Comparison of proteomic technical analysis methods and their instruments
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1 Proteomics Overview
The word "proteome" in English is Proteome, which is a combination of the words proteins and genome, meaning proteins expressed by a genome, which is the protein expressed by the genome [1] . The concept of the proteome was first proposed by Wilkins in 1994 and first published in the July 1995 issue of "Electrophoresis", referring to "all proteins expressed by a cell or a tissue genome" [2] , proteomics It is to study the composition and activity of proteins in cells from the overall level. Unlike the gene-fixed genome, the proteome as a product expressed by the corresponding genome changes with time, place, environment and other conditions. In different tissues and different cells of the same body, the types and quantities of proteins are different; even if the same tissue or cells are in different developmental stages, physiological states, or even different external environments, their proteomes are constantly changing; It is also different from normal physiological processes during pathology or treatment. Therefore, the proteome is a dynamic concept. The purpose is to analyze the dynamic changes of protein composition, expression level, and modification status in the body from a holistic perspective, to understand the interactions and connections between proteins, and to reveal the laws of protein function and life activities. Proteomics research is divided into three aspects: (1) Micro-characteristics of large-scale protein identification and post-transcriptional modification. (2) Differences show that proteomics, the study of protein expression levels, has broad prospects for the application of diseases such as tumors. (3) Studies on protein-protein interactions and post-translational modifications [3] . Analysis of the expression of different proteins can be used to compare the differences between normal and tumor tissues. Proteomics will be a marker for identifying diseases, and a mechanism can be elucidated that is being applied in more and more analyses. The human genome is much smaller than expected, and the tumor-associated genes in the genome project are now known, whereas smaller genes do not reflect a single proteome. In general, extensive post-translational modifications such as phosphorylation, glycosylation, and proteolytic processing are very common methods. Protein post-translational modifications can significantly alter the function of a protein and thus can express cellular and tissue characteristics. Therefore, one of the challenges of proteomics in the genome is to understand tissue characteristics through knowledge of protein effectors and apply it to the clinic.
2 proteomics analysis method
Proteomics can be analyzed by protein microarrays, electrophoresis and mass spectrometry for detection, identification and signature of proteins. These methods have their unique advantages and limitations, and the proteomic profiles are evaluated according to their respective abilities.
2.1 protein micro-column technology
Protein microarrays are a large number of antibodies or a large number of tissue protein samples once labeled on a glass slide for detection and analysis. This method is capable of detecting the presence of large amounts of protein or the level of expression of a large number of tissue samples, but this technique is limited in the availability of specific and sensitive antibodies. Furthermore, the specificity of the antibody must be confirmed by immunoblotting and requires an internal control, especially the microarray of the antibody has no predicted affinity and specificity. Despite this, the use of a large number of commercially available antibodies makes it possible to apply protein microarrays.
2.2 Two-dimensional gel electrophoresis
Two-dimensional electrophoresis was invented by O'Farrell in 1975. The principle is that the first-direction protein-based isoelectric point is different and is separated by isoelectric focusing. The second direction is different by molecular mass using sodium lauryl sulfate-polymerization. Acrylamide gels separate proteins in complex protein mixtures on a two-dimensional plane. This method is especially useful for proteins with similar molecular weights. Using a proteome contig, using multiple 2-DE maps with different pH gradients and overlapping molecular weights, splicing into a complete 2-DE map, greatly improving resolution and injection volume, which is useful for low-abundance proteins. Checking out is very beneficial. Individual proteins can be stained and hydrolyzed into peptides, which can be analyzed by mass spectrometry. The enzymatic map of the peptide can be analyzed from a database of proteins.
2.3 Mass spectrometry
One of the main tools of proteomics is mass spectrometry. In this method, after the gene is converted into a gas ion, the protein is analyzed according to the ratio of the feed. Solubilization and ionization techniques such as matrix-assisted laser desorption ionization provide a high level of sensitivity and precision for the detection and resolution of proteins. The high sensitivity of this technology and the simplification of the sample facilitate this technology. , but it also has limitations. Analysis of complex samples such as serum is much more difficult than detecting proteins.
Comparison of 3 proteomic analytical instruments
method | principle | advantage | Disadvantage |
Electrophoresis | The water-soluble protein moves from the negative electrode to the positive level according to the principle of the electric field, and the rate of movement depends on its charge, size and shape. | The gel is definitely stained before the protein is recognized. Gel staining itself is useless. Without the use of another detection technique, such as immunoblotting or mass spectrometry techniques, proteins cannot be accurately identified. | |
SDS-PAGE polyacrylamide gel electrophoresis | The protein migrates through an inert polyacrylamide gel. The pore size can be adjusted to block the protein. SDS is a negatively charged detergent. It can unfold the molecular chain of the protein and become a free molecule. The protein migrates to the positive electrode at different rates. | Isolation of all types of proteins, even those that are not water soluble. | The separation of one-dimensional separation methods is limited. Close to the tendency to overlap, only small molecular weight proteins can be solved. |
Two-dimensional gel electrophoresis | The first phase electrophoresis is performed by isoelectric focusing electrophoresis in a p H gradient gel depending on the isoelectric point of the protein. The second phase electrophoresis performs protein separation by gel electrophoresis (SDS-PAGE) according to the molecular weight of the protein according to the principle of protein isoelectric focusing. | Compared with a variety of protein gel electrophoresis, this method has a high resolution of separation mixture, which is convenient according to image analysis, and the protein is decomposed by about 1 ng per ml. | High-abundance proteins may be ambiguous (eg albumin, immunoglobulin). Proteins with low-abundance proteins need to be removed from the gel, digested, analyzed by mass spectrometry, and it cannot distinguish small molecule weights. Protein (<10,000 Da). Can not comply with the changing analysis. |
Two-dimensional fluorescence differential gel electrophoresis | Two-dimensional electrophoresis uses different cyan fuels to label proteins from different samples, and enzyme-multiplied immunoassay techniques occur at different wavelengths of light. Three samples can be labeled in mixed samples (test, control, reference) | The method of analyzing different proteins from a mixture is simple. The rate of protein expression can be obtained in a single gel. The change in the change between each gel or different gels is very sensitive by an internal standard. | The presence of high-abundance proteins may be ambiguous (eg albumin, immunoglobulin). Low-abundance protein-separating proteins ultimately require the removal of spots from the gel, digestion, and analysis of the peptide by mass spectrometry. Many spots are indistinguishable because of the material. The lack of it, it can not distinguish small molecular weight proteins (<10,000 Da). |
Protein chip, multi-protein chip, cytokine chip, tissue micro-column | Known proteins are localized to one surface (beads, nitrocellulose) and are detected using the principles of immunoassays. | High sensitivity and throughput, multiple analytes can be measured simultaneously. | The validity and specificity of a limited antibody requires some knowledge of previous protein expression. Analytical subtypes may not be detected. |
Matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry | Determine the precise accumulation of proteins or peptide fragments. The protein or peptide fragment is a mixture of organic acid interstitials, dried on a metal glass slide, and laser ionization destroys the peptide fragment, which allows movement in the direction of the detector in the electric field. The time to reach the detector depends on the charge and shape. The genetic information of the peptides can be obtained by mass spectrometry techniques. | The largest measured molecular weight <3000Da | |
Protein chip surface enhanced laser desorption ionization-time-of-flight-mass spectrometry | Compared with matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry, the difference is that it uses chromatographic techniques to selectively bind protein subtypes from complex samples. Flushing removes non-specific condensation proteins and other substances that can interfere with the progress of ionization. (salt, detergent, etc.) | High-throughput virus immunoassay automated; sample size is minimal; preliminary separation of raw materials can enhance the detection rate of low-abundance proteins. | It is not possible to distinguish proteins directly. Not sensitive to high molecular weight proteins (molecular weight > 20 kDa). |
Stable isotope labeling | Biological samples use different stable isotopes that use a tone change agent to target a specific amino acid. After separation and mass spectrometry, the peptides of two different samples are used in specific isotopes of different mass units, and relative quantification can be obtained. | Proteomics is more popular than other methods. Metric information is available in a large number of proteins. Identification data signals for related proteins are usually obtained. | High technical requirements, low processing capacity. Samples were trypsinized prior to testing. This reliable method is suitable for detecting large amounts of protein. |
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