Single-Cell Native Mass Spectrometry for <i>In Situ</i> Detection of Protein–Drug Complexes in Live Cells

Single-cell native mass spectrometry (MS) holds immense potential for highly sensitive <i>in situ</i> analysis of biological events within individual live cells. Herein, we present a significant advancement that enables the direct detection of protein-drug complexes in single human cells. We first established the robust and reproducible detection of enhanced green fluorescent protein (EGFP) expressed in HEK293T cells using our single-cell native MS approach. To investigate protein-inhibitor interactions with this approach, human Sirtuin 2 histone deacetylase domain (SIRT2 HDAC) was fused with EGFP and overexpressed in HEK293T cells. However, the expression level of this EGFP-SIRT2 HDAC fusion protein was too low for the detection of protein signals by single-cell native MS. To overcome this challenge, we employed a P2A peptide linker strategy to achieve coexpression of independent EGFP and SIRT2 HDAC at suitable levels within the same cell. This strategy enabled the clear identification of multiply charged ions for both proteins and, crucially, the observation of SIRT2 HDAC-drug complex ions within a single live cell. Our platform establishes a transformative approach for characterizing protein-drug interactions in their native cellular environment at the single-cell level, with potential applications in cell-cycle-resolved pharmacology and drug discovery.