Sample Preparation for Single Cell Mass Spectrometry Metabolomics Studies: Combined Cell Washing, Quenching, Drying, and Storage.

Single-cell mass spectrometry (SCMS) has become an indispensable tool for studying cellular metabolism. Owing to advancements in modern mass spectrometry (MS) techniques and demand in studies of cell heterogeneity in fundamental biological sciences and human diseases, a variety of different SCMS techniques have been developed and applied in laboratory research. As metabolites can accurately reflect cell status, SCMS metabolomics analysis of live cells is regarded as a powerful tool to provide molecular information about cells. However, a major challenge in SCMS analysis of live cells is preserving the endogenous metabolite profiles during sample preparation, transport, and measurement. Cellular metabolites undergo rapid turnover and are highly sensitive to environmental changes, making them susceptible to degradation or transformation prior to analysis. To address this limitation, we present a robust and reproducible cell preparation protocol designed to preserve cellular metabolite integrity for SCMS. The protocol integrates cell washing with a volatile ammonium formate (AF) solution, rapid quenching with liquid nitrogen (LN2), vacuum freeze-drying, and storage at -80 °C. This approach minimizes cell membrane damage while effectively halting metabolic activity. The results indicate that rapid cell quenching is vital; however, limiting storage time at -80 °C is necessary to preserve cell metabolites. The proposed protocol can potentially be used for other existing SCMS techniques for broad applications.