Objective

Introduction:

Mass spectrometry (MS) is a widely utilized label-free technology capable of measuring a broad range of analytes but lacks the throughput to support large HTS campaigns. Current commercial ESI MS-based systems using solid-phase extraction (SPE) are capable of running medium-sized screens. We have implemented a platform utilizing acoustics to load samples into a time-of-flight mass spectrometer capable of analysing more than 100,000 samples per day. 

Methods:

An acoustic transducer emits sound waves into a sample in a 384-well plate located on a moving XY-stage. High voltage is applied to a charging cone above the well, inducing charge separation in the sample. The acoustic wave ejects a mist of droplets which are drawn through a heated transfer interface into the MS, during transit droplets dry and an ion beam is generated. The extracted ion peak for substrate and product from each well are used to calculate enzyme activity.  

Results:

AMI-MS was used to build an enzyme assay to identify inhibitors from the AZ compound collection. Having established the kinetic parameters for this enzyme target, validation sets were tested to understand the variability within the assay. Once the robust nature of both the assay and this new MS technology were proven to meet the requirements of a high throughput screen the assay was moved into production. In total over 2 million samples were tested using a single automated AMI-MS platform over a 5 week period. Typical throughput of 100,000 samples per day were achieved. From the collection, 6745 compounds were identified in the first round of screening as actives - a hit rate of 0.3%. From this population of compounds, 4872 were followed up in concentration response format to confirm the activity, ~75% were confirmed as actives. The AMI-MS platform was further utilised during triaging of compounds to identify false positive samples which were likely to be contaminated with zinc. 

Conclusions:

AMI-MS proved a robust technology capable of screening a large compound collection (>2 million samples) and identifying chemical equity capable of inhibiting the enzyme target. Typically, this single instrument was capable of processing samples at a rate of 100,000 data points per day. The final hit rate in this assay was ~0.3%, of these the majority confirmed as active in follow up testing (75%).

Novel Aspect:

This is the first demonstration of AMI-MS technology successfully supporting a full collection screen against a pharmaceutically relevant target.