Organic Mass Cytometry Discriminating Cycle Stages of Single Cells with Small Molecular Indicators

Cell cycle is a significant factor toward cellular heterogeneity, so cell cycle discrimination is a precise measurement on the top of single-cell analysis. Single-cell analysis based on organic mass spectrometry has received great attention for its unique ability to profile single-cell metabolome, but the influence of cell cycle on cellular metabolome heterogeneity has been overlooked until now due to the lack of a compatible cell cycle discrimination method. Here, we report a robust protocol based on the combination of three small molecular indicators, consisting of two small molecular labels (Hoechst and docetaxel) and one cellular endogenous compound [phosphocholine (34:1)], to discriminate single cells at different cycle stages in real time by organic mass cytometry. More than 6000 HeLa cells were acquired by an improved organic mass cytometry system to build a cell cycle differentiation model. The model successfully discriminated single HeLa cells, SCC7, and Hep G2 cells, at G₀/G₁, S, and G₂/M stages with larger than 85% sensitivity and larger than 89% specificity. Along with cell cycle discrimination, obvious heterogeneity of amino acids, nucleotides, energy metabolic intermediates, and phospholipids was observed among single cells at different cycle stages by this protocol, further demonstrating the necessity of cell cycle discrimination for cellular metabolome heterogeneity research and the potential of more endogenous small molecular compounds for cell cycle discrimination.