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

Area of Science:

• Biochemistry
• Cellular Biology
• Analytical Chemistry

Background:

• Conventional optical readouts in biochemical screening face limitations in speed and directness.
• Enzymatic activity detection is crucial for drug discovery and understanding cellular pathways.
• Mass spectrometry (MS) offers sensitive and specific detection capabilities.

Purpose of the Study:

• To present a cell-based, in situ acoustic ejection mass spectrometry (Echo-MS) screening approach.
• To validate this MS-based assay for profiling the glutaminase 1 (GLS1) pathway using known inhibitors.
• To demonstrate the utility of Echo-MS for high-throughput screening (HTS) in adherent cells.

Main Methods:

• Utilized a well-characterized isotope-enriched metabolite as a tracer for cellular activity.
• Applied Echo-MS for direct detection of enzymatic activity within intact cells.
• Validated the assay in both biochemical and primary human cellular HTS formats.
• Employed known inhibitors of the human GLS1 target for proof-of-concept screening.

Main Results:

• Achieved excellent alignment between biochemical and cellular potencies of GLS1 inhibitors.
• Demonstrated the feasibility of cell-based in situ screening using Echo-MS.
• Showcased the assay's ability to profile physiologically relevant adherent cells.
• Confirmed high throughput capabilities comparable to conventional optical methods.

Conclusions:

• The presented direct high-throughput mass spectrometry activity assay is a promising cell-based screening approach.
• Echo-MS eliminates the need for detection antibodies or other labeling methods in cellular assays.
• This method facilitates efficient drug discovery and pathway analysis for targets like GLS1.