Single-Cell Membrane Molecular Cartography Enabled by Nanoengineered VUV-LDI Mass Spectrometry Imaging

Deciphering the multicomponent of cell membranes at the single-cell level is critical for understanding pathological mechanisms such as tumor metastasis, yet remains technically daunting due to the membrane's nanoscale thickness and ultralow molecular abundance. Here, we introduce a surface-assisted vacuum ultraviolet laser desorption-ionization mass spectrometry imaging (SAVUVDI-MSI) platform that overcomes long-standing challenges of cytoplasmic interference and insufficient sensitivity. Leveraging the nanoscale depth profiling capability of VUV-LDI, we achieve precise ablation of a single-cell membrane. Coupled with self-engineered VUV-laser-cleavable Au-PEG-FA/Apt nanoprobes, the system not only provides nanoprobe-mediated specific recognition and signal conversion for target membrane protein but also enhances <i>m</i>/<i>z</i> signals originating from the cell membrane by 4-10-fold. This strategy permits simultaneous spatial mapping of phospholipids and cholesterol (via direct label-free imaging) and proteins (via nanoprobe targeting)─three key membrane components─without cytoplasmic overlap. Experimental validation confirmed the aberrant overexpression of proteins and cholesterol on cancer cell membranes. The integrated multivariate statistical analytical framework enables discrimination between cancerous and normal cells based on membrane molecular signatures, establishing a robust platform for nanoprobe-enhanced pathological screening with translational potential. This study marks the inaugural application of VUV laser-enhanced nanoprobes. The SAVUVDI-MSI platform establishes a foundation for the in situ monitoring of membrane heterogeneity in pathological processes.