Grant: Discovery Grants Program - Individual

Mass spectrometry (MS) is exceptionally powerful for comprehensive biological characterization. Considering proteomics, MS offers unmatched sensitivity, specificity, and scan speed, affording rapid characterization of thousands of discrete components from a sample. Theoretically, this wealth of information can be extracted from only a single cell. However, it is emphasized that MS is but one of several steps in an intricate analytical workflow tailored to isolate, amplify, purify or modify sample components prior to MS interfacing. Consistently performing these challenging steps constitutes the primary bottleneck that limits MS as a robust tool for biological or clinical profiling. Our lab has pioneered multiple technologies for proteome sample preparation and integration to MS. We are recognized as global leaders in proteome purification, separation and preconcentration. Our detergent-based workflows have established new industry benchmarks in top-down and bottom-up proteomics. Our fundamental work on salt-mediated solvent precipitation of proteins, and membrane-based electrophoretic depletion of sodium dodecyl sulfate have each afforded new opportunities in proteomics and mark significant strides towards our long-term vision: to remove all technological barriers hindering the seamless integration of universally applicable analytical workflows with MS to permit routine clinical proteome profiling, disease diagnostics, or therapeutic developments. With our strong record of technology development for proteomics, we recognize the vast potential of an emerging approach for automated high-throughput sample manipulation: droplet-based microfluidics. Here, small (10-100 µm diameter) aqueous droplets, stabilized by surfactants, are carried in an immiscible fluid stream (eg oil). Each microdroplet serves as a vessel to manipulate a variety of compartmentalized materials including proteins, extracellular vesicles, or cells. Reagents can be introduced pre- or post-droplet formation, with downstream sensing to characterize the droplet cargo. The combination of oil + surfactant presents unfavorable conditions to interface droplet-based microfluidics with MS. In this grant cycle, we propose innovative technologies for multiplexed, microscale sample preparation and integration with MS. The synergy of technologies provides new opportunities for biological discovery. To this end, our short term objectives are: (1) to extend fundamental knowledge of proteome precipitation and digestion for high throughput processing, (2) design new, integrated electrophoretic strategies for automated protein manipulation, and (3) to re-imagine the process of droplet-based microfluidics as an MS-compatible workflow. Applications include: (a) single cell proteomics, and (b) rapid monitoring of extracellular vesicle sub-population typing, by barcode tagging and micro-droplet MS. These discoveries will immediately benefit researchers particularly in the context of human health.