Single cell protein analysis for systems biology

Abstract

The cellular abundance of proteins can vary even between isogenic single cells. This variability between single-cell protein levels can have regulatory roles, such as controlling cell fate during apoptosis induction or the proliferation/quiescence decision. Here, we review examples connecting protein levels and their dynamics in single cells to cellular functions. Such findings were made possible by the introduction of antibodies, and subsequently fluorescent proteins, for tracking protein levels in single cells. However, in heterogeneous cell populations, such as tumors or differentiating stem cells, cellular decisions are controlled by hundreds, even thousands of proteins acting in concert. Characterizing such complex systems demands measurements of thousands of proteins across thousands of single cells. This demand has inspired the development of new methods for single-cell protein analysis, and we discuss their trade-offs, with an emphasis on their specificity and coverage. We finish by highlighting the potential of emerging mass-spec methods to enable systems-level measurement of single-cell proteomes with unprecedented coverage and specificity. Combining such methods with methods for quantitating the transcriptomes and metabolomes of single cells will provide essential data for advancing quantitative systems biology.

Keywords: antibodies; gene expression and regulation; mass-spectrometry; single cell proteomics.

Figure 1. Classification of single-cell protein analysis methods based on their specificity, proteome coverage, and cell throughput

(A) Antibody-based methods, in red, are widely utilized and generally applicable to intermediate number of proteins at once. Their specificity depends on the antibody and can be rather low. The specificity of antibodies can be increased by electrophoretic separation (scWestern) or using multiple antibodies per protein (PEA). Fluorescent protein-based methods, in green, are highly specific and facilitate monitoring protein levels over time, but are limited to quantitating only a few proteins per cell because of spectral overlap. MS can increase both specificity and depth of coverage. MALDI-TOF has been used to study single cells and spatial questions for decades, but it offers only medium specificity and low proteome coverage. SCoPE-MS enables simultaneous identification and quantitation of hundreds of proteins from single cells, and demonstrates one path toward comprehensive quantitation of proteins in single cells. (B) Flow cytometry-based and automated imaging techniques, encompassing many antibody and fluorescent protein-based methods, can robustly assay tens of thousands of cells per experiment, lighting the way for statistical analysis of single cell data. For the remaining techniques of higher specificity antibodies or MS, there is generally a trade-off between specificity and cellular throughput. All values are applicable for a typically sized mammalian cell, with a diameter of ~15 μm and ~500 pg of total protein. Abbreviations: PEA, proximity extension assay; SCoPE-MS, single cell proteomics by MS; scWestern, single-cell Western blot.