New Technology Spurs on Proteomics
Companies invest in developing state-of-the-art tools
By Jennifer Fisher Wilson [The Scientist]
One recent morning at the Applied Biosystems proteomics research center
in Framingham, Mass., scientist Jason Marchese patiently used a pipettor to
place tiny samples onto a 2-inch-by-2-inch plate. He was surrounded by
technology as simple as 2-D gel electrophoresis apparatus and as cutting-edge
as a high-throughput system that uses automated robotics for multidimensional
liquid chromatography separation of proteins and an automated workstation that
uses the latest in mass spectrometry advances to identify and analyze proteins.
The only scientist working in a large room filled with machines for proteomics
research, Marchese talked about how he can load protein sample onto machines in the
afternoon and return the next day to a database full of newly identified proteins. But in
spite of major advances like this, inadequate technology still holds back the field of
proteomics. Applied Biosystems is just one of a number of biotechnology companies
working hard to provide novel tools to identify more quickly and precisely the thousands
upon thousands of proteins produced by the human body.
Now that the human genome is mapped, researchers in academia, government, and
industry have turned their attention to mapping proteins. And biotechnology companies
are responding by developing the needed tools for the job. Already a multimillion-dollar
business, proteomics is expected to grow quickly into a multibillion-dollar business,
according to market estimates.
"It was important to map the genome, but clearly, proteins are where the action is,"
says Leigh Anderson, president of the proteomics subsidiary of Large Scale Biology
Corp. in Vacaville, Calif. An early player in the proteomics industry, Anderson points to the
promise--and value--that proteins hold for new diagnostic tests, therapeutic treatments,
and medicines. But discovering and analyzing proteins is a daunting task, and there is
still a long way to go before wide-scale protein identification is complete, or even
possible. Proteins are far more numerous, fragile, and changeable than genes, and they
can be difficult to find and measure. As Anderson puts it, the question now is, "What
technology do we need?"
It's not an easy question to answer, because the field is still young and the job is so
big. Several recent technology advances have greatly improved the picture for proteomics
research. Processes that once took months may now take just a few days or less.
Biotechnology companies focusing on proteomics now hope to further reduce the
process times down to a few minutes while also improving quality.
Capturing Proteins
Just a decade ago, the only way to identify a protein was to isolate it on a 2-D gel and
then sequence it; a painstaking process that took days and sometimes weeks and could
involve a lot of material. Two-dimensional gel electrophoresis is still a workhorse tool in
proteomics, and it has become increasingly useful (see also, "2D Glasses,"). But 2-D
gels can be labor-intensive, and they do not always provide reliable quantitative
information. Additionally, they simply cannot identify certain classes of proteins, including
membrane, highly acidic or basic, and low-abundance proteins.
Now, a new, complementary technology called ICAT™ (isotope-coded affinity tags)
Reagent helps address many of the problems with 2-D gels. ICAT provides a new
method for quantification and identification of proteins from complex samples using
mass spectrometry analysis. The technology has received widespread praise
throughout the industry for being the first tool that allows both pre-fractionation and
quantification of relative expression levels of proteins, including low-abundance and
membrane proteins, in a high-throughput model. It also is capable of identifying those
proteins that 2-D gels miss, such as membrane proteins.
"It lets us do things that weren't possible with 2-D gels," explains Dave Hicks,
marketing manager for the Applied Biosystems proteomics research center. Developed
by Ruedi Aebersold at the University of Washington,1 ICAT is currently licensed by
Applied Biosystems. ICAT works by using a label that contains the stable isotope
deuterium to label all the proteins (e.g., in cell extracts) via their cysteines, mixing them
together, and digesting the samples with a protease. Peptides that contain the label are
bound and then further separated. These labeled peptides are then loaded into a mass
spectrometer and the ratio of the various peptide pairs is measured.
As an additional benefit, ICAT could theoretically make any separation technology
quantitative, including high-performance liquid chromatography and capillary
electrophoresis. Since the proteins are pre-mixed, the relative ratio stays the same no
matter how much protein is added during fractionation.
A Role for Protein Biochips?
Another new development that promises major advances in proteomics is the protein
biochip. A number of companies are developing chips in the hopes that they will become
a key tool for measuring large numbers of proteins in biological samples. "Separation
technology is the key technology today for proteomics research, especially protein
arrays," comments William E. Rich, CEO of Ciphergen Biosystems in Palo Alto, Calif.
Protein biochips draw on the technology developed for genomics, but they have proven
challenging to develop because proteins are far more difficult to analyze compared to
DNA.
Also called protein or antibody arrays, protein biochips hold the potential to measure
protein-protein interactions, protein-small molecule interactions, and enzyme-substrate
reactions. They may also allow differential profiling, such as distinguishing the proteins
of a healthy cell from those of a diseased cell. The chips hold value for identifying
potentially relevant biomarkers and for pharmaceutical discovery-based research.
Among the companies working in this area, Ciphergen has developed a protein
biochip system, the ProteinChip® System, that incorporates mass spectrometry (in
particular, surface enhanced laser desorption/ionization [SELDI]), and biochip
technology in a single, integrated platform. The chips allow researchers to capture,
separate, and quantitatively analyze proteins directly on the chip. The arrays (protein
molecular weights) are read directly by the SELDI process without radioactive or
fluorescent labels or genetically engineered tags that may interfere with the protein.
Large Scale Biology and Biosite Diagnostics Inc. of San Diego recently announced a
collaboration to develop protein biochips. Large Scale Biology plans to provide 2,000 to
5,000 protein targets from its Human Protein Index™ as well as expressed proteins
produced with its Geneware®‚ technology. Biosite will then use its high-throughput
Omniclonal™ phage display technology to generate high-affinity antibodies to the targets,
enabling the creation of antibody arrays in a variety of formats, including biochips. Based
on this, the companies intend to market a broad antibody and target package. "In a
couple of years we should be able to offer high-throughput, low-cost arrays," Anderson
says. He expects that the resulting collection of antibodies will span many of the
commercially important human proteins.
Another leader in this area is Zyomyx of Hayward, Calif., which specializes in
miniaturized protein biochip architectures containing high-density arrays. The company
has recently collaborated with MDS Proteomics in Toronto (See also, "On the Fast Track
in Functional Proteomics,") on research to identify protein pathways.
In related technology, Bio-Rad Laboratories of Hercules, Calif., recently unveiled a
platform for multiplexed, fluorescent immunoassays. Called the Bio-Plex™ protein array
system, it is intended to allow researchers to extract more data from small samples. The
platform builds on an integrated system of Bio-Rad application software, calibration and
validation protocols, along with optimized assay panels. It is intended as a tool to speed
the drug discovery process.
Continuing Advancements in Mass Spectrometry
Improved 2-D gels, ICAT, and protein biochips are important subunits advancing protein
research, certainly, but they depend on mass spectrometry (MS) technology. Now highly
accurate, and increasingly fast and automated, MS is widely viewed as the cornerstone
proteomics tool for characterizing proteins. It determines the identification of large
numbers of proteins and peptides, and it holds the promise of even more rapid
identification and characterization in the future as the technology continues to advance.
The focus today is on developing a new class of mass spectrometers that provide
higher throughput and automation and more powerful and precise protein analysis,
according to Hicks at Applied Biosystems. In one effort to achieve higher throughput,
Applied Biosystems is developing a new MS/MS system based on the matrix-assisted
laser desorption ionization (MALDI)-time of flight (TOF) technology.2
MALDI-TOF,3 first released in 1990, was the first system to enable rapid protein
identification by using a technique called peptide mass fingerprinting. The company's
newest system, the Voyager™ TOF/TOF, which is still in development, is the first
tandem-TOF platform. The system uses a high-throughput MALDI source and two TOF
mass analyzers with a timed ion selector and collision cell for both MS and MS/MS
analysis of biomolecules.
Kratos Analytical Inc. of Chestnut Ridge, N.Y., has recently released an MS system
based on MALDI-TOF technology as well. The AXIMA CFR promises a combination of
high mass accuracy, resolution, and sensitivity with high-throughput sample handling.
Using a 384-spot microtiter sample, the AXIMA-CFR generates seamless postsynaptic
density of proteins with an ion gate that allows reliable selection of parent signals for
fragmentation from complex mixtures, a camera for viewing samples using nonuniform
matrix preparations and unconventional samples, and a novel laser beam-focusing
mechanism to maximize the yield of ions extracted from the source to the flight tube.
Advances in MS/MS technology continue because MS alone is often not enough for
identifying all proteins in a sample. The electrospray ionization tandem MS (ESI-MS/MS)
method typically used for sequencing one or more protein cleavage products has led the
way to more precise protein identification. An approach involving direct MS/MS of intact
proteins for simultaneous protein identification and detection of post-translational
modification appears promising for analyzing small molecules. Applied Biosystems,
along with its joint venture partner MDS SCIEX of Concord, Ontario, is currently advancing
the Quadrupole TOF technology for this purpose. One application of the technology is the
characterization of post-translational modifications in proteins.
More Technology Needed ASAP
Despite the many new developments moving proteomics research forward at an
increasingly rapid pace, a number of barriers still hold the field back. Technologies are
needed still to better integrate the steps required for protein work and alleviate
bottlenecks that occur between sample processing, separation, identification, and
characterization. The process of loading proteins onto an MS plate, generating spectra,
and searching protein databases takes time. A number of companies--including
Genomic Solutions of Ann Arbor, Mich.; Oxford GlycoSciences of Oxford, England;
Proteome Systems of North Ryde, Australia; and Large Scale Biology--have speeded up
the process by automation.
Advances in high throughput must be balanced, however, with high-quality data,
explains Dave Walker, Bio-Rad's protein discovery business unit manager. Computer
technology also must keep up with protein discovery. "Advances in proteomics
technology must be accompanied by high-speed computing to allow fast data analysis,"
Hicks notes. A number of companies are focusing on the challenge of developing
information technology especially for proteomics, including Compaq, which is
collaborating with Celera, and IBM, which is collaborating with MDS Proteomics.
Once the technology barriers to proteomics are overcome, however, our
understanding of how proteins drive the human body may grow exponentially. Or as
Ciphergen's Rich comments, "Scientists have known for a long time that proteomics is a
necessary reality to understanding biology. If we focus on proteins now, proteomics has
potentially revolutionary applications."
Jennifer Fisher Wilson (jfwilson@snip.net) is a contributing editor for The Scientist.
References
1. S.P. Gygi et al. "Quantitative analysis of complex protein mixtures using isotope-coded affinity tags,
Nature Biotechnology, 17:994-9, 1999.
2. D. Wilkinson, "Pursuing proteomes," The Scientist, 14[12]:28, June 12, 2000.
3. B. Sinclair, "MALDI-TOF goes mainstream," The Scientist, 13[12]:18, June 7, 1999.
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