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With the introduction of the concept of proteomics, two-dimensional electrophoresis (2D gel electrophoresis) once became very popular. In recent years, due to the rapid development of mass spectrometry, the popularity of LC-MS seems to be higher. However, in some applications, two-dimensional electrophoresis is more advantageous.
Two-dimensional electrophoresis provides a bird's eye view of the entire sample, which is unmatched by mass spectrometry. It can separate the complex proteins in the sample in a holistic manner. It is currently the only method that can simultaneously separate thousands of proteins on a gel, and the purity of the separated protein components can reach more than 90%. With it, you can study and compare how the proteome changes under certain conditions.
In addition, two-dimensional electrophoresis separates intact proteins, and most mass spectrometry methods study peptides. Therefore, it is difficult for researchers carrying out mass spectrometry to determine post-translational modification events, such as whether two different post-translational modifications exist at the same time or are mutually exclusive. Of course, the cost and equipment requirements of these two technologies cannot be the same. Compared with the mass spectrometer, the equipment of two-dimensional electrophoresis is nothing special. Moreover, the mass spectrometer also requires specialized personnel to operate, without some experience can not dare to rush.
Of course, the repeatability of two-dimensional electrophoresis is also an unavoidable problem. However, with the continuous development of technology, researchers can also overcome this challenge and publish beautiful results.
Down Syndrome Study
Tracey Madgett, a senior researcher at the University of Plymouth in the United Kingdom, used two-dimensional gel electrophoresis to identify the biomarkers of Down Syndrome1. According to Madgett, the decision to use electrophoresis is based on scientific and practical considerations. First, others have used two-dimensional electrophoresis to study Down syndrome. More importantly, they have two-dimensional electrophoresis equipment, but lack the experience of LC-MS.
Madgett and colleagues studied pregnant women in the first trimester or 4-6 months of pregnancy, who are pregnant with a normal fetus or a fetus with Down syndrome. The researchers took plasma from them and processed it to carry out two-dimensional gel electrophoresis.
After each sample was processed, it was run alone, stained and analyzed. However, the differences between the gels cannot be avoided. This makes it difficult to distinguish differentially expressed proteins by comparing the spot intensity on the gel. The same position on the two pieces of glue may be two different proteins at all. How does this compare?
To overcome this problem, GE Healthcare developed a multiple analysis technique called fluorescent differential gel electrophoresis (2D DIGE) about ten years ago. The basic experimental circuit of 2D DIGE is to label protein samples with Cy2, Cy3 and Cy5 fluorescent dyes (including a protein internal standard) before electrophoresis. The labeled protein samples are then mixed and subjected to two-dimensional electrophoresis on the same gel. The electrophoresis results used three different excitation wavelengths to obtain fluorescent signals of different colors, and the difference in protein between samples was determined according to the ratio of these signals.
For the first time, 2D DIGE technology introduced an internal standard in two-dimensional electrophoresis. The internal standard of DIGE is to mix all the samples in the test in equal amounts, label it with a fluorescent dye (usually Cy2 in the smallest labeling dye), and run it with all samples. This means that protein spots in all samples will have corresponding internal standards. Since all samples undergo the same treatment during electrophoresis, researchers can accurately identify differences in protein abundance under different conditions; they only need to collect three fluorescent images and compare them using data analysis software.
Madgett and her research team used 2D DIGE to carry out biomarker analysis of Down syndrome. The researchers first used QIAGEN's Qproteome Albumin / IgG Depletion Kit to remove two high-abundance proteins, albumin and IgG. They then ran gel on three 24 cm 2D gels. The difference between the three gels was the pH during the first isoelectric focusing. The first block covers pH 4.5-5.5, the second block covers pH 5.3-6.5, and the third block covers pH 6-9. The researchers believe that the narrow range has a higher resolution than the wide range of IPG strips (such as pH 3-10).
Madgett said that through this analysis, they found 7 "promising clues" in the plasma of 4-6 months. They cut these spots and performed various mass spectrometric analyses to identify these proteins. Madgett believes that they also need to verify these clues in a larger cohort. Speaking of 2D DIGE, she said: "It's complicated, but in the end I got the results, I'm very excited. I will do it again."
New tools
As a classic technique, two-dimensional electrophoresis is still evolving. Although people want to replace it with new technology, if you want to have a comprehensive understanding of the proteome, other technologies still have a gap in resolution and sensitivity from two-dimensional electrophoresis. In recent years, from sample preparation to data analysis, there have been many new products available.
Sample Preparation
For two-dimensional electrophoresis, sample preparation is the key. Isoelectric focusing is particularly sensitive to contaminants (such as nucleic acids and lipids), so if you want to get a beautiful electropherogram, you must first remove the contaminants. Many companies provide this sample purification kit. GE Healthcare's 2-D Clean-Up Kit can efficiently remove contaminants such as salts, lipids and nucleic acids. The QIAGEN kit mentioned above can also remove high-abundance proteins.
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Sometimes, simply removing high-abundance proteins does not solve the problem. In this case, you may need to further reduce the sample complexity. If you focus on specific parts of the proteome, then you can separate complex proteins in steps, such as separating nuclear, cytoplasmic, and membrane proteins, or isolating phosphorylated or glycosylated proteins. Merck Millipore provides this step-by-step separation kit.
In addition, Life Technologies also provides a ZOOM® IEF Fractionator, which can quickly concentrate the protein to 5 isoelectric points. Its principle and operation are very simple, that is, placing a disk between the ZOOM® IEF Fractionator sample chamber (chamber) can be separated in a specific pH range. For example, if you want to separate proteins with a pH range between pH 4.6 and pH 5.4, just connect a sample tank separated by pH 4.6 and pH 5.4 Disk. This liquid-phase isoelectric focusing device facilitates sample recovery, and the Disk is disposable, reducing the possibility of cross-contamination. Bio-Rad also provides a similar device, called Rotofor Cell, with various specifications, but it seems to be slightly more complicated to use and more expensive.
Isoelectric focusing
In two-dimensional electrophoresis, in order to obtain the best electrophoresis results, we must continuously optimize the conditions of sample preparation and first-way isoelectric focusing to ensure that complex samples can be fully separated. The optimal IEF conditions for different protein samples may be very different, so they must be explored separately.
Most IEF systems on the market have multiple channels, but the conditions of each channel are the same, so only one condition can be explored at a time. The traditional IEF slot divides the total current into all channels on average. Unless all the samples have the same conductivity, the current cannot be evenly distributed. In the end, the current on each strip may not reach or exceed the ideal current. This may cause underfocusing or overfocusing.
The Bio-Rad's new PROTEAN i12 IEF system has 12 independent channels, and each channel has its own power supply. In this way, the system can simultaneously run different sample types, different focusing conditions and different pH range of the strip without worrying that different samples will affect each other. This unique ability allows you to optimize experiments in a shorter period of time, improve experiment efficiency, and obtain stable results. For laboratories with multiple two-dimensional electrophoresis topics, this is indeed good news.
GE Healthcare's Ettan 2D system also has two new members: IPGbox â„¢ and IPGbox Kit. The design of IPGbox has been improved, the base and the upper cover can be separated, more friendly and stable, and the opaque cover allows hydration in the dark, which is completely suitable for the light sensitive CyDye DIGE fluorescent dye. The design of the upper cover and the insert can ensure that the rubber strip will not dry out during the hydration process, and there is no need to cover the oil. At the same time, the single-use hydration tray and inserts also ensure that there is no cross-contamination between samples. After the hydration tray is redesigned, the highly hydrophobic material ensures that the swelling fluid will not adhere to the tray, and the hydration fluid is evenly distributed throughout the tray, ensuring the consistency of IPG strip hydration.
dyeing
For the staining of 2D gels, silver staining is a more common method, but it has many steps, long process and complicated operation. The emergence of SYPRO Ruby fluorescent dye is known as a milestone in proteome research. Its sensitivity is comparable to the best silver staining method (1 ~ 2 ng), the linear dynamic range can reach 3 orders of magnitude, and it has good compatibility with mass detection and Edman sequencing. SYPRO Ruby can detect protein in one step, it only takes 30 ~ 60 min, no destaining step is needed, which is quite convenient.
At present, many companies provide sample preparation kits, IPG strips, IEF electrophoresis devices, and second-way gel running devices, including GE Healthcare, Bio-Rad, Life Technologies, etc. But for newcomers, they may struggle with two issues.
Which adhesive strip to choose?
IPG strips are available in multiple pH ranges, from wide to narrow. For example, Bio-Rad's IPG strips cover "wide" (3-10), "narrow" (3-6, 5-8, 4-7) and "narrower" (3.9-5.1, 4.7-5.9, 5.5 -6.7, 6.3-8.3) pH range. It is recommended to perform a narrow range analysis after the wide range analysis is completed. If you are studying an unknown sample, a wide pH range will bring you a sample overview. After you know the approximate location of the protein of interest, it is recommended to use a narrower or narrower pH gradient.
What size glue to choose?
How much glue to choose is also a question. The 2D gel sizes of various companies vary from 9 cm x 7 cm to 24 cm x 20 cm, and the length of the IPG strips also ranges from 7 cm to 24 cm. In general, the larger the gel, the longer the running time, but the higher the resolution. On the other hand, too large glue is difficult to handle. This is like the front and back of a coin, it is difficult to choose. However, if you have confidence in your physical strength, then bravely use big glue.
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