Mass Spectrometry-Based Biochemical Analysis of Single Cells Beyond the Global Proteome

PROJECT SUMMARY/ABSTRACT Biological tissues exhibit a high degree of phenotypic heterogeneity and plasticity, comprising many different subpopulations of cells in various states. Quantifying this heterogeneity at the single-cell level and with molecular depth across large numbers of cells and multiple classes of molecules provides information that cannot be obtained at the bulk scale and will ultimately lead to improved diagnostics and more effective treatments. While single-cell nucleic acid sequencing approaches are having a significant impact on biomedical research, proteins, lipids and metabolites mediate the bulk of cellular function and measurement of their expression provides more direct insight into cellular phenotype. There is thus an urgent need to develop new technologies for large-scale direct proteome, lipidome and metabolome profiling at the single-cell level. To fill this gap, mass spectrometry (MS)-based profiling of protein expression in single cells has recently been demonstrated through the implementation of more efficient sample processing workflows, novel experimental designs and improved instrument sensitivity. Label-free MS-based proteomics can now quantify >3,000 protein groups per cell across >4 orders of magnitude of dynamic range. Here we propose to apply mass spectrometry to study biomolecular expression at the single-cell level beyond the global proteome. We will develop global and targeted approaches to profile posttranslational modifications in single cells, beginning with phosphorylation. We will also extend nanoflow liquid chromatography-MS capabilities for in-depth single-cell lipid profiling. Ultimately, we will develop novel means of generating complex LC gradients that utilize more than two mobile phases to efficiently profile multiple classes of biomolecules (e.g., proteome and lipidome) from the same single cell. These research directions will, in combination with mature nucleic acid sequencing strategies, provide an unprecedented view of cellular regulation from genotype to phenotype at the single-cell level.