Proteomics is the large-scale study of proteins, particularly their structures and functions.[1][2] Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997[3] to make an analogy with genomics, the study of the genes. The word "proteome" is a blend of "protein" and "genome", and was coined by Marc Wilkins in 1994 while working on the concept as a PhD student.[4][5] The proteome is the entire complement of proteins,[4] including the modifications made to a particular set of proteins, produced by an organism or system. This will vary with time and distinct requirements, or stresses, that a cell or organism undergoes.
Contents |
After genomics and transcriptomics, proteomics is considered the next step in the study of biological systems. It is much more complicated than genomics mostly because while an organism's genome is more or less constant, the proteome differs from cell to cell and from time to time. This is because distinct genes are expressed in distinct cell types. This means that even the basic set of proteins which are produced in a cell needs to be determined.
In the past this was done by mRNA analysis, but this was found not to correlate with protein content.[6][7] It is now known that mRNA is not always translated into protein,[8] and the amount of protein produced for a given amount of mRNA depends on the gene it is transcribed from and on the current physiological state of the cell. Proteomics confirms the presence of the protein and provides a direct measure of the quantity present.
Not only does the translation from mRNA cause differences, many proteins are also subjected to a wide variety of chemical modifications after translation. Many of these post-translational modifications are critical to the protein's function.
One such modification is phosphorylation, which happens to many enzymes and structural proteins in the process of cell signaling. The addition of a phosphate to particular amino acids—most commonly serine and threonine[9] mediated by serine/threonine kinases, or more rarely tyrosine mediated by tyrosine kinases—causes a protein to become a target for binding or interacting with a distinct set of other proteins that recognize the phosphorylated domain.
Because protein phosphorylation is one of the most-studied protein modifications, many "proteomic" efforts are geared to determining the set of phosphorylated proteins in a particular cell or tissue-type under particular circumstances. This alerts the scientist to the signaling pathways that may be active in that instance.
Ubiquitin is a small protein that can be affixed to certain protein substrates by enzymes called E3 ubiquitin ligases. Determining which proteins are poly-ubiquitinated can be helpful in understanding how protein pathways are regulated. This is therefore an additional legitimate "proteomic" study. Similarly, once it is determined which substrates are ubiquitinated by each ligase, determining the set of ligases expressed in a particular cell type will be helpful.
Listing all the protein modifications that might be studied in a "Proteomics" project would require a discussion of most of biochemistry; therefore, a short list will serve here to illustrate the complexity of the problem. In addition to phosphorylation and ubiquitination, proteins can be subjected to (among others) methylation, acetylation, glycosylation, oxidation and nitrosylation. Some proteins undergo ALL of these modifications, often in time-dependent combinations, aptly illustrating the potential complexity one has to deal with when studying protein structure and function.
Even if one is studying a particular cell type, that cell may make different sets of proteins at different times, or under different conditions. Furthermore, as mentioned, any one protein can undergo a wide range of post-translational modifications.
Therefore a "proteomics" study can become quite complex very quickly, even if the object of the study is very restricted. In more ambitious settings, such as when a biomarker for a tumor is sought – when the proteomics scientist is obliged to study sera samples from multiple cancer patients – the amount of complexity that must be dealt with is as great as in any modern biological project.
Scientists are very interested in proteomics because it gives a much better understanding of an organism than genomics. First, the level of transcription of a gene gives only a rough estimate of its level of expression into a protein. An mRNA produced in abundance may be degraded rapidly or translated inefficiently, resulting in a small amount of protein. Second, as mentioned above many proteins experience post-translational modifications that profoundly affect their activities; for example some proteins are not active until they become phosphorylated. Methods such as phosphoproteomics and glycoproteomics are used to study post-translational modifications. Third, many transcripts give rise to more than one protein, through alternative splicing or alternative post-translational modifications. Fourth, many proteins form complexes with other proteins or RNA molecules, and only function in the presence of these other molecules. Finally, protein degradation rate plays an important role in protein content.[10]
One way in which a particular protein can be studied is to develop an antibody which is specific to that modification. For example, there are antibodies which only recognize certain proteins when they are tyrosine-phosphorylated, known as phospho-specific antibodies; also, there are antibodies specific to other modifications. These can be used to determine the set of proteins that have undergone the modification of interest.
For sugar modifications, such as glycosylation of proteins, certain lectins have been discovered which bind sugars. These too can be used.
A more common way to determine post-translational modification of interest is to subject a complex mixture of proteins to electrophoresis in "two-dimensions", which simply means that the proteins are electrophoresed first in one direction, and then in another, which allows small differences in a protein to be visualized by separating a modified protein from its unmodified form. This methodology is known as "two-dimensional gel electrophoresis".[11]
Recently, another approach has been developed called PROTOMAP which combines SDS-PAGE with shotgun proteomics to enable detection of changes in gel-migration such as those caused by proteolysis or post translational modification.
Classically, antibodies to particular proteins or to their modified forms have been used in biochemistry and cell biology studies. These are among the most common tools used by practicing biologists today.
For more quantitative determinations of protein amounts, techniques such as ELISAs can be used.
For proteomic study, more recent techniques such as matrix-assisted laser desorption/ionization (MALDI)[11] have been employed for rapid determination of proteins in particular mixtures and increasingly electrospray ionization (ESI).
Computational predictive models[12] have shown that extensive and diverse feto-maternal protein trafficking occurs during pregnancy and can be readily detected non-invasively in maternal whole blood. This computational approach circumvented a major limitation, the abundance of maternal proteins interfering with the detection of fetal proteins, to fetal proteomic analysis of maternal blood. Computational models can use fetal gene transcripts previously identified in maternal whole blood to create a comprehensive proteomic network of the term neonate. Such work shows that the fetal proteins detected in pregnant woman’s blood originate from a diverse group of tissues and organs from the developing fetus. The proteomic networks contain many biomarkers that are proxies for development and illustrate the potential clinical application of this technology as a way to monitor normal and abnormal fetal development.
An information theoretic framework has also been introduced for biomarker discovery, integrating biofluid and tissue information.[13] This new approach takes advantage of functional synergy between certain biofluids and tissues with the potential for clinically significant findings not possible if tissues and biofluids were considered individually. By conceptualizing tissue-biofluid as information channels, significant biofluid proxies can be identified and then used for guided development of clinical diagnostics. Candidate biomarkers are then predicted based on information transfer criteria across the tissue-biofluid channels. Significant biofluid-tissue relationships can be used to prioritize clinical validation of biomarkers.
Most proteins function in collaboration with other proteins, and one goal of proteomics is to identify which proteins interact. This is especially useful in determining potential partners in cell signaling cascades.
Several methods are available to probe protein–protein interactions. The traditional method is yeast two-hybrid analysis. New methods include protein microarrays, immunoaffinity chromatography followed by mass spectrometry, dual polarisation interferometry, Microscale Thermophoresis and experimental methods such as phage display and computational methods
One of the most promising developments to come from the study of human genes and proteins has been the identification of potential new drugs for the treatment of disease. This relies on genome and proteome information to identify proteins associated with a disease, which computer software can then use as targets for new drugs. For example, if a certain protein is implicated in a disease, its 3D structure provides the information to design drugs to interfere with the action of the protein. A molecule that fits the active site of an enzyme, but cannot be released by the enzyme, will inactivate the enzyme. This is the basis of new drug-discovery tools, which aim to find new drugs to inactivate proteins involved in disease. As genetic differences among individuals are found, researchers expect to use these techniques to develop personalized drugs that are more effective for the individual.
The FDA defines a biomarker as, “A characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”.
Understanding the proteome, the structure and function of each protein and the complexities of protein–protein interactions will be critical for developing the most effective diagnostic techniques and disease treatments in the future.
An interesting use of proteomics is using specific protein biomarkers to diagnose disease. A number of techniques allow to test for proteins produced during a particular disease, which helps to diagnose the disease quickly. Techniques include western blot, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA) or mass spectrometry.[11][14] Secretomics, a subfield of proteomics that studies secreted proteins and secretion pathways using proteomic approaches, has recently emerged as an important tool for the discovery of biomarkers of disease.[15]
In what is now commonly referred to as proteogenomics, proteomic technologies such as mass spectrometry are used for improving gene annotations. Parallel analysis of the genome and the proteome facilitates discovery of post-translational modifications and proteolytic events [16], especially when comparing multiple species (comparative proteogenomics). [17]
Fluorescence two-dimensional differential gel electrophoresis (2-D DIGE)[18] can be used to quantify variation in the 2-D DIGE process and establish statistically valid thresholds for assigning quantitative changes between samples.[19]
Comparative proteomic analysis can reveal the role of proteins in complex biological systems, including reproduction. For example, treatment with the insecticide triazophos causes an increase in the content of brown planthopper (Nilaparvata lugens (Stål)) male accessory gland proteins (Acps) that can be transferred to females via mating, causing an increase in fecundity (i.e. birth rate) of females.[20] To identify changes in the types of accessory gland proteins (Acps) and reproductive proteins that mated female planthoppers received from male planthoppers, researchers conducted a comparative proteomic analysis of mated N. lugens females.[21] The results indicated that these proteins participate in the reproductive process of N. lugens adult females and males.[22]
Proteome analysis of Arabidopsis peroxisomes[23] has been established as the major unbiased approach for identifying new peroxisomal proteins on a large scale.[24]
There are many approaches to characterizing the human proteome, which is estimated to contain between 20,000 and 25,000 non-redundant proteins. The number of unique protein species likely increase by between 50,000 and 500,000 due to RNA splicing and proteolysis events, and when post-translational modification are also considered, the total number of unique human proteins is estimated to range in the low millions.[25][26]
In addition, first promising attempts to decipher the proteome of animal tumors have recently been reported.[11]
|
|||||