Most peptide synthesis services purify products using reverse-phase high-performance liquid chromatography (HPLC), though sometimes alternative methods such as ion exchange or gel filtration chromatography may be used. Custom peptides that are unusual or difficult to produce may require special services. However, nearly every peptide synthesis service ships its product with HPLC and mass spectrometry (MS) results to verify the purity level and identity of the peptide.
In an ideal world, when placing an order for synthesized peptides, of course you wouldn’t hesitate to request the highest purity that the peptide synthesis service offers. After all, isn’t it always better to use reagents that are free from impurities? Not necessarily. Sometimes using a less pure sample is a cost-effective way to perform experiments that do not require ultrapure preparation. Peptide synthesis services almost invariably offer three or more levels of purity, labeled as “>X%.” What are the differences, and what purities are best used in different types of experiments?
Lower levels of purity
Generally, the lowest purity preparation is between >50% and >70%, with an average of about >60% purity. Products at this level are referred to as “crude” or “desalted” peptides. Though unflattering by name, this purity level can serve a useful purpose while saving you money, such as in initial peptide screens. Desalted peptides are appropriate for high-throughput screenings of large numbers of peptides for the generation of leads in pharmaceutical development, for example, as well as in other applications.
A >70% pure peptide preparation, which may contain a mixture of peptides that closely resemble one another, is likely to generate an immune response that will result in the antibody that you need. However, it is also possible for impurities at this level to cause unwanted effects in the animal or even some degree of toxicity. This can be caused by organic impurities remaining in the peptide preparation after the process of peptide synthesis. If this is a problem, it might be possible to extract the offending impurities from the >70% peptide preparation instead of buying a higher purity (and possibly more expensive) preparation of the peptide.
Mid-range levels of purity
Many analytical applications use mid-range peptide purities. Generally, a >70% to >80% purity preparation is appropriate for the production, purification and testing of antibodies. These products are also useful for ELISAs, enzyme substrate studies, epitope mapping, affinity purification, bioassays or other immunological applications, and peptide screening as described above.
Peptides purified to the >85% level are appropriate for biochemistry and semi-quantitative applications, such as enzymology, tests for biological activity and epitope mapping. Other examples of applications at this level include semi-quantitative studies of enzyme-substrate interactions, phosphorylation, peptide blocking in Western blotting and cell attachment.
Higher levels of purity
The higher levels of >90% to >98% purity are designed for particular applications. This level is the most expensive, and it usually isn’t necessary for the applications described above. Peptides purified to the >90% to >95% level are best for quantitative bioassays, quantitative in vitro receptor-ligand interaction studies, biological activity with ligand binding studies, quantitative blocking and competitive inhibition assays, quantitative phosphorylation and proteolysis studies, electrophoresis markers and chromatography standards.
Usually, the highest level of >98% purity is reserved for in vivo studies, clinical trials, drug studies that use peptides as pharmaceuticals and structure-activity relationship studies. This level is also best for examining protein structure by nuclear magnetic resonance studies or protein crystallography (though these latter three applications also can be accomplished using the >95% purity preparation).
A note on packaging
Some peptide preparations may require specialized packaging to make sure they remain intact and functional. Almost invariably, the product will be delivered to you as a lyophilized powder sealed in a polypropylene or glass vial with a screw cap, sometimes packed with an inert gas to prevent oxygen or moisture from entering. Although it is fine to ship purified peptides at atmospheric temperature for relatively short durations, it is best to store the lyophilized powder at -20°C after it arrives.
The type of synthetic peptide preparation you need may be apparent. If not, speaking with scientists at several different peptide synthesis services will enlighten you considerably. Also, keep in mind that although the categories described above are generally true, there may be particular instances of overlap, in which more than one purity level may suffice. A little investigation will help you find the right peptide purity level to get the job done.
The image at the top of the page is from Thermo Scientific Pierce Protein Research Products' Peptide Synthesis.