A proteome is the full set of proteins within a biological sample like a cell, tissue, or organism. Proteomics studies all of these proteins and their interactions through various protein analysis methods.
The particular proteomic make-up of any biological entity, including individual protein abundances and locations, determines how that entity works at the molecular level. Thus, mapping a cell’s proteome provides mechanistic information on biological activities and makes it possible to study the mechanisms behind these activities as well as disease states in human health. Knowledge of the proteome may lead to a broad range of discoveries and applications including everything from new therapeutics to new ways to protect plants from drought.
Given the essential importance of the proteome, scientists have been studying it for many years using various protein analysis methods like mass spectrometry, protein sequencing and more. In this “Traditional protein analysis” series, we cover how researchers typically study the proteins that make up the proteome.
These protein analysis methods are not necessarily “ omics” scale technologies, which generally aim to measure all or a large amount of a particular type of biological molecule in a sample. Nonetheless, these methods form the foundations upon which proteomic analysis methods have been built. In a future series, we will dive into the technical concepts behind emerging omics technologies, which aim to enable scientists to study the full proteome.
In the graphics portraying the technologies below, we provide qualitative assessments of proteome coverage and ease-of-use for each. Technologies with low proteome coverage, like western blotting and flow cytometry, are generally used for targeted experiments analyzing a small number of proteins at once. Medium coverage technologies like affinity arrays can look at 100’s to 1000’s of proteins in a single experiment. Truly comprehensive, high proteome coverage technologies can analyze tens of thousands of proteins at once.
Some protein analysis methods are easier to use than others. Low ease-of-use protein analysis technologies generally require complicated, difficult, or customized sample preparation involving a lot of hands-on experimenter time, and their data may be difficult to analyze or require bioinformatics support. High ease-of-use protein analysis technologies employ simple, standardized sample preparation, include more automation, and provide simple, data-rich outputs including protein abundance. Medium ease-of-use technologies have some mix of these attributes.
In addition to assessing standard metrics like these, we also discuss some of the advantages and disadvantages of each protein analysis technique. Many protein profiling technologies have specific pros and cons that make them ideal for certain applications, but not others.
Importantly, the technology assessments here should not be viewed as definitive. Rather, our goal is to help you think about how you can best leverage these technologies for your specific experimental goals.
Antibodies are naturally produced proteins that enable organisms to protect themselves from foreign invaders. Antibodies generally bind to segments (epitopes) of other proteins. In doing so, they can prevent these target proteins from functioning. They can also recruit special immune cells to destroy the target protein or destroy the invading organism that produced the target protein.
For the purposes of protein research, scientists have different ways of creating antibodies that bind to proteins they would like to study. Researchers can use antibody-based protein analysis methods to determine the target protein’s presence, abundance, and location. Some of the antibody-based techniques researchers use to measure proteins include:
This is a low plex and low throughput protein analysis method. It involves first separating proteins by size using a porous gel. The proteins are then physically transferred from the gel to a membrane. Antibodies are applied and will bind to any target proteins stuck to the membrane. The presence of bound antibodies is then detected by imaging the membrane (directly detecting fluorescently-labeled antibodies, or using fluorescent secondary antibodies to bind to the target protein-antibody complex on the membrane).
By measuring the amount of antibodies sticking to the membrane, researchers can measure protein presence and relative abundance. Because the proteins are first separated by size, it is possible to determine the molecular weight of the target protein and multiple antibodies can be used on one membrane. Unfortunately, antibodies may also detect more than one protein due to a lack of perfect specificity. The need for an antibody specific for each target protein and the fact that antibodies may bind more than one target protein often limits how many target proteins a researcher can accurately study using this technique.
Easily customizable – Many aspects of western blotting procedures, from extraction methods to visualization techniques, can be altered and optimized for a protein of interest.
Rather than separate proteins and apply antibodies to them, researchers can do the opposite. That is, they can attach many different antibodies to specific locations on a solid surface to create an array of antibodies. An antibody microarray offers even more density, and higher coverage. To analyze proteins on antibody arrays, researchers first flow a mixture of proteins from a sample over the array. Later, they can add more reagents to the array to generate a signal indicating that a protein of interest is present. Because the signal will come from a known location with a known antibody that binds to a specific protein target, scientists can use these signals and their intensities to determine the identities and abundances of proteins in the original sample.
For some kinds of protein arrays, additional detection chemicals are not necessary. Instead, protein binding might change a physical or electrical property of the array, and this can be measured with specialized equipment for protein detection.
Neither of the above protein analysis methods provide any information about a protein’s location in a cell or in a tissue. To get that level of information with western blots or affinity arrays, researchers would first need to isolate proteins from particular cellular locations (i.e., from the nucleus or cytoplasm or from a particular section of tissue). Researchers can then analyze these extracts separately to determine what proteins they contain and where they came from. This adds significant work to the sample preparation process and is limited in resolution with respect to cellular location.
Antibodies can, however, be used to visualize and localize proteins in intact cells and tissues. One way to do this is with microscopy and a process known as immunohistochemistry. Here samples of interest are incubated with fluorescent antibodies. Often researchers use a variety of chemical treatments to fix their samples so proteins remain in their cellular locations throughout processing. After protein sample preparation, researchers can look at the cells or tissues under the microscope and see where the fluorescent signals from their antibodies are located. In this way, they can determine where their proteins of interest reside in the cells or tissues.
Protein analysis by flow cytometry uses labeled antibodies that bind to proteins expressed by subpopulations of cells. After incubating cells with antibodies, researchers insert the cells into a flow cytometer. This instrument analyzes individual cells and measures the amount of fluorescent label found on each cell. Flow cytometry also counts how many cells have a given set of labels. Specifically, the flow cytometer flows antibody-bound cells through small tubes that can only fit a single cell. A detector at the end of the tube identifies any labels found on the single cells as they pass through it. This can be done with as many colors/antibodies as a flow cytometer can distinguish.
Some flow cytometers can also be used for fluorescence activated cell sorting (FACS). When a FACS flow cytometerdetects that a cell has a particular set of labels, it will shunt the cell into a particular pool. In this way, researchers can use flow cytometry analysis to specifically collect and study subsets of cells that produce specific groups of proteins.
It is important to note that many of these protein analysis techniques do not have to be performed with antibodies. There are multiple types of biomolecules that can bind to proteins including aptamers, antibody fragments, and even other proteins. As long as a set of molecules binds to particular proteins with high specificity and binding can generate a detectable signal, scientists can use these molecules to study the proteome.
Learn about protein sequencing and mass spectrometry in the next two posts in this series.
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