Proteomics

Proteome is a complement of proteins expressed in a cell at given time and proteomics means global analysis of this protein complement. Proteomics investigates the global changes of proteins in cells and tissues in response to a stimulus (for example temperature change, drug or nutrient treatment, or growth phase). It also studies protein-protein interactions. Proteomics came into prominence after 1997 and quickly became a popular research avenue, holding much greater importance than scientists initially suspected. The main reason for this is the fact that based on the genomic sequence it is impossible to predict how the gene products (proteins) are going to behave. Proteins are regulated at the level of protein translation, subsequently they can be modified by addition of various molecules (sugar, for example). Proteins can have varying half-lives, and their intracellular distribution can be predicted only with limited certainty.

Methods

The most basic method used in proteomics is a twodimensional (2D) electrophoresis . Cellular or tissue extracts are separated on a polyacrylamide gel in two dimensions, according to their charge and size, producing a pattern of spots. Although up to 11,000 spots can be separated on one gel, a typical number is approximately 2000. Following the separation, patterns obtained from test and control samples are overlaid and analyzed to determine any changes in protein expression, their levels or modifications. Proteins that are present in one, but not the other sample are isolated from the gel. In order to identify them, proteins are digested with an enzyme (usually trypsin) and the obtained small fragments (peptides) are analyzed by mass spectrometry to produce peptide fingerprints or protein tags that can be used for identification of the unknown spot. A second method used in analysis is tandem spectrometry. When each of the analyzed peptides are further digested and re-analyzed, this approach produces some sequence information in addition to mass . It is important to realize that mass spectrometry or even microsequencing are not used to fully sequence the samples, but to create sufficient information that will identify the unknown protein by searching the databases.

An important area of proteomic studies is to identify the interactions between proteins to determine the networks created by proteins in cells. A method used for such studies is a yeast two-hybrid system, which is best compared to fishing. Scientists use a bait molecule (chimeric protein), produced from the DNA sequence of a protein of interest fused to a sequence of a DNA-binding part of a known transcriptional activator, to identify what protein (prey molecule) interacts with their protein of interest. The process of identification of interacting complexes involves observing a color change resulting from an activation of a reporter gene. This gene is activated by a formed complex due to the fact that a prey molecule contains a coding sequence of a protein that might interact with the bait fused to an activation domain of the same transcriptional activator used for creating the bait. As of 2002, a number of commercial companies (for example, Hybrigenics) have developed a large scale automated yeast two hybrid screen.

An alternative method for studying protein interactions is creating tagged proteins, introducing them into cells, and subsequently using the tag to isolate the protein complexes formed in cells. The complexes are then separated on a gel according to their size and individual proteins are isolated, and identified by mass spectrometry.

Computational tools in proteomics are very important as the data generated requires image analysis, peptide and protein tag analysis, extensive database searching, and further investigation involving for example protein modification analysis.

Use of proteomics

Proteins play the most important part in creating cells and tissues, and directing their functions. It should be possible to identify the protein signatures of various diseases, especially at their onset to help in diagnosis and treatment. Protein signatures are particularly valuable in drug design and clinical trials. Scientists also have more basic interests in proteome, and proteomics is used to study bacteria , plant, and animal cells in order to understand how the proteins change during a particular treatment or phase of growth.

Future application of proteomic analysis is highly dependent on further technological developments to streamline analysis of clinical samples. The most obvious challenge for proteomics is developing protein chips similar to DNA microarrays. Also, new methods are required for studying the protein-protein interactions and large protein complexes.

Resources

books

Palzkill, Timothy. Proteomics. Boston: Kluver Academic Publishers, 2002.

Pennington, S.R., and M. J. Dunn, eds. Proteomics: From Protein Sequence to Function. Oxford: Bios and New York: Springer, 2001.

Westermeier, Reiner, and Tom Naven. Proteomics in Practice. Berlin: Wiley-VCH Verlag GmbH, 2002.

periodicals

Dove, Alan. "Proteomics: Translating Genomics into Products?" Nature Biotechnology (March 1999): 233–236.

Kumar, Anuj, and Michael Snyder. "Protein Complexes Take the Bait." Nature (January 2002): 123–124.

Rappsilber, Juri, and Matthias Mann. "What Does it Mean to Identify a Protein in Proteomics?" Trends in Biochemical Sciences (February 2002): 74–78.

organizations

Swiss Institute of Bioinformatics, CMU. Rue Michel-Servet 1, 1211 Genève 4, Switzerland, 41–22–7025858. <http://www.expasy.ch>..

other

Protein Prospector [cited November 16, 2002]. <http://prospector.ucsf.edu>.

Human Proteome Organization [cited November 16, 2002]. <http://www.hupo.org/.>.

Agnieszka Lichanska

KEY TERMS

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Chimeric protein

—Protein containing at least two different parts derived from two separate genes, but expressed as a single protein.

DNA-binding domain

—Part of a protein that interacts with DNA.

Electrophoresis

—Separation of nucleic acid or protein molecules in an electric field.

Peptides

—Low molecular weight molecules formed from two or more amino acids linked together by a peptide bond.

Polyacrylamide

—Branched polymer of acrylamide, used to make gels for electrophoresis.

Reporter gene

—Gene that encodes easily assayable product (protein), for example luciferase, green fluorescent protein, or chloramphenical acetyltransferase (CAT). It is fused to a promoter region of gene that is being tested.

Transcriptional activator

—A protein that induces transcription of a gene if stimulated, contains a DNA-binding domain and an activator domain