Dual Desalting Electrospray Strategy for In-Cell Mass Spectrometry to Reveal Novel Sphingolipid Metabolism in an Epithelial–Mesenchymal Transition

The metabolome offers a direct snapshot of cell function and can respond to external changes within a very brief time scale of seconds or minutes. In situ in-cell mass spectrometry, with minimal pretreatment, enables direct analysis in a nonvolatile salt environment. However, it is challenging to obtain abundant metabolomes due to the inherent incompatibility of nonvolatile salts with mass spectrometry. Here, we developed a dual desalting electrospray ionization mass spectrometry (dd-ESI MS) technology for in-cell MS measurement to obtain a comprehensive and native cellular metabolome in nonvolatile salt buffers. The salt ions and metabolites were initially separated through the mild electrophoretic effect of induced nanoelectrospray ionization (InESI). In the following electrospray process, the complex interactions between aqueous droplets and methanol droplets further enhanced the desalting effect. Compared with nanoESI, dd-ESI MS exhibited stronger salt tolerance and higher sensitivity for cell metabolome analysis in PBS buffer. Interestingly, we observed a significant enrichment of the sphingolipid metabolism pathway during the epithelial-mesenchymal transition, a metabolic pathway not previously confirmed by metabolomics techniques. In addition, the transcriptome analysis also revealed consistent gene changes, further confirming the validity of our findings. dd-ESI MS enabled the acquisition of a more comprehensive and native metabolome, providing novel insights into complex physiological processes.