The majority of single-cell analysis studies focus on nucleic acids. The central dogma of molecular biology states that DNA is transcribed into RNA and RNA is translated into proteins, which are the functional molecules within a cell. However, biology is not that straightforward. Post-translational modifications can alter the structure and function of proteins, thereby altering cell phenotype. Studying proteins in their various forms has been suggested to provide a more faithful representation of cell phenotype. We therefore require proteomics to fully understand cellular functions.
Single-cell proteomics methods
At a single-cell level, there are two main approaches available for analysing the proteome. These are antibody-based methods and mass spectrometry-based methods (see Figure 1).
Flow cytometry is a traditional method for measuring proteins in a single cell suspension and commonly uses fluorescent-tagged antibodies that bind to the protein of interest on the surface of cells.
Mass cytometry is an advancement of flow cytometry, which also combines elemental mass spectrometry. The antibodies used in mass cytometry are metal-tagged and can bind to proteins of interest on the surface or within cells. Protein detection is then performed using mass spectrometry. The use of metal isotopes dramatically increases the number of protein targets that can be simultaneously analysed compared to flow cytometry. By combining laser ablation with the mass cytometry workflow, spatial imaging of tissue sections can also be achieved – a process termed imaging mass cytometry.
Mass spectrometry-based proteomics
Mass spectrometry provides a sensitive, nontargeted, method for the analysis of the whole proteome by identifying molecules based on their mass-to-charge ration (m/z). Typically, mass spectrometry methods are optimised for bulk analysis of cells.
Miniaturised sample preparation techniques in the form of microwell plates, such as nanoPOTS, have enabled proteomic analysis of single cells using mass spectrometry. However, a major bottleneck of these techniques is the limited throughput – both from the number of wells available for sampling and the length of time it takes to analyse a single cell.
In response to the challenges within single-cell proteomics, Single-Cell ProtEomics by Mass Spectrometry (SCoPE; and it’s second iteration SCoPE2) was developed. SCoPE multiplexes single-cell proteomics by using isobaric tandem mass tags (TMT). By employing this method, thousands of proteins from thousands of individual cells can be profiled and quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) at a significantly faster rate than with label-free methods
Spatial proteomics has been explored with the introduction of Deep Visual Proteomics (DVP), a method that incorporates advanced microscopy, artificial intelligence, and ultra-high-sensitivity proteomics to spatially characterise the proteome of individual cells.
By combining imaging technologies with unbiased proteomics to quantify the number of expressed proteins in a given cell, DVP is an important addition in the toolbox of spatial-omics technologies