Objective

Keywords: acoustic loading; mass spectrometry; high-throughput screening; kinase; ADP Glo

Introduction:

We have implemented a platform utilizing acoustics to load samples into a time-of-flight mass spectrometer capable of analyzing more than 100,000 samples per day. In this poster we demonstrate the use of our platform to screen a kinase and compare the data with a classical high-throughput screening technology. Since acoustic mist ionization mass spectrometry (AMI-MS) is a direct infusion system, kinases are a challenge due to the enzyme’s requirement for MgCl₂.

Methods:

An acoustic transducer generates a nebulized spray from the wells of 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 nebulized spray is drawn through a heated transfer interface into the MS and an ion beam is generated. To utilize the platform with a kinase enzyme, ammonium phosphate at high pH is added to precipitate out the Mg²⁺ ions.  

Results:

AMI-MS was used to build a kinase enzyme assay to identify inhibitors from a subset of the AZ compound collection. We established a protocol using ammonium phosphate as a stop solution at the end of the assay to remove magnesium chloride and reduce suppression in the mass spectrometer. Having established the kinetic parameters for this target we screened a 60,000 compound subset using both ADP Glo™ kits and AMI-MS endpoints. There was a strong correlation between both assay formats, over 2000 compounds were identified as active in both formats. Surprisingly, there were only small clusters of compounds which appeared active in only one of the two assay technologies (38 unique to AMI-MS and 101 unique to ADP Glo™).  

Conclusions:

Our data demonstrates that AMI-MS is able to screen kinase targets where there is a requirement for magnesium chloride despite this being a direct infusion MS technology. AMI-MS compared very favorably with traditional kinase screening technologies but proved significantly cheaper as there was no requirement to purchase detection kits.

Novel Aspect:

This is the first example of AMI-MS being used to screen a kinase target. The data presented clearly demonstrates that AMI-MS is comparable with existing kinase technologies.