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May 15, 2010 (Vol. 30, No. 10)

Mass Spectrometry's Role in Discovery on the Rise

Technological Advances Continue to Increase the Versatility of This Reliable Lab Workhorse

  • Yellow Fever Vector

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    Researchers at the University of Arizona are studying Aedes aegypti, the mosquito responsible for transmitting yellow fever and dengue fever. The inset shows a dissected mosquito in a droplet of protease inhibitor cocktail, showing the mid-gut as well as ovaries removed from the abdomen.

    Vicki Wysocki, Ph.D., professor of chemistry at the University of Arizona, Tucson, provided a proteomics analysis of iron-related ovarian proteins from Aedes aegypti, the mosquito responsible for transmitting yellow fever and dengue fever. As the mosquito requires an iron-containing blood meal to carry out oogenesis, Dr. Wysocki and her colleagues reasoned that the iron transport proteins could be a target for vector intervention. Accordingly, they searched for iron-regulated and iron-transport proteins in the ovaries of mosquitoes raised on different diets.

    This was another example of the bottoms-up approach, in which the ovaries of the insects were dissected and separated on one-dimensional gels. The bands were excised, digested with proteases, and subjected to liquid chromatography followed by mass spec analysis. The identified proteins were then surveyed on the protein database.

    A large number of proteins was identified. Ferritin was one of the proteins found in the ovaries of the mosquitoes raised on the iron-containing diet, suggesting that it is upregulated in the presence of hemoglobin in artificial diets. The presence of the enzyme aconitase, known to require iron for activity in other organisms, was also detected.

    “We found that proteins expressed in the ovaries of mosquitoes varied significantly depending on the diets these mosquitoes received,” explained graduate student Michelle Huang, who was involved with the study. “The proteins expressed included essential proteins performing developmental, structural, and ribosomal functions.”

    Interestingly, iron-transport proteins did not pass the filtering criteria that the team had designed as part of the experimental protocol. “Some of these proteins could represent targets for population control. We are currently expanding on these experiments for more confident identification.”

  • Blocking The Memory Thief

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    Shimadzu Scientific Instruments’ LCMS ion trap time-of-flight (LCMS-IT-TOF) mass spectrometer

    Michael Bowers, Ph.D., professor in the department of chemistry and biochemistry at University of California, Santa Barbara, reviewed the structure and assembly of prion protein fragment PrP106-126. All prion-based disorders are characterized by the accumulation of an abnormally folded form of the prion protein as β-sheet-rich amyloid fibrils in the brain. This pile-up of aggregates in neuronal cells is believed to be responsible for the memory loss that is the hallmark of these devastating conditions.

    The early events in the formation of these β-sheets are not well understood. The development of effective vaccines or molecules that could block the aggregation process will require the intimate knowledge of these changes. Dr. Bowers and his team employed ion mobility spectrometry combined with mass spec to delineate one of the key domains involved in amyloid formation.

    The team’s observations on monomeric and oligomeric structures of aggregating and nonaggregating forms of the critical region were crunched using replica exchange molecular dynamics. This computer simulation model analyzes the interactions of atoms and molecules over time by approximations from physics, giving a view of the kinetic motion of the particles. The Bowers analysis also employed replica exchange, a Monte Carlo simulation approach that runs parallel replicas of the simulation, selecting the most probable outcomes.

    Based on the data collected and the analyses conducted, the team concluded that ordered β-hairpin monomers form the nucleus of the aggregates rather than the converse hypothesis, which states that the process is driven by disordered monomers. This ordered collection of β-structured aggregates then progress to the fibrils responsible for the actual disease. Since observations of the behavior of different forms of the peptide argue for intermolecular rather than intramolecular initiation of oligomer formation, the understanding of the mechanism of the β-sheet-rich amyloid fibrils is moved to a new level.

    The analysis performed by the Bowers lab has a broad range of applications outside the field of Alzheimer investigation. A thorough understanding of the dynamics of chemical interactions is a powerful informative tool that will aid in rational drug design and could serve as a means of modeling effective antigens for vaccines.

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