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.