論文ID: A0080
The rapid development of next-generation sequencing techniques has enabled single-cell genomic and transcriptomic analyses, which have revealed the importance of heterogeneity in biological systems. However, analytical methods to accurately identify and quantify comprehensive metabolites from single mammalian cells with a typical diameter of 10–20 μm are still in development. The aim of this study was to develop a single-cell metabolomic analytical system based on highly sensitive nano-liquid chromatography tandem mass spectrometry (nano-LC/MS/MS) with multiple reaction monitoring. A packed nano-LC column (3-μm particle-size pentafluorophenylpropyl particles of dimensions 100 μm × 180 mm) was prepared using a slurry technique. The optimized nano-LC/MS/MS method showed 3–132-fold (averaged value, 26-fold) greater sensitivity than semimicro-LC/MS/MS, and the detection limits of several hydrophilic metabolites, including amino acids and nucleic acid related metabolites were in the sub-fmol range. By combining live single-cell sampling and nano-LC/MS/MS, we successfully detected 18 relatively abundant hydrophilic metabolites (16 amino acids and 2 nucleic acid related metabolites) from single HeLa cells (n = 22). Based on single-cell metabolic profiles, the 22 HeLa cells were classified into three distinct subclasses, suggesting differences in metabolic function in cultured HeLa cell populations. Our single-cell metabolomic analytical system represents a potentially useful tool for in-depth studies focused on cell metabolism and heterogeneity.
