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.
Delving into the proteome starts with exploring proteins themselves. For instance, a scientist may find cells that carry out a particular function and want to know what protein or proteins give them that function. To figure this out, they can extract the proteins from the cells and separate the proteins in fractions based on a variety of characteristics such as size and hydrophobicity. Then, a researcher can test each protein fraction to see if it still has the function of interest. If it does, the researcher can repeat the process, making the protein fractions smaller and smaller, hopefully isolating an individual protein with the function they’re interested in.
Even after isolating a protein, researchers still don’t know the identity of the protein, what gene encodes it, or how that protein carries out its function. That’s where protein sequencing methods come in.
Proteins are composed of chemical building blocks called amino acids. There are 20 amino acids with a variety of chemical properties. These are attached to one another in a linear fashion to form full proteins, and their precise order and abundance in a protein gives the protein its specific structure and function. Protein sequencing methods can determine the order and abundance of all the amino acids that make up a protein.
Sequencing proteins can give scientists information about:
So how do researchers determine protein sequences? Traditionally, protein sequencing is done with Edman degradation. In this protein sequencing method, a purified protein is incubated with a chemical that attaches to an amino acid on one of its ends. Manipulating reaction conditions causes this chemical and the terminal amino acid to break off. The chemical-terminal amino acid compound can then be extracted from the rest of the protein.
Researchers can use a variety of analytical techniques to determine which of the 20 amino acids is contained in the extracted compound. Repeating this process many times over reveals the full amino acid sequence of a protein and thus its identity.
The Edman degradation protein sequencing method is very slow and can only be used to sequence short fragments of full proteins called peptides. Nonetheless, by breaking apart a purified protein and sequencing its component peptides, researchers can determine the full sequence of the original protein.
Researchers are currently developing variations on Edman degradation to sequence many proteins at once. While Edman degradation is slow, one of its key advantages is it does not require prior knowledge of a protein’s sequence to determine its full identity.
Antibodies, on the other hand, require known, well-validated protein targets to be useful for protein identification. And, as you’ll learn in the next post in this series, mass spectrometry relies on prior knowledge of proteins to determine protein sequence as well. Thus, while protein sequencing may be a bit old school, it’s still very useful when researchers have limited information!
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