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Feature Articles : May 1, 2007 ( )
Protein Characterization Entering New Era
Novel Technology and Integrated Methods to Facilitate Detection and Research
Although mass spectrometry has facilitated protein identification and characterization via various techniques, many new approaches are becoming available. The success of protein and peptide therapeutics is helping to drive technology forward. According to a February 2007 market report by Data Monitor, the protein engineering market is forecast to be $118 billion in 2011, or 12% of pharmaceutical sales.
Imaging Alternative to Gels
Label-Free Intrinsic Imaging™ (LFII) provides detection of molecules without labels, dyes, stains, or radioactivity. “This technolog is an alternative to 1-D acrylamide gel systems,” explains Stuart Hassard, Ph.D., head of biology and co-founder of deltaDot (www.deltadot.com). “This allows us the extra resolution needed to see post-translational modifications with superb reproducibility. The standard deviation we observe ensures results are real data and not artifacts.”
The technology works by using multipixel UV detectors to image biomolecules as they traverse a detection window. Separations are analyzed via two algorithms designed to look for exotic subatomic particles. The General Separation Transform algorithm combines data from the pixels, preserving peak shape information of the electropherograms while simultaneously maximizing the signal-to-noise ratio. The Equiphase Vertexing Algorithm uses a velocity map of the molecules passing over the detector to perform vertexing. Analysis of the map provides significant improvement in performance and enables multiple injections into the same capillary to increase throughput (30 samples/hour), according to Dr. Hassard.
Compared to conventional capillary electrophoresis, the company’s Peregrine HPCE system, measures each molecule 512 times instead of just once. Resolution data approaches that of mass spectrometry in certain applications, such as post-translational modifications. Reproducibility reflects less than 1% relative standard deviation, and the user sees the molecule, not any attached label, equaling direct quantification. The amount of energy absorbed by the molecule is directly proportional to the number of peptide bonds. Applications of the system include protein and small molecule analysis and molecular sizing.
Developing Better Antibodies
Researchers at Centocor (www.centocor.com) are also working on characterizing small molecules early in drug development by utilizing a variety of mass spectrometry platforms. “This approach started somewhat when people weren’t sure of what compound they had in development,” explains Jennifer Nemeth, Ph.D., senior research scientist. “Not all companies will characterize antigens. That’s where the potential novelty comes in.”
She says her mass spectrometry group’s focus on early characterization is key to saving up to a year of work on the wrong molecule. “The minute something comes out of purification, we screen it first.” She adds that antibodies are now key in drug development because their structure is consistent and makes for easy production. However, when discovering a new target, “it’s best to know the tertiary structure of your antigen and determine epitope location.”
Her group now routinely analyzes crystal structures on each antigen and antibody complex. “One of our goals is to map out the location of all modifications in a molecule and then make knock-outs to enable use of the molecule for crystallography studies.”
This approach is being applied to all the company’s programs earlier in the development process; instead of a year prior to IND filing, it is now being done three to four years earlier. “We’re investing more upfront, with the idea we’ll spend less money in the long run,” states Dr. Nemeth.
Building a Better Microscope
Technology needs improvement in this rush to characterize proteins. Conventional microscopy is limited when used for imaging protein crystals and protein crystallization experiments. Emerald Biosystems (www.emeraldbiosystems.com) developed a microscope, Detect-X™, that displays high-contrast images of protein crystallization setups in crystallization plates. The fully automated polarization and UV fluorescence microscopy captures, processes, and displays high-contrast images.
“This instrument allows you to filter the massive amounts of crystallization experiments down to those likely to give you good results in the x-ray diffraction experiment,” points out Peter Nollert, Ph.D., director of crystallography. Additional potential applications include imaging dynamic processes, clinical applications, and material science.
Crystallographers often use conventional microscopes to detect transparent crystals by their facets or edges. Since most protein crystals lack color, this is difficult to do. Dr. Nollert says Detect-X displays colorless proteins as if they were colored and allows users to discover crystals hidden by precipitate. “We convert a qualitative image into a quantitative measurement,” adds Dr. Nollert. “All the proteins get a color; they become red or green, and the background stays blue or black. It’s easier to see positive crystallization results.” A UV lamp detects fluorescence emitted by protein crystals, allowing differentiation between salt and protein crystals.
To stain colorless crystals, the microscope images two physical parameters associated with crystal matter: birefringence and crystal axis orientation. Illumination from oblique angles provides high-edge contrast images.
Similarly, for characterizing glycoprotiens, an integrated approach is providing more specific information than traditional methods, according to researchers at Wyeth (www.wyeth.com).
Joseph McClennan, Ph.D., department of characterization and analytical development, says his group combines a top-down mass spectrometry approach with a bottom-up approach. “We can get more specific information about what the amino acid sequence is as well as do a site-specific determination of where post-translational modifications are located.”
The top-down method looks at an intact protein or protein subunit and provides information on the heterogeneity and modifications. The bottom-up technique includes peptide mapping with mass spectrometry identification. “When you combine these two approaches, it gives you a lot of confidence that you are characterizing and observing a lot of different things about the proteins,” adds Dr. McClennan.
Traditional methods, such as SDS-PAGE and chromatography, would usually drive all the characterization. However, his group is using the top-down, bottom-up approach initially to determine what type of traditional analytics are used. “It’s much more of a flow—integrating all these things to get the covalent glycoprotein structure.”
It enables faster screening and, if integrated into development structure, it provides faster results. His group has shown this approach also works well for antibodies, and they have started using it for complex recombinant glycoproteins.
Surface Plasmon Resonance
In dealing with biomolecular interactions, researchers at ICx Technologies (www.icxt.com) developed surface plasma resonance (SPR) biosensors for processes such as antibody selection and screening, drug discovery, ligand binding specificity, and gene regulation.
“Our surface is not a hydrogel,” notes John Quinn, Ph.D., CSO, “so you can immobilize things on the surface that behave like they do in solution and retain all biological activity.” He reports that the company licensed the surface plasmon technology but developed the chemistry, software, and microfluidics in-house. Dr. Quinn explains that the 15-nanoliter scale of the microfluidics allows for high mass transport rates and ensures kinetic information and high data quality.
One of the advantages to the SPR system is that it can analyze whole cells. It isn’t necessary to purify the receptor from the cell membrane. The sample is injected across the surface, and the vesicles are captured on the surface. “We have data sets showing high reproducibility of injections and we have data sets showing concentration assays.”
Dr. Quinn says one of the main features was the data set on kinetics. The model conformed exactly to the data set, validating the company’s approach. “In our model fitting, we find that the actual empirical data matched these models very closely.” The analysis software, Qdat, uses the entire data set.
“This technology provides structure function. It also allows thermodynamic analysis—you can repeat kinetic analysis at different temperatures to provide you with the type of bonding involved,” says Dr. Quinn.
There are currently two instruments available: an entry-level device and a semiautomated device, the SensiQ. The company is currently developing a fully automated instrument.
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