Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

Abstract

Cellular heterogeneity is important to biological processes, including cancer 1,2 and development 3 . However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem Quantification of TMT-labeled peptides relies on reporter ions (RI) whose levels reflect both peptide abundances and noise contributions, such as coisolation interference and background noise 8,9 .To evaluate the contribution of background noise to single-cell RI quantification, we estimated the signal-to-noise ratio (SNR), Extended Data Fig. 1. The estimates indicated that RI intensities are proportional to the amount of labeled single-cell proteomes, and very low for channels left empty.These data suggest that the signal measured in single cells exceeds the background noise by 10-fold or more. As an added SNR control for every TMT set, SCoPE-MS leaves the 130N channel empty, thus simultaneously avoiding isotopic cross-contamination from the carrier cells in channel 131 and having a channel that reflects the background noise.To evaluate the ability of SCoPE-MS to distinguish different cell types, we prepared two labelswapped and interlaced TMT sets with alternating single Jurkat and U-937 cells, two blood cancer cell lines with average cell diameter of only 11 µm (Fig. 1b). The levels of all 583 proteins quantified in single cells were projected onto their principle components (PC). The three-dimensional projections of single-cell proteomes clustered by cell type (Fig. 1c), suggesting that SCoPE-MS can identify cell types based on their proteomes. Next, we identified proteins whose levels vary less within a cell type than between cell types. Among the 117 proteins showing such trends at FDR < 2%, we plotted the distributions for seven in Fig. 1d. Some of these proteins are expected to be cell type specific, such as the higher abundance of Complement C3 in the U-937 cells, which are myeloid lineage precursors for macrophages. The consistency of protein fold-changes between Jurkat and U-937 cells is also reflected in the positive correlations among fold-changes estimated from different cells and TMT channels, Extended Data Fig. 2.Next, we quantified single-cell proteome heterogeneity and dynamics during ES cell differentiation. To initiate differentiation, we withdrew leukemia inhibitor factor (LIF) from ES cell cultures and transitioned to suspension culture; LIF withdrawal results in complex and highly heterogeneous differentiation of epiblast lineages in embryoid bodies (EB). We used SCoPE-MS to quantify over a thousand proteins at FDR = 1 % (Extended Data Fig. 3a) and their pair-wise cor- relations (averaging across single cells) in days 3, 5, and 8 after LIF withdrawal (Fig. ...