|Topic:||The Fine Art of Peptide Synthesis .|
Image: Marlene Viola Synthetic peptides, generally less than 20 amino acids in length, are essential reagents for a wide range of studies and one of the fundamental tools of proteomics researchers. Applications include generating antibodies against specific epitopes, eluting specific proteins from affinity columns, testing the biological functions of enzymes, and screening for potential drug targets. Synthetic peptides are the protein equivalent of synthetic oligonucleotides, and to some exten
By Alan Dove | September 16, 2002
Synthetic peptides, generally less than 20 amino acids in length, are essential reagents for a wide range of studies and one of the fundamental tools of proteomics researchers. Applications include generating antibodies against specific epitopes, eluting specific proteins from affinity columns, testing the biological functions of enzymes, and screening for potential drug targets. Synthetic peptides are the protein equivalent of synthetic oligonucleotides, and to some extent the two technologies have had parallel histories.
But where DNA synthesis is routine, peptide synthesis is anything but. Its inherent complexities have ensured that the technique has remained more craft than science, and re-searchers must therefore consider more than just the bottom line when shopping for peptides. As a result, academic core facilities have been able to maintain their foothold in the peptide-making business, even as researchers go elsewhere for oligonucleotides.
HARDER THAN IT LOOKS As with oligonucleotides, the technology that made synthetic peptides accessible to researchers was a liquid-handling robot. In the mid-1980s, Applied Biosystems of Foster City, Calif., and Advanced ChemTech of Louisville, Ky., two companies that remain dominant players in the field, developed the first automated synthesizers. These instruments could synthesize only a single peptide at a time; many modern systems now can handle multiple syntheses simultaneously.
Inside the synthesizer, peptides are built one amino acid at a time. The synthesis starts at the peptide's C-terminus and proceeds to the N-terminus, exactly the reverse of protein translation in cells. In order to minimize unwanted reactions, the process uses specially modified amino acids, which have their amino groups chemically protected. After each residue is linked to the growing chain, its protective group is removed to allow the next amino acid to attach to it.
Though the process is similar in principle to oligonucleotide synthesis, the tremendous chemical diversity of the 20 standard amino acids makes each peptide synthesis unique. "You only have four bases in DNA, and they usually will couple to each other. You don't have as many problems with steric hindrance, ... deprotection, and side-chain reactions [as with peptide synthesis]," explains Janet Crawford, director of small-scale peptide synthesis for the Biotechnology Resource Laboratory at Yale University. Indeed, while molecular biologists have become accustomed to ordering any oligonucleotide sequence they desire, Crawford says that "we get some [peptide sequences] that are not synthesizable, or some of the membrane-spanning stuff is extremely hydrophobic and very difficult to analyze."
Once the peptide has been created, it must be analyzed and in many cases extensively purified, steps that can take considerably longer than the synthesis itself. In addition, many experiments require modified peptides, with phosphate groups, artificial side-chains, dye markers, or linking structures chemically attached. Mark Lively, professor of biochemistry at Wake Forest University and president of the Association of Biomolecular Resource Facilities, calls peptide synthesis an art form, adding that "every single product is different in properties and in steps required to process it."
Apparently, it is an art form with a growing audience. Anita Hong, founder of Anaspec of San Jose, Calif., says demand for peptides has followed the boom-bust-boom pattern common to many biotechnologies: "In the '80s ... it was a hot field and lots of people were looking at peptide drugs, but then in the '90s people started thinking that peptides cannot be used as drugs. Now there's more and more interest in peptides not just as drugs but also to study the mechanisms of drug [action]." Anaspec now fills orders for about 100 peptides a week and, like many other peptide synthesis services, has experienced a steady increase in demand over the past few years.
CAN THE CORE CENTERS HOLD ON? The increasing demand for peptides might be good news for academic core facilities, which have been squeezed in recent years as commercial suppliers have undercut both their prices and turnaround times for oligonucleotides. "There are many commercial houses that make [peptides], but their prices have not decreased by and large to the extent that oligo synthesis has," says Lively. In fact, peptide prices have remained relatively flat in recent years, as inflation has been offset by the increasing availability of cheaper imported starting reagents.
In addition, the unpredictability of peptide synthesis makes it difficult for companies to count on reaping economies of scale. As a result, core facilities, which need not turn a profit, may be able to provide a peptide for considerably less than a commercial supplier. "My purification is a little bit slower than an outside company," says Crawford, "[but] if you go outside, generally it will cost you three times as much." Crawford estimates that a typical peptide synthesis in her facility takes about two weeks, followed by a month to purify the crude peptide. Commercial peptide synthesizers generally take two to three weeks to complete both the synthesis and the purification.
Though they are clearly able to compete much more effectively in peptide synthesis than in oligonucleotide synthesis, core facilities are far from thriving. Instead, Lively defines success in negative terms: "Where there's a significant interest in peptide synthesis, those [core] labs are not dying as rapidly as the oligo labs are."
Most core labs are heavily subsidized, making them easy targets for funding cuts. Even if core services are offered at a reduced cost, an institution might cut expenses even more by eliminating the core lab and ordering peptides from commercial suppliers. Yale's thriving core facility is an exception, covering its costs entirely through user fees, with no subsidies from the university. In fact, the nonprofit facility provides peptides to academic and corporate researchers outside of Yale as well, though Crawford says surging demand has now forced her to close her doors to new outside business.
In the long term, even being self- supporting may not be enough to ensure a core facility's survival. "We don't provide grant overhead for space, so a university doesn't find us very attractive. We can be a selling point when they're trying to get new investigators, [but] we're bad the rest of the time," says Crawford.
ICKING THE PROPER PEPTIDE PROVIDER Deciding where to buy peptides entails more than just comparing prices; peptide synthesis is complex, and enormous variability can occur in the final product. Lively explains that peptides are "unlike oligos, where ... molecular biologists don't even care about the purity state, they just assume if it's in a package with a nice typed label it must be good. For peptides, you really do need to have good characterization." At a minimum, a peptide should come with a high-performance liquid chromatogram showing the sample's purity, and a mass spectrometry analysis demonstrating that it is the correct size for the sequence that was ordered.
Since every peptide is unique, it makes sense to shop around every time. A company or core facility that provides fast service and good quality for one peptide might be completely flummoxed by another. A facility that specializes in a particular type of peptide may even direct customers elsewhere if it is not up to the task. "I always determine whether this is a peptide I want to make, or is it a peptide they're better off getting somewhere else," says Lively.
In many cases, a peptide that has been used by other researchers and reported in the literature may be available for purchase off the shelf, rather than requiring custom production. This option is generally more expensive on a per-microgram basis, but the rapid turnaround time and ready availability of a well-defined product (far from guaranteed in a custom synthesis) make it an attractive choice. Anaspec offers several off-the-shelf peptides. Other companies frequently stock available peptides, even if not specifically advertised.
Some peptide synthesis companies are beginning to offer related services. Cell Essentials of Boston will synthesize a peptide and then produce antibodies against it. This service allows researchers to skip directly to the reagents they need. A few telephone calls could save weeks of aggravation.
Peptide buyers need to consider purity and quantity when examining their options. To make antibodies, a "machine grade" product that is about 85% pure is generally sufficient, and is much less expensive than purer peptides. The same grade can often be used for eluting proteins from an affinity column. Biological studies that depend on specific peptide-protein interactions, however, generally require peptides that are 99% pure.
Many facilities produce peptides in only one or a few concentrations. An instrument configured to produce 0.25 mmol of product will yield about 250 mg of a 1000-kDa peptide. This is an enormous quantity for some applications, yet insufficient for others. Researchers needing to screen large numbers of peptides to identify drug targets, for example, might want to shop for a lab capable of carrying out smaller-scale syntheses. Typical prices run from about $200 (US) per peptide for simple, small-scale synthesis and machine-grade purity, up to thousands of dollars for large quantities of a high-purity peptide requiring complex synthesis.
Whether ordering from a core lab or a private company, researchers should plan to spend some time discussing their specific needs with the facility's representative. Proteins will probably always be a challenge to study, but finding someone with the right expertise could make the process considerably easier.
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