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W. Hayes McDonald PhD: Vanderbilt University, Nashville, TN Oak Ridge National Laboratory |
Keywords:
Mass spectrometry, LC-MS, MudPIT, proteomics, tandem MS, post translational modifications
Description of Research:
What could we do if we could measure the amounts and localization of every protein within a cell? Chemical modification states? Other proteins with which it interacts? Activity? These lofty goals fall under the somewhat nebulous moniker of proteomics. Despite significant progress over the past several years these goals have only been realized to a limited degree. Because of the complexity of the challenge, proteomics has been primarily a technology-driven endeavor with many different devices and strategies being brought to bear on the problem.
Mass spectrometers, devices used to measure the mass of atoms and molecules, have played a central role in these developments. As these devices have developed, they have become faster, more sensitive, and capable of making even more complex measurements. One strategy enabled by these newer generation mass spectrometers is "shotgun proteomics". A mixture of proteins to be analyzed is first digested en masse with a protolytic enzyme, such as trypsin, to make an even more complex mixture of peptides. These peptides can be readily separated using high performance liquid chromatography (HPLC or LC) and then individually sequenced using the mass spectrometer through tandem mass spectrometry (MS/MS). Even more complex mixtures of proteins can be analyzed by adding another dimension to the LC separation (LC/LC). One implementation of this multidimensional LC strategy was developed in John Yates's laboratory and is known as MudPIT (Multidimensional Protein Identification Technology).
MudPIT has proven useful for a broad range of analysis, from just a few proteins all the way up to the many thousands of proteins present with in a cell. My work has focused primarily on the analysis of protein complexes. These molecular machines including well known ones such as the ribosome, RNA polymerase, the spliceasome, proteosome, etc. perform much of the "business" of the cell. In addition to the previous examples, many if not most of the proteins within the cell interact with other proteins to some extent. To understand proteins and how they are regulated one must not only identify its partners but also find how they change under different cellular conditions.
In addition to changes in their binding partners, much of protein function is regulated through post-translational chemical modifications (PTM). By digesting a protein with multiple enzymes it is possible to generate peptides covering a larger portion of the protein. Coupling this digestion strategy with MudPIT allows one to pinpoint both the type and position of the PTMs on the protein. Without the ability to describe protein complexes, map PTMs, and then follow changes in response to various cellular stimuli, it is impossible to fully understand the biology taking place within a cell. As we are able to take these measurements more rapidly and with less starting material, we will be well on our way to fulfilling the promise of proteomics.
Selected Publications: