Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Let’s say you need some purified peptides for your next experiment. Should you make your own or order them from a peptide-synthesis service?
Synthesized peptides have a range of applications, including antibody production, antibody purification and epitope mapping. They’re used as peptide hormones and enzyme substrates and also in the construction of peptide microarrays.
So there’s a good chance you’ve used a peptide as some point in your research. But have you ever made one yourself? Perhaps not, but you probably have the skills to do it, and it might be cheaper than outsourcing the job. On the other hand, doing the work in-house takes time you might otherwise spend on the research you really want to do. And ordering synthetic peptides may be more affordable than you think.
“When deciding whether to synthesize one’s own peptide or pay a peptide company, one must consider whether the savings [are] worth the time and effort, even if one has the necessary peptide-synthesis background,” says Raman Afshar, business development manager at AnaSpec, a company that provides synthesis services. When you factor in the advantages of having your peptides synthesized, purified and QC’d by experts who know what they’re doing, a service may well end up saving you considerable time and frustration.
Benefits of outsourcing
The most obvious benefit of a peptide-synthesis service is convenience. Hervé Le Calvez, president and CEO of peptide-synthesis firm Abbiotec, says his company routinely makes 10 to 20 amino-acid peptides at a time using a peptide synthesizer, followed by purification by HPLC.
“Peptide-synthesis services already have well-established and regular production protocols for regular and efficient peptide synthesis, purification and quality control,” says Stacey Hoge, market segment manager at Sigma Life Science, Custom Products. Not many labs do this routinely while also concentrating on other areas of research, so outsourcing the job can save you time that you’d rather spend doing science.
Another benefit of using a peptide-synthesis service is consistency. Many services monitor the quality of their peptides using liquid chromatography-mass spectrometry (LC-MS), producing high-quality peptides that would be time consuming to make and verify yourself.
“Given that peptide synthesis is a complex, multifaceted process, problems can arise,” says Connie McGee, project specialist for custom peptides at Thermo Fisher Scientific. Peptide-synthesis services “possess the tools, expertise and sourcing capabilities to troubleshoot and produce the peptide in a timely manner.”
Expert advice is another plus. “Using a peptide-synthesis service allows a staff scientist experienced in peptide design to aid the researcher in appropriate peptide design,” says Dan Kenney, director of scientific business development at Rockland Immunochemicals. Staff scientists can help users optimize such variables as peptide solubility, length, immunogenicity, post-translational modifications, purity and quantity. A scientist who specializes in peptides likely will know how to head off pitfalls before you encounter them, which can save you time and money.
And don’t forget about reproducibility, says Hoge. If your results rely on the peptides, it is vital that competing or collaborating labs be able to count on peptides being the same from batch to batch.
Labeling or special modifications
Although simple peptides may be easier to synthesize in-house, you might consider a service if you need peptides with a specific label or special modifications. Le Calvez says Abbiotec’s scientists specialize in long peptides of 40 to 100 amino acids, and they can append such tags as biotin, fluorescent dyes or colorimetric groups onto a peptide’s N- or C-terminus. “We have also shown our expertise in non-natural amino acids present in opioid-related peptides secreted by amphibians, such as dermorphin,” says Le Calvez.
In addition to adding labels for detection, another complicated part of peptide engineering is the addition of chemical modifications. Modifications that AnaSpec routinely adds to its peptides include acetylation, biotinylation, citrullination, glycosylation, methylation, phosphorylation, PEGylation and small-molecule conjugations, says Afshar. The company also can attach cyclic peptides, or peptides used in fluorescent resonance energy transfer (FRET), he says.
Purification strategies
Purification is an essential step in peptide production, because after synthesis the crude sample may be only about 40% to 50% pure. The sample “contains many by-products which are a result of deletion or truncated peptides, as well as side products stemming from cleaved side chains or oxidation during the cleavage and deprotection process,” says Afshar. After purification, the peptide sample may be 90% to 98% pure, depending on the nature of the peptide, its modifications (if any) and the purification strategies.
Most peptide-synthesis services purify synthesized peptides using HPLC, usually with a reverse-phase or ion-exchange strategy. Reverse-phase HPLC separates peptides according to their hydrophobicity; ion-exchange methods separate peptides by their charge interactions with the column materials.
At Abbiotec, peptides are purified by reverse-phase HPLC. “A crude peptide corresponds to a purity of 70% with salt and solvent contaminants (sodium acetate and trifluoroacetic acid), whereas [an] HPLC-purified peptide has a purity of 90% to 98% free of contaminants,” says Le Calvez. Although antibodies can be produced with a purity of more than 70%, he suggests using peptides with a purity of more than 90% for most other applications. Exceptions include in vivo studies and perhaps other specific types of in vitro assays (depending on the nature of the experiment) in which a purity of more than 98% would be beneficial.
The costs of synthesized peptides usually are rated according to purity grade and peptide length, and can range anywhere from hundreds to thousands of dollars. For example, Le Calvez says that at Abbiotec, “most of our prices fall into the $12-15 per amino acid range for 70-90% grade purity, and $15-25 per amino acid for 95-98% grade purity, for routine sequences.” McGee says peptides can run from $22 for “the simplest of peptides” to $2,000 and up for longer, modified molecules.
That may sound expensive, but remember: When it comes to peptides, primary synthesis is only part of the equation. You may find that time saved and quality assured more than balance the cost of outsourcing.