Multi-dimensional bio mass cytometry: simultaneous analysis of cytoplasmic proteins and metabolites on single cells

Single-cell multi-dimensional analysis enables more profound biological insight, providing a comprehensive understanding of cell physiological processes. Due to limited cellular contents, the lack of protein and metabolite amplification ability, and the complex cytoplasmic environment, the simultaneous analysis of intracellular proteins and metabolites remains challenging. Herein, we proposed a multi-dimensional bio mass cytometry platform characterized by protein signal conversion and amplification through an orthogonal exogenous enzymatic reaction. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology was applied in the quantification of endogenous intracellular protein glycer-aldehyde-3-phosphate dehydrogenase (GAPDH) through exogenous luciferase Nanoluc (Nluc). The simultaneous detection of GAPDH and hundreds of metabolites at the single-cell level was realized for the first time. Semiquantitative analysis of GAPDH together with single-cell metabolomes under <i>S</i>-nitrosoglutathione (GSNO)-induced oxidative stress was investigated. Bioinformatics analysis revealed 16 metabolites that correlated positively with GAPDH expression upon oxidative stress, including long-chain fatty acids (palmitoleic acid, myristic acid, <i>etc.</i>) and UDP-<i>N</i>-acetylglucosamine (UDP-GlcNAc). Potential synergetic functions of GAPDH and UDP-GlcNAc-mediated oxidative stress responses were also elucidated. Our work proposes a novel strategy for the simultaneous quantitative analysis of single-cell intracellular proteins and metabolites, deepens the understanding of inherent anti-oxidative stress response mechanisms, and provides the molecular fundamentals for the study of inherent biological processes.