Dems Sars Hbv Hcv and HIV1

3.1.1. Description

The classical proteomics platforms include 2DE and MS [85]. 2DE is employed to separate proteins in a mixture in the first dimension by their isoelectric points and then in the second dimension by molecular mass. The resulting gel can be stained with a variety of protein dyes to reveal a pattern of spots. In the first dimension, isoelectric focusing (IEF) is performed by using IPG strips which are based on the use of bifunctional immobiline reagents, a series of 10 chemically well-defined acrylamide derivatives that copolymerize with the acrylamide matrix, to generate extremely stable pH gradients forming a series of buffers with different pK values between 1 and 13. Subsequently, linear or nonlinear wide (IPG 3-12), medium (IPG 4-7), narrow (IPG 4.5-5.5), and ultra-narrow (IPG 4.9-5.3) pH range IPGs can be cast [95]. We suggest the reader to refer to the review by Gorg et al. for more details about IPG strip rehydration, sample application, and IPG strip equilibration [96]. The second dimension consists of using SDS-PAGE to separate proteins according to their molecular weight. However, the analysis of low-molecular-weight (<15 kDa) and high-molecular-weight (>150 kDa) proteins is challenging since there is no standard 2DE system that effectively allows separation of proteins over the entire range between 5 and 500 kDa. A common approach is to combine several gels optimized for different molecular weight ranges instead of using a single standard 2DE system.

3.1.2. Application for virus studies

Current methods for the diagnosis of HCC rely on serological markers such as a-fetoprotein (AFP) [97] and certain liver enzymes as well as Des gamma car-boxyprothrombin (DCP) [98]. This type of diagnosis lacks the sensitivity to detect HCC at an early stage when therapy can be more effective. To find markers of disease progression, 2DE was employed to resolve and compare proteins present in serum obtained from individuals infected with HBV or HCV and with varying risks for the development of HCC [99,100]. In several studies, proteins expressed at different levels among diseased individuals as compared to those of healthy ones were identified as markers for disease progression as well as proteins with different N-glycosylation patterns [99-101]. In another study, 2D-MS was also employed to analyze altered plasma proteins due to SARS-CoV infection. Thirty-eight different plasma proteins from SARS patients were identified, most of which were associated with acute phase proteins [23].

3.1.3. Advantages

One advantage of 2D gels is their resolution since they can resolve as many as 2000 proteins simultaneously and proteins can be detected at greater than 1 ng in one spot [96]. 2DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. In addition, 2DE produces a map of intact proteins, which reflects changes in protein expression level, different isoforms, or PTM. In fact, a great advantage of this methodology is its capability to study proteins that have undergone some form of PTM (such as phosphorylation, glycosylation, or limited proteolysis) that can be detected visually on the 2DE gels as they appear as distinct spot trains in the horizontal and/or vertical axis of the 2DE gel. This is in contrast with other methods, including LC-based methods, which perform analysis on peptides, where molecular weight and pI information is lost, and stable isotope labeling is required for quantitative analysis [96].

3.1.4. Drawbacks

Although 2D gel electrophoresis is a standard technology, it suffers from several problems that may limit its utility. These include issues with reproducibility, as well as the inability to separate hydrophobic proteins, which are poorly soluble. Although the use of IPG strips increases the reproducibility of 2DE, various problems with 2D separation remain such as streaking, poor focusing, and the variable occurrence of gaps [85]. Although 2DE allows for high resolution of individual spots, a single spot may not correspond to a single protein, since proteins can comigrate as a single spot on a 2D gel [102]. Furthermore, 2DE requires milligram quantities of protein, reflecting the low sensitivity of this method. To further enhance the utility of 2DE-MS, enrichment of samples for low-abundance proteins by improved methods is required. Enrichment can include prefractionation of samples, as well as more sensitive detection and quantitation methods, or the use of alternative methods including laser capture microdissection [103] for heterogeneous tissues. In most of the studies mentioned previously, the resolution problem was overcome by narrowing the pH range allowing for greater focusing. However, the reduced pH range in IEF can lead to the elimination of a large number of proteins that may be informative. Comparing hundreds of protein spots across gel images taken from a large number of different samples is extremely time-consuming, even with specialized software. For this reason, although 2D electrophoresis is a promising tool, it is not very practical for clinical application. The challenge is to develop this technique into a system capable of automation, high throughput, and high sensitivity.

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