proteins was typically the main difficulty. Thus various parameters were systematically tested and adjusted to overcome these instabilities. This resulted in moderate, but improved, spray stability. Moreover, the new adjustments were applied to further enzymatic assays (chitinase, chymotrypsin and acetylcholinesterase). In doing so, they were tested whether they are applicable in universal manner. These assays reveal additional findings like a partial reduction of enzymatic activity in the nanoESI setup. The observed complicacies in spraying pure aqueous solutions and the partial enzyme inactivation will be discussed in detail as well as possible approaches to overcome them.