Integrated Microscale Analysis System for Targeted Liquid Chromatography Mass Spectrometry Proteomics on Limited Amounts of Enriched Cell Populations

Limited samples, such as those that are in vivo sourced via biopsy, are closely representative of biological systems and contain valuable information for drug discovery. However, these precious samples are often heterogeneous and require cellular prefractionation prior to proteomic analysis to isolate specific subpopulations of interest. Enriched cells from in vivo samples are often very limited (<10(4) cells) and pose a significant challenge to proteomic nanoliquid chromatography mass spectrometry (nanoLCMS) sample preparation. To enable the streamlined analysis of these limited samples, we have developed an online cell enrichment, microscale sample preparation, nanoLCMS proteomics workflow by integrating fluorescence activated cell sorting (FACS), focused ultrasonication, microfluidics, immobilized trypsin digestion, and nanoLCMS. To assess the performance of the online FACS-Chip-LCMS workflow, 5000 fluorescent labeled cells were enriched from a 5% heterogeneous cell population and processed for LCMS proteomics in less than 2 h. Within these 5000 enriched cells, 30 peptides corresponding to 17 proteins spanning more than 4 orders of magnitude of cellular abundance were quantified using a QExactive MS. The results from the online FACS-Chip-LCMS workflow starting from 5000 enriched cells were directly compared to results from a traditional macroscale sample preparation workflow starting from 2.0 X 10(6) cells. The microscale FACS-Chip-LCMS workflow demonstrated high cellular enrichment efficiency and high peptide recovery across the wide dynamic range of targeted peptides. Overall the microscale FACS-Chip-LCMS workflow has shown effectiveness in efficiently preparing limited amounts of FACS enriched cells in an online manner for proteomic LCMS.