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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

  • The “Ion Chemistry and Mass Spectrometry Conference” held at Lake Arrowhead, CA, earlier this year, covered many advances in mass spectrometry that have allowed unprecedented analysis of molecular events occurring in normal and pathological biomolecular systems.

    Among the enabling technologies highlighted at the meeting was work done by Joseph Loo, Ph.D., professor of chemistry and biochemistry of the University of California, Los Angeles. “We have developed electrospray ionization mass spectrometry methods with improved sensitivity for detecting and measuring protein noncovalent complexes,” he said.

    His group is presently also concerned with obtaining structurally relevant information on protein-ligand interactions. In addition, the team, which previously reported a mass spec-based method for localizing ligand binding sites, has now extended this work to address nucleotide binding, specifically aiming at the site of ATP-binding to adenylate kinase.

    Mass spectrometry is an outstanding tool for investigating molecular interactions—it is sensitive, specific, and fast, particularly when combined with electrospray ionization, which ionizes macromolecules without disrupting weak, noncovalent interactions. The extremely precise molecular mass measurement provides direct evidence for protein-ligand or nucleotide-ligand associations. Indeed, the interactions may be retained during the transition from liquid to gas phase, thus permitting the size of the complex and the binding stoichiometry to be accurately assessed.

    Classically, mass spec analysis uses a “bottom-up” strategy, in which molecules are fragmented with proteases, then their molecule weights are assessed, and an overall picture of the original protein is assembled. However, Dr. Loo and his colleagues are exploiting a “top-down” approach to ligand-protein dynamics in which intact complexes are introduced into the mass spectrometer, so information describing the native complex is retained.

    Even molecules that bind relatively weakly to one another can maintain their association in the gas phase, the relationships being fixed by the solution-phase positioning of the component molecules. Dr. Loo has taken advantage of the stability of electrostatic interactions to determine the adenosine 5´ triphosphate binding site of adenylate kinase.

    “We have also discovered new reagents for increasing protein charge for electrospray ionization mass spectrometry that markedly improve the efficiency of tandem mass spectrometry of proteins.”

    A number of neurological disorders are the result of abnormal protein aggregation, including Alzheimer, Parkinson, Creutzfeld-Jacob, and Huntington diseases. Normal protein components are modified in ways that force their aggregation, blocking normal neuronal impulse transmission. An understanding of this process, obtained through the use of electrospray ionization mass spectrometry, could contribute to the design of therapeutic molecules for these conditions.

    Dr. Loo has used data from his top-down mass spec strategy to probe the sites of compounds binding to amyloid b protein. Amyloid b self-aggregates into a variety of neurotoxic structures from fibrils to oligomers, which are the more neurotoxic of the two. A potential anti-Alzheimer therapy could be to develop low molecular weight compounds that block the formation of these toxic aggregates.

    “In the development of novel inhibitors, localization of their sites of interaction on the target of interest is a critical first step in the drug discovery process. Our mass-spectrometry studies can augment the existing body of knowledge for a given biochemical complex, and they can provide key preliminary insight on the structure of supramolecular assemblies, especially for complexes not amenable to x-ray crystallography or NMR.”

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