Proteomics: Introduction



Proteomics, the study of the total protein complement of a genome, is essential if we are to fully exploit the knowledge acquired as a result of mapping the entire human genome. The key strength to proteomics is that a DNA sequence alone is enough to figure out complex biological systems present within living cells. Functional proteomics takes the field one step further, mapping the activity of these proteins as they respond to changes in their environment, in response to drug treatment or in a disease state. This will be a particular challenge with respect to protein kinases as they are expected to be present within the cell in very small amounts.

Being able to measure activity of proteins as an entire pathway responds to a target drug, disease state, or other stimulus is far more powerful than genomics, or any other technique invented before proteomics. Here, you can actually see what the proteins do, rather than what proteins are being made. This means you can account for inhibition pathways, activation/deactivation by phosphorylation, and all sorts of other cellular mechanisms you really can't measure using any other technique. Proteomics also allows you to do this quickly, frequently, reproducibly, and cheaply.

Skill Sets Needed in Proteomics

The most common form of proteomic analysis utilizes two-dimensional gel electrophoresis (2-DE) as its basic method. Historically, the most significant and troublesome problems with 2-DE has been the difficulty obtaining reproducible 2D gel patterns. This has been largely due to pH gradient drift during the isoelectric focusing stage of the separation procedure. As we move from genome to proteome, the demand for high-quality reagents has been met, and many tools have been developed to enhance reproducibility and to allow the process of 2-DE to become more automated and less labor intensive. The challenge now lies in the development of methods for the detection of specific proteins of interest, such as low-abundance proteins, and in procedures for the analysis of these proteins. Low-abundance proteins, which are often the molecules of interest, are obscured by the high concentration of structural and metabolic proteins. As a result, there is a dire need for enrichment protocols, which maximize the likelihood of detection of these low-abundance species.

The quantity of information generated by 2-DE technology, when combined with the variety of enrichment and detection techniques being developed, can be overwhelming. The detailed analysis of protein expression level and migration pattern, combined with assessment of phosphorylation state, must be carefully analyzed using available 2-DE software packages and catalogued using specialized databasing formats. Bioinformatics is therefore an essential component of the proteome project, allowing efficient organization of data output, spot-linked identification of protein migration, and query-based searches for protein profiles.

At Kinetek, we're using a novel protocol using 2-DE technology and bioinformatics look at kinase proteomics, (or Kineomics?). We use Kineomics? in three steps of our drug discovery program: We use it to identify protein kinases believed to play a role in disease; to validate those kinases as the elements causing the disease; and to validate the specificity of protein kinase inhibitors ("hits") identified in a high throughput screening program. We believe that, largely thanks to the use of this tool, we will be able to rapidly and efficiently identify, using cell based and animal models of cancer and diabetes, protein kinases with abnormal enzymatic activity in the disease state. Some of these abnormally activated protein kinases will be validated as causal of disease, and therefore "targets". Once the proteomics program has determined these targets, they are then submitted for use in the in vitro enzymatic assays of our high throughput inhibitor screening program.

The Future of Proteomics

It is clear that the field of proteomics is open to scientists from a variety of backgrounds. The many steps in the process employ cell culture, animal model assessment, protein biochemistry, protein microsequencing, and bioinformatics. The ability of this technology to integrate these many facets of science ensures that the field will remain on the cutting edge of research and discovery. The ease of application of this work to diverse areas of study defines its role as a research tool, which can resolve the complexity of the cell's signaling machinery, independent of the model system upon which it is applied. It is for this reason that proteomics is finally coming of age, and why functional proteomics is being applied to drug and target discovery programs worldwide.

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