DOI: 10.1126/science.opms.p0600004

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As scientists delve more deeply into proteomics, they focus more on studying the natural characteristics and interactions of proteins. Ikunoshin Kato, president and chief executive officer at Takara Bio, says the most significant challenge for proteomics today is “an efficient protein expression system, which can produce large amounts of correctly folded recombinant protein. This is critical for structural and functional studies.” He adds, “The protein’s purity is also important, especially for structural analysis.” Proteins of interest can be screened against thousands of compounds for biological activity or function to explore their properties.

Protein scientists turn to a number of tools and techniques to analyze protein-protein interactions. Methods like chromatography and 2-D gel electrophoresis help researchers separate and purify mixtures of proteins. Techniques like two-hybrid systems, surface plasmon resonance (SPR), and mass spectrometry (MS) help uncover interactions between proteins, thereby leading to a better understanding of how cells function. Methods like nuclear magnetic resonance (NMR) shed light on the 3-D structure of proteins and can also be used to study protein folding and dynamics. In the end, proteomics could reveal much more about how proteins work, as well as helping biomedical scientists take advantage of proteins as tools.

Express Yourself
A variety of systems—ranging from bacteria to cell-free systems—can express recombinant proteins. Different systems provide various benefits and challenges. For example, Escherichia coli provides rapid expression but may not produce a functional protein due to issues surrounding posttranscriptional modification. Proteins from eukaryotic organisms, on the other hand, are more likely to be functional, because they conserve the processes of transcription, translation, and posttranscriptional modifications. In comparison to cell-based systems, cell-free protein expression systems offer several advantages: easy introduction of labels into proteins, the possibility to express toxic or apoptotic proteins, and compatibility with studies that require coexpression of proteins. Protein expression systems are offered by companies like ATCC, Invitrogen, Promega,Qiagen, and Takara Bio.

Takara Bio’s Single Protein Production (SPP) System, for example, expresses proteins in bacteria. Kato says that this system was developed in collaboration with Masayori Inouye, professor in the Department of Biochemistry at the University of Medicine and Dentistry of New Jersey. Kato calls this “a novel system for generating large amounts of soluble and pure protein for structural analysis studies.” He adds, “The SPP System utilizes the E. coli toxin protein, MazF, and cold shock technology.” The MazF protein—called an mRNA interferase—cleaves single stranded RNA at adenosine-cytosine-adenosine (ACA) sequences. In this system, the transcript of interest, which should not contain any ACA sequences, and MazF are co-expressed in E. coli. The MazF cleaves most everything but the transcript of interest. “The result is a high level—up to 90 percent of the newly synthesized protein—of target protein expression with relatively low cellular protein background,” says Kato.

In an E. coli system, however, some proteins do not fold properly. To enhance the proportion of correctly folded proteins, scientists at Takara Bio again collaborated with Inouye on the pCold DNA Vectors. Kato calls these “a series of unique protein expression vectors for generating large amounts of soluble and pure protein in E. coli. The pCold Vectors provide increased in vivo protein yield, purity, and solubility of expressed recombinant proteins at lowered incubation temperature.” He adds, “They can be used in conjunction with Takara’s Chaperone Plasmid Set to further increase the amount of correctly folded protein.”

For expression in mammalian cells, New England Biolabs offers its RheoSwitch Mammalian Inducible Expression System. “The key to this system is that it is precisely inducible,” says Chris Taron, head of gene expression research at New England Biolabs. “This system relies on a specific interaction between an engineered nuclear receptor and a synthetic inducer called RSL1, which triggers a chain of events that lead to protein expression.” Taron adds that using a nonnative receptor and synthetic inducer leads to little cross-talk with native cellular receptors. “The induction is proportional to the amount of RSL1 that you add,” says Taron. “You can fine tune the level of expression like a rheostat, or like turning up or down the volume knob on a radio.”

Taron notes, however, that all questions about protein expression cannot be answered in the same system. So New England Biolabs offers other systems, such as the K. lactis Expression Kit, which is a yeast-based expression system. Taron says, “K. lactis was developed in the food industry in the 1980s as a means of producing proteins at industrial scale.” The same system can now produce high levels of proteins for research.

To keep track of which proteins are being expressed, scientists often use luciferase as a reporter. Another recent product from New England Biolabs—the Gaussia Luciferase Assay Kit—provides a new reporter. Taron says, “Gaussia luciferase behaves fantastically in secretory systems and is more sensitive than other available luciferases.”

Purifying Proteins
As Kato mentioned, the purity of a protein impacts results. Various methods are used to enrich or purify a protein of interest. One of the most powerful of these methods is affinity chromatography, also called affinity purification, where the protein of interest is purified based on its specific binding properties to an immobilized ligand. This process can be performed with products from several companies, including EMD Biosciences, GE Healthcare, Pierce, and Qiagen.

Brian Johnson, market segment manager at Pierce says, “Affinity purification can provide 10,000-fold purification in a single step. It takes advantage of a highly specific interaction between a protein and a binding partner, such as an antibody.” This process can be used in many ways, including purifying a specific protein or class of proteins, such as glycoproteins or cell surface proteins.

Pierce also makes the Pinpoint Cell Surface Protein Isolation Kit. Johnson says, “This kit labels cell surface proteins with biotin that has a cleavable spacer arm. It allows you to pull out proteins labeled with biotin and specifically elute the labeled cell surface proteins.”

Jutta Drees, protein product manager at Qiagen, says, “The addition of a His tag is the most commonly used method for affinity purification of recombinant proteins. Since their launch, Qiagen’s Ni-NTA matrices have set the standard for highly selective purification of His-tagged proteins.” She adds, “Our most recent addition to the Ni-NTA range is the easy-to-use Ni-NTA Fast Start Kit, which purifies up to 10 milligrams of protein in as little as 90 minutes.” For purification of nonrecombinant proteins, Qiagen has developed Qproteome affinity-based kits for isolating phosphoproteins, glycoproteins, and plasma membrane proteins.

Getting the best result from proteomic studies, though, depends on sample handling, according to Eric Fung, vice president of medical and clinical affairs at Ciphergen Biosystems. Fung says, “We seek a balance between running enough samples to keep the statisticians happy and doing extensive fractionation since the more you fractionate, the more information you can get. But doing this on lots of samples in high throughput is very challenging.” To pull out the desired proteins, Fung says that scientists can use Ciphergen’s ProteinChip Arrays. These provide a variety of surface chemistries that allow researchers to optimize protein capture and analysis. The surface chemistries of the arrays include a series of classic chromatographic chemistries and specialized affinity capture surfaces. Fung says, “The different chromatographic surfaces generate an additional degree of fractionation.”

These arrays are often used for comparative protein profiling. For example, Fung says that a scientist can take samples from patients with a disease and compare the proteins to patients without the disease. “These arrays can also be used for protein identification or to look at protein-protein interactions or different types of protein modification,” says Fung.

Cataloging the Characteristics
Once a protein is captured, scientists characterize it. “Characterization is a broad term,” says Dave Hicks, senior director of proteomics at Applied Biosystems. “In general, it means having a full understanding of a protein, including its full amino acid sequence, the splice edits, and the identification of all posttranslational modifications.”

Protein scientists can characterize proteins with a wide range of approaches, like two-hybrid systems, MS, NMR, and SPR. Each of these offers a somewhat different view into protein-protein interactions. The two-hybrid method, for instance, can identify protein-protein interactions, protein cascades, and mutations that affect protein-protein binding. Two-hybrid systems are available from companies like Clontech (a Takara Bio Company), Invitrogen, and Stratagene.

Various forms of MS also characterize proteins. For example, 2-D gel profiles can be further analyzed using MS and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) techniques. Several companies manufacture these instruments including Applied Biosystems, Bruker Daltonics, and Thermo Electron.

Hicks says that one of Applied Biosystem’s most popular MS systems for proteomics is the 4800 MALDI TOF/TOF Analyzer. “This is often used in protein expression analysis, with and without tagging chemistries,” he says. Hicks adds that the QSTAR Elite Hybrid LC/MS/MS System is often used for protein identification and characterization studies. For studies of phosphorylation and other posttranslational modifications, Hicks says that scientists might use the 4000 Q TRAP LC/MS/MS System. He says, “This system can be used in protein quantitation studies, looking at expression and quantity across multiple samples, or even studying multiple proteins in multiple samples, all at high throughput.”

To increase sensitivity and facilitate analysis of low-abundance proteins in MALDI analysis, Anke Cassing, associate protein business director at Qiagen, says, “We looked at how to put the maximum amount of sample on the MALDI target. By providing on-target processing—for example, purification of phosphopeptides, sample desalting, and concentration—Mass·Spec·Focus Chips eliminate sample losses due to ‘offline’ processing. Users can pipette large sample volumes directly onto the chip, enabling analysis of dilute samples without the need for a concentration step using a pipette tip device.” Mass·Spec·Focus Chips are available for Applied Biosystems, Bruker Daltonics, Waters, Shimadzu, and Thermo Electron instruments. For high-throughput MALDI analyses using ABI 4700, QSTAR, and Voyager instruments, Qiagen has developed Mass·Spec·Turbo Chips, which are high-density, ready-to-use MALDI targets that carry up to 1,600 matrix spots.

Binding the Biologics
Today’s proteomics goes beyond the structure and identity of proteins. Stefan Löfås, vice president and chief scientific officer at Biacore, says, “We see an increasing interest to learn more about protein function, especially through binding and interactions. This is really determining how proteins talk with each other.”

Some scientists follow protein interactions using SPR detection, which can monitor proteins as they interact with various targets: proteins, carbohydrates, lipid bilayer vesicles, nucleic acids, and even whole cells. Moreover, the information appears in real time, thereby giving the affinity and kinetics of the binding event. The events are detected as molecules in solution interact with a partner attached to a gold surface. This interaction causes a change in refractive index that causes a change in the SPR signal.

In the past year, Biacore introduced several new systems based on SPR detection. For example, the Biacore T100 is what Löfås calls “our new flagship system.” He says, “It compiles all of the knowledge and experience we have gained into the most modern and versatile system for research through quality control, including various proteomics applications.” He adds, “For in-depth analyses, this instrument can even smoothly extract the thermodynamic parameters driving an interaction.”

For higher productivity, scientists can use the Biacore A100. Löfås says, “In comparison to Biacore T100, this generates the same type of data with the same level of quality and sensitivity, but, with parallel processing and multiplex analysis capability. Biacore A100 can monitor up to thousands of protein interactions per day.” For those working with large numbers of samples who want to rapidly select interactions of interest for in-depth study, Löfås recommends the Flexchip, which offers an array format that “can profile up to 400 interactions at the same time.”

Other companies also use SPR on arrays. Timothy G. Burland, president and chief executive officer at GWC Technologies, says, “Our SPRimagerRII instrument is an SPR system with an array format so you can take a picture of the array.” Burland notes several examples of using this SPR instrument. For instance, his company has collaborated with Neoclone to show “that you obtain the same affinity values for antibody-antigen interactions whether you make antibody arrays using purified antibody or the corresponding ascites fluid. In other words, you don’t need to purify the ascites fluid to use antibodies as probes on our arrays.” He also notes that scientists in the laboratory of Lloyd Smith at the University of Wisconsin used the SPRimagerRII to show the presence of specific cell receptors on baby hamster kidney cells. Likewise, Robert M. Corn, at the University of California, Irvine, used SPR imaging arrays to characterize transcription factors and antibodies.

To help scientists create other proteomic applications, GWC Technologies produced its SpotReadyT16 and SpotReadyT25, which have 16 and 25 gold spots, respectively, for attaching probes. Burland says, “Some people only need about a dozen probes per array to study protein function. With SpotReady arrays, you use a pipette to spot half a microliter or less per probe.” He adds, “This is quick and does not require a robot for spotting.”

Structural Specifics
“NMR has been used for some time to study protein structure,” says Steve Smallcombe, manager of applications laboratories at Varian. NMR places a molecule in a strong magnetic field, irradiates the molecules with radio frequency waves, and then detects changes in the spin of atoms. “NMR is very nice,” says Smallcombe, “because you see signals from all of the atoms in a protein.” Also, NMR can reveal time dependent phenomena. Judit Losonczi, product marketing manager at Varian, says, “NMR can provide an understanding of protein dynamics, folding, and kinetics.” NMR systems are offered by companies like Bruker BioSpin, Jeol, and Varian.

“A downside to NMR has been that it is less sensitive than MS,” says Smallcombe, “but we increased NMR’s sensitivity with a cryogenic probe—essentially an antenna in the instrument’s magnet—that is cooled down to 25 Kelvin. This technology provides improved sensitivity.” He adds that Varian’s Salt Tolerant Cold Probe can be used in physiological conditions.

Smallcombe adds that most NMR in the past used proteins in solution. “However, proteins in membranes cannot be studied this way,” says Smallcombe. Nonetheless, Varian’s new Bio-MAS probe allows for studies of membrane bound proteins in a solid state.

Building Better Bioinformatics
“The current situation in proteomics reminds me of the struggle with microarrays for gene expression about seven or eight years ago,” says Lee Weng, director of applied research at Rosetta Biosoftware. “At that time, the focus was on perfecting the instrument, getting the resulting numbers right.” Now, with more confidence in the results from proteomic instruments, Weng says that scientists can focus on extracting information. “We need to get the useful information,” Weng says, “so that we can do something with it.” Some companies—including Applied Biosystems, Beckman Coulter, Bio-Rad Laboratories, Caliper Life Sciences, and Syngene—develop core instruments for proteomic research along with integrated software systems. Other companies—including Accelrys, Elsevier MDL, and Rosetta Biosoftware—offer software suites for proteomics research.

Weng says that, last year, Rosetta Biosoftware introduced its Elucidator Protein Expression Data Analysis system. This system can capture large volumes of data from various MS instruments and then manages and analyzes the results. “The Elucidator does data preparation and manages data in very large-scale, label-free quantification studies,” says Weng. “Sometimes, hundreds of samples are analyzed all together, and each can have several gigabytes of information.”

This system can be used in a wide range of proteomics research. For instance, Weng says, “Output from this system can be a list of biomarker candidates that scientists can go after and map to pathways to understand disease. We can also see what protein has been affected by a drug or compare protein expression in normal versus diseased samples.”

Defining the Details
“It’s easy to find the stable interactions,” says Johnson of Pierce, “but harder to get the weak and transient ones.” Still, understanding the finer points of proteomics might resolve many questions in both basic and applied biological research. “The problem is nailing down the details,” says Johnson. As proteomics scientists hammer out the specifics, the applications of this field will continue to grow.

Mike May (mikemay{at}mindspring.com) is a publishing consultant for science and technology based in Minnesota. Gary Heebner (gheebner{at}cell-associates.com) is a marketing consultant with Cell Associates in St. Louis, Missouri, U.S.A.

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