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Department of Chemistry Seminar
August 28, 2017 @ 4:10 pm - 5:00 pm
Dr. Ryan Kelly from PNNL will present a seminar hosted by Prof. Brian Clowers in Fulmer 201 at 4:10pm.
Title: Extending Mass Spectrometry-Based Proteomics to the Single Cell Level
Abstract: Despite the power of mass spectrometry (MS)-based proteomics for understanding cellular function, the large amounts of material required for in-depth studies (typically thousands to millions of cells) hinder many applications. Analytical platforms based on ultrasensitive liquid chromatography coupled to MS are not the bottleneck, as single-cell sized samples have been accessible for over a decade. Rather, sample preparation procedures have been incapable of preparing and delivering such small samples to these analytical platforms. Conventional procedures based on pipetting and multiwell plates suffer from large sample losses due to adsorption to surfaces and poor reaction kinetics, such that orders of magnitude more starting material are required than should otherwise be necessary. We have developed NanoPOTS: Nanowell-based Preparation in One pot for Trace Samples, in which a robotic platform dispenses cells and reagents into photolithographically patterned nanowell reaction vessels with subnanoliter precision. Sample preparation utilizes a novel workflow that eliminates the need for multiple reaction vessels and cleanup steps to process cellular tissue into purified tryptic peptides. Compared to the typical tens-of-microliter volumes for proteomic sample preparation, the ~200 nL nanowells minimize sample losses to surfaces and maintain elevated sample concentrations for efficient digestion. To date, we have identified >3,000 proteins from just 10 cells, which is a level of proteome coverage not previously achieved from fewer than 10,000 cells. We will describe the sample preparation and analytical platforms that have made this advance possible, including ultra-low flow nanoLC and Orbitrap Fusion Lumos MS. We will also describe the proteome coverage achieved when analyzing individual mammalian cells.