Human tissues contain a variety of cell types and subtypes with distinct functions, and understanding heterogeneity at the single cell level is of great importance for biomedical research. Although MS-based proteomic analyses are capable of quantifying thousands of proteins, the extension to single cell studies has been largely ineffective. This is primarily due to protein and peptide losses during sample processing. To address this, we have developed nanoPOTS (Nanodroplet Processing in One-pot for Trace Samples) to efficiently process and analyze single mammalian cells. Cultured HeLa cells, primary lung cells, and spiked circulating tumor cells (CTCs) isolated from whole blood were FACS-sorted or isolated by laser microdissection into nanoPOTS chips based on cell morphology or immunohistochemistry staining. All processing steps were carried out in nanowells having a total processing volume &lt;200 nL to reduce protein losses to surfaces. A custom robotic nanopipetting system was used to dispense reagents and collect prepared samples. The processed samples were analyzed by nanoLC/MS. We first used FACS-sorted HeLa cells to evaluate the sensitivity of present system. We are able to identify an average of 669 protein groups (n=3) per HeLa cell, with 332 quantifiable protein groups having valid LFQ values in at least 2 samples. We also tested whether the single cell proteomic platform could discriminate cell types isolated from human tissues. Antibody-labeled epithelial and fibroblast cells from the lung tissue of a healthy donor were used for the evaluation, and the two cell types were well clustered without overlap. Statistical analysis revealed a panel of proteins that were enriched for each cell type, which could serve as protein signatures. Finally, we analyzed spiked CTCs isolated from whole blood and could differentiate the single CTCs from white blood cells based on their proteomic signatures.<br/>

In-depth proteome profiling of single cells including circulating tumor cells by nanodroplet sample processing and ultrasensitive LC-MS

2019 Spring National Meeting

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