Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC-MS

Abstract

We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analyzed by ultrasensitive nanoLC-MS. An average of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single-cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells.

Keywords: cell typing; droplets; microfluidics; single-cell proteomics; ultrasensitive LC-MS.

Figure 1.

a) Cells are FACS-sorted into nanowells. Cells are lysed and proteins are extracted, denatured, reduced, alkylated, and finally digested into peptides in the nanoPOTS chip. Peptides are separated and sequenced with ultrasensitive nanoLC-MS. b) Fluorescence micro-graphs show the precise collection of defined numbers of HeLa cells in nanowells. Nanowell diameters are 1 mm. c) Representative plot showing 2825 peptides identified from a single HeLa cell.

Figure 2.

a) Number of unique peptides and b) protein groups identified from triplicate analysis of 0 (blank), 1, 3, and 6 cells based on MS/MS only and MS/MS with MBR. Pairwise correlation of log10-transformed protein LFQ intensities between c) 1 and d) 6 cell samples. Pearson correlation coefficients were labeled with color coded background.

Figure 3.

a) Unsupervised PCA based on label-free quantification of proteins expressed epithelial and mesenchymal cells from human lung. b) Volcano plot of differentially expressed proteins. Epithelial cell Replicate 2 was excluded for this analysis.