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.
Mass spectrometry, also called mass spec, is one of the primary tools researchers turn to for proteomic analysis. Mass spectrometers can identify specific proteins with high resolution, and they’re a fundamental tool for many protein researchers.
While there are many versions of mass spectrometry, they generally involve:
Using mass spectrometry, a researcher can get a list of proteins in their sample. Proteomics researchers can also use mass spectra to measure the relative abundance of the identified proteins.
As you might imagine, some proteins can generate very similar spectra and/or may not be separated well because they have similar masses and charges. In addition, protein spectra signals from high abundance proteins can often drown out signals from low abundance proteins. Thus, even with extensive optimization, mass spectrometry can only measure a fraction of the proteome.
Researchers can complement mass spectrometry with a variety of techniques that make the protein spectra easier to analyze. For instance, before putting them through the mass spectrometer, researchers can pass their proteins or digested proteins (peptides) through separation columns. These columns are tubes containing porous materials that separate proteins or peptides based on properties like size or hydrophobicity. Separations can be accomplished off-line, where a single sample is separated into multiple fractions and each fraction is analyzed separately.
Alternatively, with in-line separation, a researcher adds a single sample to the separation column and the proteins or peptides exiting the column can directly enter a mass spectrometer. Protein or peptide analytes are sequentially analyzed by the mass spectrometer based on how they interact with the column. As only a fraction of the proteins or peptides in the sample exit the column at any point in time, the mass spectrometer can focus analysis on a smaller number of analytes increasing detection and quantitation resolution.
While there are many mass spectrometry techniques for proteomics that can ultimately make the resulting spectra easier to analyze, they introduce biases into the protein detection process. For instance, some proteins may be incompatible with certain columns. Thus, these time-consuming processes may limit the amount of data researchers get from mass spectrometry in one experiment.
Despite its limitations for proteomics, mass spectrometry has been, for many years, the main tool available to researchers wishing to analyze more and more of the proteome. The technique has been used to make many biological discoveries and develop many therapeutics. It is continually being improved through instrumentation developments, innovative methods, and improved software for data analysis. To overcome some of the drawbacks of mass spectrometry for proteomics, many researchers are developing new technologies that will make it easier to comprehensively analyze the proteome. We discuss the concepts behind some of these up-and-coming proteomics technologies, including our own platform, in other “Proteomics” posts.
Learn about protein sequencing as well as antibodies and other affinity reagents in the previous two posts in this series.
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