Frequency Modulation-Boosted Electrolaunching Ionization Mass Spectrometry Enables Mass-Selective Single-Cell Metabolomics

Abstract

Compared with conventional bulk cell analysis, single-cell chemical profiling provides unprecedented insights into the molecular foundations of cellular heterogeneity, thus facilitating a deeper understanding of disease pathogenesis. Intact living-cell electrolaunching ionization mass spectrometry (ILCEI-MS) eliminates sample diffusion losses during ambient ion transmission, leading to a substantially enhanced detection sensitivity and optimized sample utilization efficiency. Here, we developed a mass-selective single-cell metabolomics approach based on induced ILCEI-MS through the application of frequency-modulated AC voltages. The induced AC voltage eliminates physical contact between cellular samples and electrodes, preventing electrical interference, significantly enhancing ionization efficiency and matrix tolerance of cellular analytes, and enabling mass-selective detection through AC frequency modulation. Using this method, we successfully introduced single intact GL261 cells into the mass spectrometer for analysis, achieving high single-cell detection throughput (∼45 cells per minute) and extensive ion coverage (∼400 distinct ions per cell). Frequency modulation of the AC voltage revealed a preference in the detection of ions with different m/z ratios: lower frequencies favored the detection of ions with relatively higher m/z, while higher frequencies were more effective for ions with lower m/z. We further provided a theoretical explanation for this AC frequency-dependent mass-selective phenomenon. This selectivity facilitates targeted ion analysis, enabling more comprehensive profiling of single cells. Furthermore, the platform was applied to analyze various cell types, achieving differentiation among different cells and their subtypes, thereby demonstrating the potential of this method for cellular heterogeneity studies.