MS Assay Development for Acetylcholinesterase

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In the following example we describe the implementation of a mass spectromet-ric assay for acetylcholinesterase (AChE) [22]. AChE plays an important role in the nervous system. This enzyme rapidly hydrolyzes the active neurotransmitter acetylcholine into the inactive compounds choline and acetic acid. Amongst others, low levels of acetylcholine in the synaptic cleft are associated with Alzheimer's disease [23, 24]. Patients afflicted by this disease may benefit from inhibition of AChE activity thereby increasing ACh level.

Traditionally, plants are a rich source of AChE inhibitors. People from the Caucasus used bulbs of snowdrops (Galanthus sp.) to treat forgetfulness [25]. The active compound in this plant has been isolated and called galanthamine. Other plant-derived AChE inhibitors used for treatment of Alzheimer's disease include Huperzine A from Huperzia serrata and Rivastigmine (Excelon). The latter is a derivative from physostigmine isolated from the calabar bean, Physos-tigma venenosum.

In order to develop an MS-based screening method for AChE, we used a continuous-flow fluorescence assay [26] as the starting point and adapted the assay conditions to MS-compatible conditions using the assay format described in Fig. 5.1. In this assay, the synthetic non-fluorescent AChE substrate 7-acetoxy-1-methyl quinolinium iodide (AMQI) is hydrolyzed into the highly fluorescent 7-hydroxy-1-methyl quinolinium iodide (HMQI). First, it was assessed whether AChE was still active in volatile buffer and whether ionic strength influenced AChE activity. Batch measurements indicated that the reaction proceeded most efficiently in 50 mM potassium phosphate whereas AChE activity proceeded at a somewhat slower rate in 10 mM ammonium hydrogencarbonate; the addition of 180 mM sodium chloride to the 10 mM ammonium hydrogencarbonate did not influence enzyme activity as compared with the 10 mM ammonium hydrogencar-bonate buffer. Although somewhat slower in volatile buffers, enzyme activity is sufficiently high for assay purposes.

Figure 5.7 demonstrates the implementation of the assay and shows the readout in the MS that was obtained for injections of the AChE inhibitor galanth-amine at 0, 1, and 10 mM. Figure 5.7a shows the extracted ion chromatogram of galanthamine, Fig. 5.7b shows the extracted ion chromatogram of HMQI (prod-

Acetylcholinesterase Proteins

Fig. 5.7 AChE-catalyzed hydrolysis of the fluorescent substrate AMQI in volatile buffer monitored by mass spectrometry. Line 1: Start of the substrate pump delivering AMQI. Line 2: Start of the enzyme pump delivering AChE. Peak 3: Injection of 0.1 ||M galanthamine. Peak 4: Injection of 1.0 |M galanthamine. MS instrument: Q-ToF2 (Waters) equipped with a Waters Z-spray electrospray (ESI) source. (a) Mass chromatogram of m/z 288 (galanthamine); (b) mass chromatogram of m/z 104

(choline); (c) mass chromatogram of m/z 146 (acetylcholine). Assay conditions: the carrier solution consisted of 95% 10 mM ammonium bicarbonate, pH 7.8, 5% methanol; the AChE solution (0.25 units AChE mM) was prepared in 10 mM ammonium bicarbonate, pH 7.8. The substrate solution consisted of 30 mM acetylcholine dissolved in 97.5% 10 mM ammonium bicarbonate, pH 7.8, and 2.5% methanol; all reagents were pumped at a flow of 20 mL min-1.

Fig. 5.7 AChE-catalyzed hydrolysis of the fluorescent substrate AMQI in volatile buffer monitored by mass spectrometry. Line 1: Start of the substrate pump delivering AMQI. Line 2: Start of the enzyme pump delivering AChE. Peak 3: Injection of 0.1 ||M galanthamine. Peak 4: Injection of 1.0 |M galanthamine. MS instrument: Q-ToF2 (Waters) equipped with a Waters Z-spray electrospray (ESI) source. (a) Mass chromatogram of m/z 288 (galanthamine); (b) mass chromatogram of m/z 104

(choline); (c) mass chromatogram of m/z 146 (acetylcholine). Assay conditions: the carrier solution consisted of 95% 10 mM ammonium bicarbonate, pH 7.8, 5% methanol; the AChE solution (0.25 units AChE mM) was prepared in 10 mM ammonium bicarbonate, pH 7.8. The substrate solution consisted of 30 mM acetylcholine dissolved in 97.5% 10 mM ammonium bicarbonate, pH 7.8, and 2.5% methanol; all reagents were pumped at a flow of 20 mL min-1.

uct trace), whereas Fig. 5.7c shows the extracted ion chromatogram of AMQI (substrate trace). The line marked with the number 1 indicates the position at which the substrate pump was switched on, whereas the line marked with the number 2 indicates the position at which the AChE pump was started. When the substrate pump was switched on, a clear increase in the substrate trace was observed. However, also a sharp increase in the product trace was evident, indicative of autolysis of the substrate.

Upon starting the AChE pump, a ready decrease in the substrate and a matching increase in the product trace was observed. Injections of galanthamine resulted in a negative peak in the product trace and a positive peak in the substrate trace, accurately matching the peaks observed in the galanthamine trace.

As AMQI was both an expensive and unstable artificial substrate, it was replaced by the native substrate of AChE, acetylcholine that is both cheap and

196 5 Continuous-flow Systems for Ligand Binding and Enzyme Inhibition Assays Based 100 q a TIC

readily detected by MS. As acetylcholine and its product choline were both readily detected by MS, cheap, and acetylcholine being the native substrate of AChE, this substrate was chosen for further studies.

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