3.2 Single cell proteomics
As scRNA-seq allows to examine the heterogeneity of the transcriptome between cells, single cell proteomics enables quantification of proteomic heterogeneity. In contrast to scRNA-seq, where RNA can be amplified, proteins cannot be, which leads to lesser starting material in single cell proteomics experiments. There are many methodologies that attempt to overcome this limitation by absolute quantification of a small number of proteins or through multiplexed measurements.43 Due to the wide variety in methodologies, there is no centralized workflow for exploring the single cell proteome. Instead, the workflow chosen by the researcher should be specific to the research question being addressed. As such, the field of single cell proteomics can be broken down into two subsections- ‘absolute quantification’ which involves targeted and untargeted methodologies, and ‘multiplexing based methodologies’ to carry out multiple protein measurements within the same experiment. As is the case with all quantification methodologies, there are tradeoffs in single cell proteomics as well. While one technique may have a wider dynamic range, it can suffer from low resolution, or a versatile multiplexing assay may lose sensitivity for the protein targets. Quantification with SCP also reduces the reliance on high affinity antibodies to capture the target protein using traditional assay like ELISA, which is a real limitation, especially considering non-specific antibody binding as well as antibody availability. In the direction of for miniaturizing these immunoassays, microfluidic technologies have emerged at the forefront for absolute quantification-based techniques. Microfluidics now allow for experiments to be run in the microliter and even picolitre volumes, which is essential for detecting the low protein concentrations released from a single cell.48 For single cell western blotting (scWestern), Hughes et al. have developed a method that has been multiplexed for 11 protein targets and supports up to 1000 concurrent blots in only four hours.49 When a low starting number of cells is used (<200), FACS sorting can be applied in tandem to improve upon resolution for single cells. The overall workflow of a typical scWestern blot is housed in a 30 µm thick photoactive polyacrylamide gel which rests atop a glass microscope slide with an array of 6270 wells. Every step of the procedure from single cell sorting, cell lysis, target protein capture, and fluorescence detection are compiled within this apparatus. The group concluded that scWesterns represent a viable singe cell protein assay capable of high throughput quantitative analysis coupled with multiplexing ability. The technique can address target molecular mass via protein electrophoresis, as well as subsequent high affinity antibody probe binding. The information when combined, generates a high confidence protein identification and specificity, representing a powerful diagnostic tool for single cell proteomics analysis. A miniaturized version of ELISA was presented by Shirai et al., where they created a single-molecule ELISA apparatus using micro/nanofluidic technology. The device can use sample volumes in the picolitre (pL) range and has the capability to chemically process and capture single molecular targets.50 Both techniques represent significant stepping stones in the fields of microfluidics and single cell proteomics with vast applications in medicine and systems biology research. Even though most immunoassays rely upon fluorescent-based readouts, there are newer methods such as single-cell barcode chip (SCBC) which do not require fluorescence.51
Absolute quantification of proteins from a single cell can also be conducted in an untargeted manner. This bypasses the need for pre-defined protein targets and their corresponding high affinity antibody conjugates, which most of the targeted single cell proteomics approaches rely upon. Similar to the miniaturization of immunoassays to accommodate single cell methodologies, bottom-up proteomics workflows have also been adapted to the single cell scale with different considerations during the workflow.52 For example, when extracting the proteins from single cells, it is imperative to limit the non-specific absorption from materials that are housing the sample. Another consideration comes in with the use of enzyme trypsin. Since trypsin follows Michaelis-Menten Kinetics, the digestion rate is significantly hampered when using small substrate amounts (i.e., total protein within a single cell) when compared with the amount of substrate in a global bottom-up proteomics workflow (i.e., total protein from many cells pooled together). The ramifications of both these factors on the quantitative readout can be controlled by minimizing sample volumes. A more in-depth review on sample processing and emerging technologies in nano proteomics can be found here.53,54,55
Specht et al. describe that using SCoPE-MS, they were able to detect the underlying heterogeneity in macrophage populations differentiated from monocytes using the agonist phorbol-12-myristate-13-acetate (PMA).56 They concluded that macrophage heterogeneity exists within each cell’s proteome independent of cytokine induced differentiation processes. The group assessed whether cell type can be assigned based on the abundance of proteins specific to monocytes or macrophages by color-coding cells based on the median abundance of differentially abundant proteins. They also assessed the protein fold changes, by averaging the fold changes of protein abundance in single cells (monocytes and macrophages) and comparing that to the fold changes in bulk samples for each cell type (mixing the lysates of single cells). This supports the utility of SCoPE2 as an accurate method for determining protein fold changes in single cells. Their findings also support the known biological functionality of M1 and M2 - polarized macrophages cell types suggesting that SCoPE2 provides an excellent framework for quantifying relative protein abundance at the single cell level and facilitates identification of cell-type specific proteins to assess population heterogeneity. This is one example of how single cell proteomics workflows can provide meaningful biological insight into heterogeneity, which is an important aspect to consider for any systems biology approach as heterogeneity is not accounted for in the omics approaches at global level.
Single cell analytics also involves the use of multiplexing methodologies allowing multiple analyses to run simultaneously. This is important for systems biology research as cellular signaling dynamics are realized by the concerted functioning of many proteins. Some of the techniques can even retain spatial information, which is another fundamental aspect of cell signaling. Multiplexing has been traditionally limited by spectral overlap of fluorophores that are conjugated to the detection antibodies used for reading out data in a highly multiplexed experimental workflow. This limitation is bypassed using antibodies conjugated to metal isotopes which do not have spectral overlap, in a technique known as mass cytometry (CyTOF).48 This technique involves the vaporization of a tissue sample region using a laser beam, where the ejection or plume released by contact with the laser is aerosolized, atomized, ionized, and entered into a time-of-flight mass spectrometer to quantify the isotope abundance.57 Using the quantified isotope abundance, the “spots” of the vaporized material can then be mapped back to the original coordinates on the tissue section, with the generation of high dimensional map. This provides targeted proteomic information mediated by the detection antibodies while maintaining spatial resolution. The technique can be used with up to 40 unique isotope labeled antibodies to detect up to 40 different proteins. Since multiplexed mass cytometry-based imaging (IMC) provides information regarding cell composition, phenotype, and spatial organization, all of which dictate cellular signaling, IMC represents an important area of research for systems biology.