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May 15, 2013 (Vol. 33, No. 10)

Improving Proteomics Approaches

  • Click Image To Enlarge +
    The cell membrane is chemically heterogeneous (left). Lipid rafts, enriched in cholesterol and exhibiting local lipid ordering (light gray), sequester certain kinds of membrane biomolecules (red); while other biomolecules (green) prefer a more disordered, phospholipid-rich lipid environment (dark gray). (Right) The natural partitioning preference of membrane biomolecules within a mixture (yellow) can be exploited to separate species that prefer raft-like regions from those that do not. This separation is carried out using a supported lipid bilayer platform that is patterned with two different lipid phases. [Cornell University]

    If molecules required for a signaling event are held within the raft, they’re available to carry out that function. Likewise, if outside molecules are required, and the cell can control their partitioning into and out of the raft region, the cell has a means to control how signaling events occur.

    Dr. Daniel developed a technique for purifying lipid raft resident molecules from nonraft species through a biological membrane-based platform that achieves 2D separations based on chemical affinity for a particular membrane composition. She likened the technique to traditional extraction except that instead of oil and water, materials partition into one or another flat membrane material in close contact. Ionic strength and pH are controlled as they would be in electrophoresis.

    The membrane sheets (one raft, one nonraft) are positioned parallel, within the same plane and with a 2Dp (line) interface. “But instead of a 2D interface you have a line formed by two planar sheets butting up against one another.” When a mixture is loaded and forced through, components migrate into the region with which they enjoy the higher affinity.

    The membrane sheets (one raft, one nonraft) are positioned parallel, within the same plane and with a 2Dp (line) interface. “But instead of a 2D interface you have a line formed by two planar sheets butting up against one another.” When a mixture is loaded and forced through, components migrate into the region with which they enjoy the higher affinity.

  • Purifying PARP-Associated Proteins

    Guy Poirier, Ph.D., Canada chair in targeted proteomics at Laval University, described a high-throughput affinity purification—actually a profiling of the poly(ADP-ribose)-related response to DNA damage signaling.

    When DNA damage occurs inside cells, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize anionic poly(ADP-ribose) (pADPr) scaffolds that bind to several proteins, resulting in formation of pADPr-associated multiprotein complexes. These complexes were for many years believed to be covalent—similar to more common post-translational modifications such as phosphorylation and methylation. Subsequently, the noncovalent interactions have emerged as central in events downstream from DNA damage, such as cancer.

    Professor Poirier uses a combination of affinity purification and mass spectrometry-based proteomics to isolate pADPr-associated complexes, and assess protein dynamics with respect to pADPr metabolism.

    His approach involves a substrate trapping strategy that uses a mutant but inactive poly(ADP-ribose) glycohydrolase (PARG), as mutant can act as a physiologically selective “bait” for isolating specific pADPr-binding proteins. Along with more standard antibody-mediated affinity purification methods, Prof. Poirier employs a pADPr macrodomain affinity resin to recover pADPr-binding proteins and their complexes.

    “Only one PARP was known before the human genome was sequenced,” Prof. Poirier explained. “Now we know of six major PARPs, and several homologs.” PARP activity can rise up to 100-fold during DNA damage, with concentrations in the micromolar range.

    PARP activity has been implicated in several diseases, including cardiovascular, neurodegenerative diseases, and cancer.

    PARP repairs single-strand breaks that occur with frightening regularity in dividing cells. If these breaks persist during cell replication (as they might during times of low PARP activity), the single-strand breaks become double-strand breaks and the cell dies. A PARP inhibitor, therefore, could sustain DNA damage in susceptible cells, causing tumor cells to die.

    The key to designing a therapeutic PARP inhibitor, many believe, is to identify PARP-binding proteins. A decade ago around 60 such proteins were known. “Today we know more than 1,000,” Prof. Poirier said. Thanks to the sensitivity of new mass spectrometry instruments and the availability of peptide/protein databases, experimental timelines have shrunk exponentially. “We can now see more than 100 proteins during a one-hour run. That would have taken several years not too long ago.”

  • Protein Crystal Structures

    Established in 2004, the private-public University of Toronto partnership, the Structural Genomics Consortium (SGC), has deposited more than 1,300 structures into its protein database. Susanne Gräslund, Ph.D., discussed various high-throughput methods to generate these proteins within a range of expression hosts. Since 2011, the organization has also been involved in a large-scale project to generate recombinant antibodies to epigenetics proteins.

    With a U.K. charity as its base, SGC relays funds from public and private donors to member labs in Toronto and Oxford, U.K. The group also maintains satellite labs worldwide. Approximately half of SGC’s members are dues-paying industrial firms. “It’s important to maintain that public-private balance,” Dr. Gräslund commented.

    SGC’s core mission is to solve crystal structures of proteins relevant to human health, which explains the strong pharma/biopharma connection.

    As part of its mandate, SGC has begun investigating chemical probes, mostly small molecules, that bind to protein targets. “These are not necessarily drug candidates, as the leap from binding to drug is very large,” Dr. Gräslund explained. “Rather, researchers use these molecules as starting points for drug discovery.”

    The source of this molecular largesse is chemical libraries, whose access is provided by member companies. SGC also maintains academic collaborations, for example with the University of Colorado. A potential drawback to commercial partners is the unique agreement by which SGC publishes results freely and openly.

    The consortium’s third charge is making monoclonal antibodies using purified proteins as antigens, and subsequently selecting synthetic recombinant antibodies using phage-display technology.

    Binding is determined through in vitro assays and cell-based assays. “We do a lot of co-crystallizations as well,” Dr. Gräslund said. Most of the proteins employed in this work have already been solved, in their native unbound state, by crystallography. “Small molecules that bind sit in a specific locus, altering the protein’s conformation relative to its unbound state. Conformational changes cause shifts in thermal melt temperature.”

    SGC is open to additional collaborations, both academic and corporate, and has space to host visiting scientists with common interests in proteomics. The consortium also holds formal courses in protein purification and crystallization.


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