Targeted proteomics technology for accurate quantitative single-cell proteomics

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ABSTRACT Multi-omics characterization of a broad spectrum of small subpopulations of cells between tumors and within individual tumors at the single-cell resolution is crucial to achieve understanding of a complete disease biology. Furthermore, biologically important clinical specimens are available in low quantity (e.g., <10 tumor cells), requiring advanced single-cell technologies for effective analysis. However, single-cell proteomics technologies are lagging far behind other omics technologies. Antibody-based immunoassays are used primarily for targeted single-cell proteomics, but they have inherent limitations (e.g., low multiplex), and generally lack quantitation accuracy. Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative for broad accurate quantification. However, current single-cell MS can only allow for relative quantification of ~870 proteins from single mammalian cells. There are three major challenges in single-cell MS for accurate quantitative single-cell proteomics: 1) ineffective processing of single cells, 2) insufficient MS sensitivity and low sample throughput, and 3) lacking well-characterized universal internal standard (UIS). To address these challenges, we propose to develop a single-cell MS system for rapid accurate analysis of single-cell proteome. The feasibility is strongly supported by our recent progress in many aspects of technology development (e.g., introducing the `carrier' concept for effective processing of small numbers of cells including single cells, and developing disruptive MS technologies to improve MS detection sensitivity and specificity) as well as our extensive experiences in high- resolution liquid chromatography (LC) separation for sensitive detection and targeted proteomics analysis for absolute quantification of signaling pathway proteins. The single-cell MS system will be developed through 1) establishing super-SILAC (stable isotope labeling with amino acids in cell culture) as both proteome carrier and UIS, 2) incorporation of proteome carrier super-SILAC (cSILAC) into the sample preparation workflow for robust processing of single cells, and 3) leveraging cutting-edging LC and MS technologies developed at PNNL with integration of ultralow-flow LC separation, high-efficiency ion source (the combination of an emitter array technology and sub-ambient pressure ionization with nanoelectrospray), and ultrafast high-resolution ion mobility separation for significantly improving both MS sensitivity and sample throughput. Super-SILAC will be characterized as UIS for absolute quantification with crude peptide standards, whose purity will be cost- effectively accurately determined using a combined lanthanide labeling and ICP-MS method. With 96-well plate-based cSILAC preparation and well-characterized UIS, the new single-cell MS system is expected to allow for rapid accurate quantification of a large fraction of human proteome (~60%) in single cells with ~120 samples per day. We anticipate that the new MS system will eventually become a convenient indispensable tool not only for quantitative single-cell proteomics but also for routine analysis of very small samples (e.g., rare cells). In turn, it will make substantial contributions to current biomedical research.