Single-Cell Proteomics

Abstract

The inability to make broad, minimally biased measurements of a cell's proteome stands as a major bottleneck for understanding how gene expression translates into cellular phenotype. Unlike sequencing for nucleic acids, there is no dominant method for making single-cell proteomic measurements. Instead, methods typically focus on either absolute quantification of a small number of proteins or highly multiplexed protein measurements. Advances in microfluidics and output encoding have led to major improvements in both aspects. Here, we review the most recent progress that has enabled hundreds of protein measurements and ultrahigh-sensitivity quantification. We also highlight emerging technologies such as single-cell mass spectrometry that may enable unbiased measurement of cellular proteomes.

Keywords: multiomic; multiplexing; proteome; targeted proteomics; untargeted proteomics.

Figure 1.. Properties of an Absolutely Quantitative Immunoassay.

(A) Each assay has a dynamic range over which the measured output correlates with the concentration of analyte. (B) The correlation between analyte and output can be estimated using a standard curve with known analyte concentration. (C) The limit of detection (LOD) is the concentration of analyte that is three standard deviations (SDs) above the measured output in the absence of analyte. (D) The smallest difference between two values that can be reliably measured is the resolution. Similar to LOD, the resolution is determined by the SDs of measured values.

Figure 2.. Single-Cell Absolute Quantification Methods.

(A) Single-cell western blotting uses a series of microwells embedded in gel in order to trap individual cells. Upon lysis, the protein is electrophoretically separated directly in the gel. The separated proteins are photocaptured in the gel to enable probing and washing with detection antibodies. (B) The single-cell barcode chip measures single-cell proteins using an array of immobilized antibody strips. Cells are captured in chambers and enclosed with the antibody array. Upon lysis or secretion, proteins will bind to the array and are detected with a second fluorescent antibody, with the location of the fluorescence indicating the identity of the targeted protein. (C) The proximity ligation assay (PLA) detects protein by using pairs of DNA-functionalized antibodies. When both antibodies are bound to the same target, the proximity facilitates ligation. By encoding protein abundance into DNA, one can measure both a protein and mRNA in the same cell. Elements of the figure were generated with BioRender. Abbreviations: ddPCR, droplet digital PCR; PA, polyacrylamide.

Figure 3.. Highly Multiplexed Single-Cell Protein Measurements.

(A) Cytometry time of flight (CyTOF) allows dozens of proteins to be measured in the same cell through mass spectrometry [39]. Data can be analyzed via traditional flow cytometry methods, such as biaxial plots, or more advanced methods designed to accommodate the additional dimensionality of CyTOF. (B) CITE-Seq (cellular indexing of transcriptomes and epitopes by sequencing) enables the measurement of even more protein targets than CyTOF by replacing the metal isotopes with DNA barcodes [47]. They are designed such that they integrate into DNA-sequencing protocols, providing the option to analyze mRNA and protein simultaneously. (C) Histocytometry uses an eight step (i–viii) analysis method to enable single-cell interrogation while simultaneously maintaining the spatial arrangement of each cell [51]. Elements of the figure were generated with BioRender. Abbreviations: FU, fluorescent unit; SPADE, spanning-tree progression analysis of density-normalized events.

Figure 4.. Single-Cell Mass Spectrometry (MS) Methods.

(A) MS requires several steps between cell lysis and analysis that must be optimized in order to be compatible with single-cell samples. At a minimum, proteins must be digested into peptides, undergo cleanup to be MS compatible, and be separated from one another. (B) The nanoPOTS (nanodroplet processing in one pot for trace samples) platforms miniaturize each sample preparation step into 200-nl droplets atop a microwell plate. A nano-liquid chromatography (nano-LC) system then separates and injects each sample with minimal dilution. (C) The single-cell ProtEomics by mass spectrometry (SCoPE-MS) platform uses isobaric labeling and tandem mass spectrometry to both increase the sample throughput and improve detection by adding carrier and reference samples. These additions increase the total amount of peptide, making it easier to measure and quantify, while the isobaric labels enable quantification of the peptide in individual cells. Peptides ions selected in the first MS analysis (MS1) are fragmented and passed to the second MS analysis (MS2) for quantification and identification. Elements of the figure were generated with BioRender. Abbreviation: mPOP, minimal ProteOmic sample preparation.