Proteomics/Protein Chips/Types
Types:
Types
[edit | edit source]The two main types of protein chips are analytical and functional. With analytical protein chips, the proteins being studied are in the solution that is washed over the chip.[1] Analytical chips are primarily used to identify the contents of an analyte. With functional protein chips, the proteins being studied are attached to the chip.[1] Functional chips are primarily used to study interactions between the protein of interest and other molecules.
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Example of an analytical protein chip. |
Example of a functional protein chip. |
Analytical
[edit | edit source]Analytical chips are classified according to the capture molecule that is affixed to the chip. The molecule can be very specific as to the types of proteins it binds to. Examples of these specific molecules include antibodies, antigens, enzymatic substrates, nucleotides and other proteins. Analytical chips can also contain molecules that bind to a range of proteins. These molecules are similar to the ones used in liquid chromatography. The techniques include reverse phase, cation exchange and anion exchange.[1]
Reverse phase protein chips, also known as reverse phase protein arrays (RPA), are related to analytical microarrays and are used to identify different levels of expression of proteins.[2] RPAs have become well used to the point where they may even be considered a separate type of protein chip all together. RPAs are high-throughput technology that involves the use of two pre-existing technologies known as laser capture microdissection (LCM) and microarray fabrication. LCM visualizes stained tissue cells of interest under a microscope in real time. Once visualized, the cells are then isolated and lysed and placed into the spots of the microarray. An antibody that can be detected by a fluorescent is used to probe the slides. RPA allows for the protein to be immobilized in order to be analyzed instead of the typical protein microarrays which immobilizes the antibody probe. This is how it got the name ‘reverse phase’. This process allows that the protein need not be labeled since the protein lysate has already been denatured.[3]
Advantages of RPAs over other types include the ability to run different test samples in each individual array spot and only require a single antibody to probe an entire array slide.[4] This use of a single antibody eliminates the need to run multiple analytes, and instead, a single one is measured and then compared to the different test samples that were applied to the individual spots.[5] Use of Reverse Phase Protein Microarrays and Reference Standard Development for Molecular Network Analysis of metastatic Ovarian Carcinoma. Molecular & Cellular Proteomics, 4(4), 346-355. This process is optimal for cell populations with low cell count due to its ability to run a single analyte for a greater number of spots in the array, for example, all of the proteins present in the cell.
A major use of RPAs is to view the different stages of progressing cancer along with studying signaling transduction pathways. They can be used to determine different activation statuses of proteins over a set amount of time or due to certain treatment conditions. [2]
Functional
[edit | edit source]Unlike analytical chips, there is only one type of functional chip. Functional chips are used to discover additional information and properties about a particular protein. These properties include binding strength, biochemical functions and protein-protein interactions.[1]
The major methods used to characterize an organism's proteome often result in the denaturing of the sample thus ruling out any functional studies. Current functional analysis methods are mostly in vivo techniques which have inherent variabilities.[6] The benefits of functional analysis using these chips is that proteins can be identified and studied in vitro while they are still biochemically active and in their multimeric complexed form.
There are many challenges when developing a functional protein array including creating an expression clone library, actual protein production which includes isolation and purification, adaptation of microarray technology, stabilizing the proteins on the array, and keeping the concentrations of the protein constant between slides as well as between spots on the same slide.[6]
Functional arrays have many uses including the complete characterization of an organism's whole proteome. A study by Zhu et al. developed a yeast proteome array that contained proteins from 93% of the organism's 6280 protein-coding genes.[7] Other experiments include large scale screenings of proteomes for phospholipid-binding and calmodulin-binding specificities.
Protein chips enable us to study biochemical interactions on an unprecedented scale. Thousands of proteins can be screened for protein-protein, protein-nucleic acid, and protein-small molecule interactions simultaneously. The in vitro nature of the method ensures that it has advantages over current functional assay methods, and its parallel, quantitative format propels it above many other techniques in the field.
References
[edit | edit source]- ↑ a b c d Twyman R.M. Principles of Proteomics; BIOS Scientific Publishers: Oxon, U.K., 2004; Chapter 9.
- ↑ a b Hall, D.A., Ptacek, J., Snyder, M. 2007. Protein Microarray Technology. Mech Ageing Dev., 128(1), 161-167.
- ↑ Charboneau, L., Scott, H., Chen, T., Winters, M., Petricoin, E.F., Liotta, L.A., Paweletz, C.P. 2002. Utility of reverse phase protein arrays: Applications to signaling pathways and human body arrays. Briefings in Functional genomics and Proteomics, 1(3), 305-315.
- ↑ Tibes, R., Qiu, Y., Lu, Y., Hennessy, B., Andreeff, M., Mills, G.B., Kornblau, S.M. 2006. Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells. Molecular Cancer Theory, 5(10), 2512-2521.
- ↑ Sheehan, K.M., Calvert, V.S., Kay, E.W., Lu, Y., Fishman, D., Espina, V., Aquino, J., Speer, R., Araujo, R., Wulfkuhle, J.D., 2005.
- ↑ a b Bertone P, Snyder M. Advances in functional protein microarray technology. FEBS, 2005; 272(5400-5411).
- ↑ Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A, Bertone P, Lan N, Jansen R, Bidlingmaier S, Houfek T et al. (2001) Global analysis of protein activities using proteome chips. Science 293, 2101–2105