Proteomics is a functional science, the main subject of study of which is the proteome. A proteome is a whole set of proteins that are produced or modified by an organism or system. Proteomics is a science that studies the types of protein, and therefore it has helped to discover many new species of this compound - much more than was known before its emergence as a science. The amount of protein, as it turned out, depends on time and the various requirements or stresses that cells or organisms are exposed to. Proteomics is an interdisciplinary field that is largely predetermined by the latest genome research projects. It covers the study of proteomes from a general level of protein composition, structure, and activity. Functional proteomics is often called the most important component of functional genomics.
Subject of study
Defining proteomics is not as easy as it might seem at first glance. This science usually involves large-scale experimental analysis of proteins and proteomes, but is often used to study the possibilities of protein purification.
After genomics and transcriptomics, proteomics is the next step in the study of biological systems. It is much more complicated than genomics, because the genome of the body is more or less constant, while the proteome differs from cell to cell from time to time. Individual genes are expressed in different types of cells, which means that even the basic set of proteins that are produced in the cell must be identified.
Study history
Proteomics of studying the structure of proteins is a recent trend in biochemistry. In the past, protein studies were carried out using RNA analysis, but it turned out that the structure of RNA does not correlate with the protein content. It is known that mRNA is not always translated into protein, and the amount of protein produced for a given amount of mRNA depends on which gene is transcribed, as well as on the current physiological state of the cell. Proteomics is a science that confirms the presence of protein and provides a direct estimate of the amount present.
Subsequent changes
Not only does extracting the protein from mRNA cause damage, but in addition, many proteins also undergo a wide range of chemical modifications after this process. Many of these post-translational modifications are crucial for protein function.
Phosphorylation
One of these modifications is phosphorylation, which occurs with many enzymes and structural proteins during cell signaling. The addition of phosphate to certain amino acids, most often serine and threonine, mediated by serine / threonine kinases, or more rarely tyrosine mediated tyrosine kinases, causes the protein molecule to become a target for binding or interaction with a different set of other molecules that recognize the phosphorylated domain.
Since protein phosphorylation is one of its most studied modifications, many “proteomic” efforts are aimed at determining the set of phosphorylated proteins in a particular cell or tissue type under certain circumstances.
Ubiquitination
Ubiquitin is a small protein that can be attached to specific substrates by enzymes scientifically called E3 ubiquitin-ligases. Determining which proteins are poly-ubiquitinated helps to understand how the movement of the molecules of this substance is regulated. Similarly, once a researcher determines which substrates are ubiquitinated by each ligase, it is useful to determine the set of ligases expressed in a particular cell type.
Additional changes
In addition to phosphorylation and ubiquitination, proteins can be subjected (in particular) to methylation, acetylation, glycosylation, oxidation and nitrosylation. Some proteins undergo all of these changes, often in time-dependent combinations. This illustrates the potential complexity of studying the structure and function of a protein.
Individual proteins are produced under different conditions. A cell can create different sets of proteins at different times or under different conditions, for example, during development, cell differentiation, cell cycle or carcinogenesis. A further increase in proteome complexity, as already mentioned, implies that most proteins can undergo a wide range of post-translational modifications.
Therefore, research in the field of proteomics is a difficult task in the future, even if the topic of studying this science will still be limited. For more ambitious tasks, such as when looking for a biomarker for a particular cancer subtype, a proteomist scientist may choose to study several serum samples from several cancer patients to minimize confounding factors. Thus, complex experimental constructs are sometimes necessary to account for the dynamic complexity of the proteome.
Differences from Genomics
Proteomics provides different levels of understanding than genomics for many reasons:
- The level of gene transcription gives only a rough estimate of its level of translation into protein. The mRNA obtained in abundance can be quickly degraded or transformed not in the most efficient way, as a result of which a small amount of protein is formed.
- As mentioned above, many proteins experience post-translational modifications that greatly affect their functionality. For example, some proteins are not active until they become phosphorylated. Methods such as phosphoproteomics and glycoproteomics are used to study post-translational modifications.
- Many transcripts lead to the appearance of more than one protein, through alternative splicing or alternative post-translational modifications.
- Many proteins form complexes with other proteins or RNA molecules and act only in the presence of these other molecules. The degree of protein degradation plays an important role in its content.
Reproducibility
One of the main factors affecting the reproducibility of experiments in proteomics is the simultaneous elution of many other peptides that can be measured by mass spectrometers. This leads to stochastic differences between the experiments due to tryptic peptides dependent on the given methods. Although early large-scale analyzes of the yeast proteome showed significant variability in the results between different laboratories, apparently due in part to technical and experimental differences between them, reproducibility was improved in later mass spectrometric analysis, especially when using mass spectrometers.
Research methods
In proteomics, there are many methods for studying proteins. As a rule, they can be detected using antibodies (immunological analyzes) or using mass spectrometry. If you are analyzing a complex biological sample, you must either use a very specific antibody in a quantitative blot analysis (qdb) or biochemical separation.
Detection of protein using antibodies (immunological assays)
Antibodies to specific proteins or their modified forms have been used in biochemistry and cell biology studies. They are among the most common tools used today by molecular biologists. There are several specific methods and protocols that use antibodies to detect protein. For decades, an enzyme-linked immunosorbent assay (ELISA) has been used to detect and quantify biological samples. Western blot can be used to detect and quantify individual proteins, where at the initial stage a complex organic mixture is separated using SDS-PAGE, and then the protein of interest is identified using antibodies.
Modified proteins can be studied by developing antibodies specific for this modification. For example, there are antibodies that only recognize certain proteins when they are tyrosine phosphorylated, known as phosphospecific antibodies. In addition, there are antibodies specific for other modifications. They can be used to determine the set of proteins that have undergone modification.
Proteomics in medicine
The detection of diseases at the molecular level is the driving force behind the new revolution in diagnosis and treatment. Digital immunoassay technology has improved the detection sensitivity of molecules to the so-called attomolar range. This opportunity gives us the potential to discover new advances in diagnostics and therapy, but such technologies have been categorized as manual procedures that are not well suited for effective daily use.
Although the detection of protein with antibodies is still very common in molecular biology, other methods have been developed that do not rely on the antibody. These methods offer various advantages, for example, they can often determine the sequence of a protein or peptide, they can have a higher throughput than an antibody, and sometimes they can identify and quantify proteins for which there are no antibodies.
Proteomics methods
One of the earliest protein analysis methods was Edman degradation (introduced in 1967), where a single peptide undergoes several stages of chemical degradation to determine its sequence. These methods have largely been superseded by technologies that provide higher throughput. Different directions of proteomics depend on methods.
Basic separation methods
The analysis of complex biological samples requires a reduction in their complexity. This can be done using one-dimensional or two-dimensional separation. More recently, online methods have been developed in which individual peptides were separated using reverse phase chromatography and then directly ionized using the ESI method.
Hybrid technology
There are several hybrid technologies that use the purification of individual antibody-based analytes and then perform mass spectrometric analysis to identify and quantify them. Examples of these methods are the MSIA (mass spectrometric immunoassay) method developed by Randal Nelson in 1995 and the SISCAPA (stable isotopic standard capture with antipeptide antibodies) method introduced by Lee Anderson in 2004.
Comparative proteomic analysis can reveal the role of proteins in complex biological systems, including reproduction. For example, triazophosome insecticide treatment leads to an increase in the content of brown seedlings (Nolaparvata lugens (Stål)) - male auxiliary iron proteins (Acps), which can be transmitted to females through mating, which leads to an increase in the fertility (i.e., fertility) of women. To identify changes in the types of auxiliary gland protein (Acps) and reproductive proteins obtained from male grasshoppers, the researchers conducted a comparative proteomic analysis of sleeping males of the species N. lugens. The results showed that these proteins are involved in the reproductive process of adult females and male grasshoppers of N. lugens.
High performance proteomics
Proteomics is a science that has steadily gained momentum over the past decade. Many of the approaches developed by this science are absolutely revolutionary, while some are based on old scientific methods. Methods based on mass spectrometry and microcells are the most common technologies for large-scale study of proteins.
Mass spectrometry and profiling
Currently, two methods of mass spectrometry are used for protein profiling. The more well-known and widespread method uses high-resolution two-dimensional electrophoresis to separate proteins from different samples in parallel with the subsequent selection and staining of differentiated expressed proteins that must be identified by mass spectrometry. Despite the achievements in 2DE and the overall sophistication of this method, it also has its limits. The main problem is the inability to identify all the proteins in the sample, given their variability and other unique properties.
The second quantitative approach uses stable isotope labels for differentiated protein labels from two different complex mixtures. Here, proteins in a complex mixture are first labeled with isotopes and then cleaved to produce labeled peptides. Then the labeled mixtures are combined, and the peptides are separated by multivariate liquid chromatography and analyzed using tandem mass spectrometry. Isotope Coded Labels (ICATs) are commonly used isotope labels. In this scientific method, cysteine residues of proteins are covalently attached to the ICAT reagent, thereby reducing the complexity of mixtures excluding residues other than cysteine.
Proteomics, genomics, metabolomics - new areas in biology, characterized by complexity and innovativeness. Not everyone can study them.