Developing a Cell Quenching Method to Facilitate Single Cell Mass Spectrometry Metabolomics Studies.

Single-cell mass spectrometry (SCMS) has emerged as a powerful tool for analyzing metabolites in individual cells, including live cells. However, cell metabolites have a rapid turnover rate, whereas maintaining metabolites' profiles of live cells during sample transport, storage, or extended measurements can be challenging. In this study, a cell preparation method, which integrates cell washing by volatile salt solution, rapid liquid nitrogen (LN) quenching, freeze-drying in vacuum, and freezer storage at -80 °C, to preserve cell metabolites for SCMS measurement is discussed. Experimental results revealed that LN quenching preserved the overall cell metabolome, whereas storage at -80 °C for 48 h slightly changed the metabolite profiles in quenched cells. However, metabolites in unquenched cells were changed regardless of low-temperature storage. The influence of omission of quenching and low-temperature storage on cell metabolites and relevant pathways were investigated. Results from this work indicate that cell quenching is necessary, but low-temperature storage time should be minimized to preserve cell metabolites. The method developed in the current work can be readily adopted by SCMS techniques with storage remaining largely unaltered, allowing for extended SCMS studies.