An Ultra-High Throughput Cell-Based Mass Spectrometry Flux Assay as a Pathway-Selective In Situ Screening Approach

Acoustic ejection mass spectrometry (Echo-MS) is a viable biochemical screening platform enabling direct detection of biologically relevant enzymatic activities with throughput approaching or meeting the cycle time of conventional optical readouts. Here, we present a cell-based in situ approach for the application of an MS-based screening technique using a well-characterized isotope-enriched metabolite as a tracer of cellular activity through the glutaminase 1 (GLS1) pathway. This assay was validated as a proof-of-concept screen in biochemical and primary human cellular high-throughput screening (HTS) formats using known inhibitors of the human GLS1 target. The results generated displayed an excellent alignment between biochemical and cellular potencies. The direct high-throughput mass spectrometry activity assay presented has promise as a cell-based screening approach for profiling of physiologically relevant adherent cells, eliminating the need for detection antibodies or other labeling methods.