Peptides, Made to Order

The use of synthetic peptides in life science research is growing as researchers in a wide variety of disciplines find applications for these reagents. Many of these peptides are easily produced by automated peptide synthesis, which enables the economic and relatively easy production of peptides in large quantities. As a result, many widely used, biologically active peptides are commercially available as pre-made catalog reagents.1 But when experimental approaches call for peptides that are not on the market, researchers have two basic options: make the peptides from scratch, or employ the services of a custom peptide facility. Those who use peptides as tools in their studies but are not focused on or experienced with the synthetic processes per se often choose to outsource the fabrication of these reagents. For academic researchers, many universities and institutes maintain their own core facilities for peptide synthesis. When such facilities are not available, or when they do not offer the exact services needed (e.g., a specific type of synthesis or desired quality control measure), an investigator can turn to one of the many external commercial sources. This profile details the various options that should be considered when choosing a custom peptide facility, including some general questions that should be addressed. Specific information about companies providing custom peptide synthesis services is presented in the accompanying table. Researchers familiar with using custom services know that reputation, built from consistent results and word-of-mouth from other scientists, is perhaps the best way to estimate the quality of work done by a particular company. Researchers should also evaluate how closely each candidate matches the desired services, timeline, and budgetary constraints. There are many companies to choose from, ranging from those focused on quick turnaround times and low prices for routine syntheses, to those specializing in more complex syntheses. Time spent up front comparing commercially available options may prove to be time well spent in the long run. This is especially true if one's work requires the use of numerous custom peptides over time, or if the studies require peptides that are difficult to make. The chemical makeup of the desired peptides, the relative quantities needed, and the types of applications they will be used for can all influence the type of company chosen for a given project. One of the first issues to consider is product yield. If only a few experiments are expected, milligram quantities of product may suffice, but a large series of studies will require correspondingly larger amounts of peptide. Not every company can accommodate these larger yields: Although some vendors offer samples in kilogram or even "industrial" quantities per run, most companies offer peptides in the milligram to "multigram" (hundreds of grams) range. A second consideration is purity and quality control. Whereas some companies emphasize extensive quality control measures, and may even offer full money-back or replacement guarantees, others focus on low prices and fast delivery. Peptides can always be purchased crude and desalted, but otherwise unpurified after synthesis. However, many companies also offer purification services. Most offer several purification tiers, each associated with progressively higher fees. The choice of purity level is generally dictated by the application. Those in need of peptide antigens for polyclonal antibody generation may find that "immunological grade" peptides (ranging from 60 percent to greater than 75 percent) are of sufficient purity. In contrast, researchers employing these specialty reagents for monoclonal antibody production or bioassays may need more highly purified versions. For other applications, intermediate (75-85 percent), highly purified (greater than 95 percent), or ultra pure (greater than 98 percent) peptides may be required. Most companies provide analytical HPLC and mass spectrometry analyses for quality control. Some also offer amino acid analysis or peptide sequencing. Certain clinical and pharmaceutical applications require the use of good manufacturing practices, and a number of companies produce peptides according to these standards. Pep Talk Peptides are complex molecules with sequence-dependent physiochemical properties. Because all peptides are not created equal, not all companies are capable of synthesizing a particular peptide, either because of its length, chemical composition, or required modifications. Most basic synthesis applications call for solid-phase peptide synthesis (SPPS),2 for which two basic chemistries are widely used. One employs 9-fluorenylmethyloxycarbonyl (Fmoc) for N-a-amino protection, and the other uses tertiary butoxylcarbonyl (t-Boc). In the latter, a relatively strong acid (usually hydrogen fluoride) is required to remove the final product from the resin. The milder conditions of the Fmoc chemistry have made it the preferred choice in many peptide laboratories. However, the t-Boc chemistry may be advantageous in some cases, and a mixed t-Boc/Fmoc approach is sometimes used to synthesize a single peptide containing certain functional groups. Custom peptide houses generally prefer to use the Fmoc approach when possible. Some, but not all, are willing to employ t-Boc chemistry when the application calls for it. More complex peptides may require significant changes in the SPPS protocol. For example, the construction of peptides with multiple disulfide bonds requires different sets of protecting groups on cysteines.3 Also, peptides rich in hydrophobic residues can be difficult to synthesize due to decreased coupling efficiency caused by the formation of inter-chain b-sheet aggregates during synthesis. If modifying the sequence is not an option, different reaction conditions, protection schemes, or resin types may help increase coupling efficiencies.2 Some modifications are more straightforward to implement than others. Although most companies advertise a number of specific modification services, they can usually perform modifications in addition to those listed. The relative numbers and diversity of services listed in promotional materials may, at least in some cases, reflect the company's general comfort level with more challenging projects. When ordering peptides that are not readily synthesized via conventional automated solid-phase methods under standard conditions, it is a good idea to ask whether the particular company has experience with the specific synthesis strategies needed. In many cases, these peptide synthesis services employ chemists who can provide the expertise necessary to tackle new types of projects. Peptide length is another issue. Especially short (less than five residues) and long (greater than 50 residues) peptides can be difficult to make. In some cases, formats other than stepwise SPPS, such as solution chemistry or "fragment assembly," are called for. In fragment assembly, individual peptide strands are made separately, then covalently linked to form the desired product.2 In a subclass of fragment assembly strategies known as "convergent synthesis," fully protected fragments are coupled while one is still resin-bound. Another general approach is to couple the purified fragments in solution via chemoselective ligation. Schemes for generating complex peptides can incorporate more than one type of synthetic format. One consideration that may not be obvious is the company's "focus." For example, some companies focus primarily on custom peptide services and related products, whereas others have a broader bent toward organic chemistry, and offer other types of syntheses as well. Some companies meet different specialty needs, such as the synthesis of radioactively labeled custom peptides. Others provide peptide libraries, arrays, and peptide mimetics, which are non-peptidyl compounds designed to behave like a peptide. Still other companies focus on providing an economical source of peptides readily produced via automated synthesis. Although these companies may be willing to attempt some of the more difficult syntheses, this is not their major focus. Companies with expertise in molecular biological approaches offer yet another alternative: custom cloning, expression, and even purification of recombinant proteins. In some cases, companies that initially focused on other technologies have expanded to include custom peptide synthesis as well. For example, a number of immunological companies have found that many customers appreciate the convenience of being able to order their peptide antigens and subsequent antibody-generation projects from the same company. Similarly, numerous companies that specialize in peptides offer products of interest to those involved in immunological studies. For example, multiple antigenic peptides (MAPs), which consist of small immunologically inert cores of branching lysine dendrites onto which multiple copies (usually four or eight) of a peptide antigen (or antigens) are attached, are becoming common fare. Most custom peptide houses will conjugate proteins or peptides to carrier proteins such as bovine serum albumin or keyhole limpet hemocyanine, and many will conjugate proteins to other moieties as well. The Bottom Line The obvious consideration is cost--peptides can be expensive reagents. Because different companies use different pricing strategies, it is better to consider each company's policy in its entirety rather than to focus on just one aspect. Some companies do not advertise set price ranges, but rather prefer to provide quotes upon request. Companies may charge per residue or use a stepladder approach, giving a set price for peptides falling within several different ranges. Higher purity and modifications will generally result in additional charges. Some analyses are standard procedure; others cost extra. For example, most of the companies that offer amino acid analysis charge an extra fee for this service. Another issue is whether the price quoted is per synthesis, or per product. For complex peptides, multiple syntheses might be necessary to make the desired quantity. In other words, be sure to read the fine print. Courtesy of Biosource InternationalRibbon diagram of interleukin-8 Though this article focused on commercial options for custom peptide production, a researcher can, of course, choose to equip his or her own lab for peptide generation. "Do-it-yourselfers" are free to experiment with different synthesis strategies when applicable, and may optimize each peptide production protocol. Further, they can choose their own purification and analysis methods, institute their own "quality control" standards, and work within their own time frame. The Scientist will report on commercially available peptide synthesizers in a future article. The reasons for using custom peptides are as varied as the life sciences themselves. For example, synthetic analogues of biomolecules enable researchers to uncover the relationships between molecular structure and biological or pharmacological activity.3 In some cases, compounds with activity, selectivity, or stability superior to the natural product can be produced at the industrial scale. The magic of chemistry means that investigators need not be confined by the structures of naturally occurring molecules or their closely related analogues. Indeed, in their book about peptide and protein synthesis,3 Paul Lloyd-Williams, Fernando Albericio, and Ernest Giralt write, "In the field of protein de novo design it might be said that 'anything goes.' L-Amino acids can be combined with D-amino acids; natural amino acids can be combined with non-natural ones; and peptide sequences that are unknown in nature can be investigated. ...We can hope that, among other things, [such endeavors] will provide us with a better knowledge of the rules that control protein folding and allow us to produce compounds with tailor-made properties." In the meantime, however, scientists no longer have to rely on nature to provide interesting reagents for them. Thanks to custom peptide synthesis facilities, they can make these products for themselves. Deborah A. Fitzgerald (df@sciwriters.com) is a biotechnology writer based in Birmingham, Ala.; Jennifer S. Guimbellot (JGuimbellot@peds.uab.edu) is a freelance writer based in Birmingham, Ala. References 1. P. Diehl, "Biologically active peptides: Who makes them and who sells them?" The Scientist, 12[18]:16, Sept. 14, 1998. 2. J. Borgia, G. Fields, "Chemical synthesis of proteins," Trends in Biotechnology 18:243-51, 2000. 3. P. Lloyd-Williams et al., Chemical approaches to the synthesis of peptides and proteins, Boca Raton, Fla.: CRC Press, 1997. Supplemental Table Click to view the PDF file: Custom Peptide Synthesis Services (Research-scale quantities)

Peptides, Made to Order

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