Viability-Informed Single-Cell Mass Spectrometry for More Comprehensive Metabolic Analysis

Area of Science:

• Cellular Biology
• Metabolomics
• Mass Spectrometry

Background:

• Cell viability is critical for cellular function, yet metabolic studies often ignore differences between live and dead cells.
• This oversight can lead to misinterpretations of biological systems in mass spectrometry-based research.
• Existing methods lack the ability to simultaneously assess cell viability and metabolic profiles at a single-cell level.

Purpose of the Study:

• To develop a high-throughput, label-free technique for single-cell mass spectrometry that incorporates cell viability information.
• To simultaneously measure cell viability and metabolic profiles, enabling a more accurate understanding of cellular states.
• To identify intrinsic markers for distinguishing live from dead cells within mass spectrometry data.

Main Methods:

• Developed a viability-informed single-cell mass spectrometry (ViSCMS) technique.
• Utilized phosphocholine (PC) 34:1 for event confirmation and glutathione (GSH) as an intrinsic viability marker.
• Validated ViSCMS against traditional AO/PI staining for viability assessment.

Main Results:

• ViSCMS demonstrated high concordance with AO/PI staining for viability rates (mean bias: 0.33%).
• Achieved excellent reproducibility (SD < 3%) across diverse cell lines and viability levels.
• Successfully subtyped six colorectal cancer (CRC) cell lines, a feat only possible when considering cell viability.
• Observed clear viability-dependent metabolic shifts in HCT116 cells treated with an anticancer drug.

Conclusions:

• ViSCMS provides a robust and accurate method for simultaneous single-cell viability and metabolic profiling.
• Accounting for cell viability is essential for accurate metabolic subtyping and understanding cellular responses, particularly in cancer research.
• The technique offers novel insights into tumor cell adaptation to chemotherapy stress at the single-cell level.