Single-Cell Native Mass Spectrometry for In Situ Detection of Protein-Drug Complexes in Live Cells

Area of Science:

• Biochemistry
• Cell Biology
• Analytical Chemistry

Background:

• Single-cell native mass spectrometry (MS) offers sensitive in situ analysis of cellular events.
• Detecting protein-drug interactions within individual cells is crucial for pharmacology.

Purpose of the Study:

• To develop and validate a single-cell native MS approach for direct detection of protein-drug complexes in live human cells.
• To overcome challenges in detecting low-abundance proteins for interaction studies.

Main Methods:

• Established single-cell native MS for enhanced green fluorescent protein (EGFP) detection in HEK293T cells.
• Utilized a P2A peptide linker strategy for coexpression of EGFP and Sirtuin 2 histone deacetylase domain (SIRT2 HDAC).
• Analyzed protein expression levels and identified protein-drug complex ions within single live cells.

Main Results:

• Successfully detected EGFP in single cells using native MS.
• Overcame low expression issues of a fused EGFP-SIRT2 HDAC protein via P2A coexpression.
• Achieved clear identification of multiply charged ions for both proteins and observed SIRT2 HDAC-drug complex ions in single live cells.

Conclusions:

• The developed platform enables direct detection of protein-drug complexes in single live cells using native MS.
• This approach provides a transformative method for characterizing protein-drug interactions in native cellular environments.
• Potential applications include cell-cycle-resolved pharmacology and advancing drug discovery.