RESUMO
Single-cell analysis uncovers phenotypic differences between cells in a population and dissects their individual physiological states and differences on all omics levels from genome to phenome. Spectrometric observation allows label-free analysis of the metabolome and proteome of individual cells, but is still mainly limited to the analysis of mammalian single cells. Recent progress in mass spectrometry approaches now enables the analysis of microbial single cells - mainly by miniaturizing cell handling, incubation, and improving chip-coupling concepts for analyte ionization by interfacing microfluidic chips and mass spectrometers. This review aims at distilling the enabling principles behind microbial single-cell mass spectrometry and puts them into perspective for the future of the field.
Assuntos
Metaboloma , Microfluídica , Animais , Espectrometria de Massas/métodos , Análise de Célula Única/métodos , MamíferosRESUMO
We introduce the coupling of droplet microfluidics and ion mobility spectrometry (IMS) to address the challenges of label-free and chemical-specific detection of compounds in individual droplets. In analogy to the established use of mass spectrometry, droplet-IMS coupling can be also achieved via electrospray ionization but with significantly less instrumental effort. Because IMS instruments do not require high-vacuum systems, they are very compact, cost-effective, and robust, making them an ideal candidate as a chemical-specific end-of-line detector for segmented flow experiments. Herein, we demonstrate the successful coupling of droplet microfluidics with a custom-built high-resolution drift tube IMS system for monitoring chemical reactions in nL-sized droplets in an oil phase. The analytes contained in each droplet were assigned according to their characteristic ion mobility with limit of detections down to 200 nM to 1 µM and droplet frequencies ranging from 0.1 to 0.5 Hz. Using a custom sheath flow electrospray interface, we have further achieved the chemical-specific monitoring of a biochemical transformation catalyzed by a few hundred yeast cells, at single droplet level.
Assuntos
Espectrometria de Mobilidade Iônica , Microfluídica , Espectrometria de MassasRESUMO
The label-free and sensitive detection of synthesis products from single microbial cells remains the bottleneck for determining the specific turnover numbers of individual whole-cell biocatalysts. We demonstrate the detection of lysine synthesized by only a few living cells in microfluidic droplets via mass spectrometry. Biocatalyst turnover numbers were analyzed using rationally designed reaction environments compatible with mass spectrometry, which were decoupled from cell growth and showed high specific turnover rates (â¼1 fmol/(cell h)), high conversion yields (25%), and long-term catalyst stability (>14h). The heterogeneity of the cellular reactivity of only 15 ± 5 single biocatalysts per droplet could be demonstrated for the first time by parallelizing the droplet incubation. These results enable the resolution of biocatalysis beyond averages of populations. This is a key step toward quantifying specific reactivities of single cells as minimal functional catalytic units.