Development of acoustic ejection mass spectrometry for label-free cellular based assays for solute carrier transporter targets in drug discovery

Abstract:
Solute carrier transporters (SLCs) mediate many essential physiological functions and therefore have been implicated is many different disease states, making them highly interesting targets for exploitation within the pharmaceutical industry. SLCs are highly specialised proteins that translocate substrates such as sugars, amino acids and inorganic ions across cellular membranes, which can allow for a multitude of techniques to monitor the uptake of these substrates as a measure of potential candidate compounds to modulate transportation. The most commonly used techniques focus on labelled substrate or inference of current change across the membrane, whereas mass spectrometry is uniquely placed to directly measure the uptake of the specific substrate of interest. Targeted mass spectrometry methods have previously been used for biochemical assays but application in cellular assay space provides more biologically relevant results. The use of acoustic ejection mass spectrometry was explored to monitor substrate uptake in a higher throughput setting compared to LCMS, with minimal sample preparation post-cell lysis. The AE-MS technique was able to read 384 well plates within 20 minutes, required only 40uL of total sample volume, an ejection volume in the low nL range and had minimal carryover effects. Targeted multiple reaction monitoring (MRM) methods allowed for highly selective and sensitive measurements of defined substrates. Automation of sample preparation alongside the AE-MS method improved assay robustness and feasibility to profile compounds for hit validation within a screening cascade. Additional work used the proposed workflow to answer biological questions earlier in the drug discovery pipeline. AE-MS data was also compared to data produced via non-direct techniques, with extremely high correlation of results. The overall workflow for monitoring substrate uptake could be applied a variety of SLCs with minimal additional method development for detection.