Peptide Chemistry Penetrates A Growing Range Of Disciplines

Peptides--the amino acid chains that make up proteins--were long relegated to a sideline of the much larger field of protein chemistry. But over the last few decades, their importance has grown as the understanding of these biological building blocks has become essential to a multitude of sciences, from agriculture to therapeutics to the Human Genome Project. As peptide chemistry continues to make its way into other disciplines, it behooves researchers from these disciplines to gain a basic grounding in the techniques of peptide chemistry. One of the first scientists to recognize the potential of peptide chemistry was Frederick Sanger, who in 1944 embarked upon a 12-year project to unravel the amino acid sequence of the protein molecule insulin. Sanger eventually solved this puzzle by breaking the insulin molecule into two peptide chains, breaking them in turn into still smaller chains, figuring out their sequences, and then overlapping the sequences of the constituent chains. Sanger won the Nobel Prize for chemistry in 1958 for his work, and the science of peptide chemistry came into its own. Today's biochemists and molecular biologists are armed with a sophisticated array of equipment enabling them to separate and purify peptides; to take them apart, amino acid by amino acid; and to even synthesize useful peptides created to fit their own specific applications. Peptides are a diverse class of molecules. They can function as cofactors to enhance enzymatic catalysis, or as chemical messengers, such as enteropeptides and neuropeptides, which influence the function of the endocrine and nervous systems. Synthetic peptides have become entrenched in our everyday life--the dipeptide derivative aspartame (NutraSweet) is a daily staple of dieters everywhere. Peptide hormones--such as vasopressin, used to treat metabolic disorders--have been created synthetically for use as therapeutics, along with other peptide drugs and vaccines. The simplicity of peptides has made them extremely useful to researchers who work with them in the study of protein structure, to look more closely at the various "motifs"--sheets and turns, zippers and fingers--that make up a protein's three-dimensional structure. Regardless of the application, one of the first steps in peptide analysis is to separate the peptide of interest from a biological mixture or collection of peptides. In the late 1970s, gel electrophoresis reigned in the science of protein separation. Today, capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) are the most popular methods. These techniques are used for such tasks as isolating peptides and their component amino acids from physiological fluids, sorting the mixture of peptides that results from protein digestion, or purifying a sample following peptide synthesis. In HPLC, a peptide mixture is passed through a column under pressure. The components are then sorted on the basis of their interaction with the column material--for example, they may be separated by size (size-exclusion chromatography) or by charge (ion-exchange chromatography). Whereas HPLC relies on the chemistry of chromatography, CE is based on the same principles of gel electrophoresis that researchers have used for years. The difference is that capillary electrophoresis is carried out in narrow-bore capillary tubes with an inner diameter of only 25 to 50 micrometers (The Scientist, Sept. 3, 1990, page 27). Samples are injected into the capillary and move through the tube as voltage is applied. Because the capillaries dissipate heat effectively, very high voltages (up to 30,000 volts) can be applied. This translates into very rapid and highly efficient separations. In a very short time, with only nanoliters of sample, capillary electrophoresis can resolve peptides that differ only slightly on the molecular level. Researchers interested in peptide separations see these two techniques as complementary--they can use HPLC for initial separations involving protein digests or synthesis reactions, and then CE to characterize the peptide fractions recovered from the HPLC separation. A number of companies market fully automated versions of both HPLC and CE that allow for online, real-time detection of the peptide products. Once the purified peptide is in hand, researchers have at their disposal a number of technologies that can be used alone or together to aid them in its characterization. Amino acid compositional studies are often performed following peptide synthesis to confirm the peptide's amino acid sequence, or as part of the much larger task of protein structural analysis. According to Tracy Zoellner, marketing specialist for Applied Biosystems Inc. of Foster City, Calif., compositional analysis is an important complement to more detailed ways of looking at peptides, such as sequencing. It can be used, Zoellner says, "to confirm a sample's identity, for example, or in case the N-terminus is blocked and the peptide can't be sequenced." The arduous and lengthy task of amino acid compositional analysis has been simplified by the development of machines that automate the four key steps in amino acid analysis--hydrolysis, derivatization, separation (using an online HPLC), and data collection. Since Sanger and Swedish chemist Pehr Edman separately developed methods of sequencing amino acids in the 1950s, sequence analysis--the determination of the linear arrangement of amino acids in a peptide or protein--has become perhaps the most widely used technique in peptide chemistry. Via a method known as the Edman degradation, peptides are reacted with phenylisothiocyanate (PITC) under acidic conditions. This selectively labels the amino terminal residue and cleaves it off, without disrupting the rest of the chain. The labeled residue is then identified chromatographically. This degradation and analysis process continues cyclically until a peptide sequence is determined. In 1967, Edman and Geoffrey Begg, both working at St. Vincent's School of Medical Research in Victoria, Australia, created an automated sequencing machine. Their invention, subsequently marketed by Beckman Instruments Inc. of Fullerton, Calif., touched off an explosion in protein biochemistry by making it both possible and practical for researchers to obtain structural data on all sorts of proteins--enzymes, structural proteins, and hormones, to name only a few. Today researchers can combine fully automated gas, liquid, or solid-phase sequencers with online computers to determine the exact sequence of amino acids in any given peptide. The uses of this kind of information seem limitless. From the amino acid sequence, researchers can investigate the structure and function of peptides, either alone or as part of a larger protein, by generating computer models to predict how a particular sequence may fold, for example. This technology has found a niche with molecular biologists, who create oligonucleotide probes by reversing the genetic code of a peptide's amino acid sequence. These probes can then be used to clone and sequence otherwise elusive genes. New microsequencing techniques can be implemented to determine the sequence of peptides only available in tiny amounts--in the submicrogram range. Sometimes amino acid analysis and sequencing may not provide researchers with a complete picture of a given peptide. Mass spectrometry, an analytical technique that determines the exact molecular weight of a sample, helps researchers detect anomalies introduced by prior analyses of the peptide. In nearly all instances, the first step in determining the composition of a peptide or protein is acid hydrolysis, a process that destroys the peptide linkages between individual amino acids. The harsh conditions under which these reactions are performed can destroy amino acids that are particularly susceptible to hydrolysis, while not completely cleaving the bonds between others. Synthesized peptides in particular may be subject to reactions that create modified amino acid residues. In addition, subsequent sequencing reactions may be hindered by the incomplete removal of blocking groups. Mass spectrometry can help expose these problems. Mass spectrometry is based on the principle that a charged particle will be deflected when passed through a magnetic field. The degree of deflection is dependent upon the particle's momentum, which in turn is dependent on its mass. Since this method detects only charged particles, peptides are first bombarded by electrons before passing through the magnetic field. The researcher then determines the precise molecular weight of the peptide--down to a single atom--by observing the amount of deflection of each particle, using a spectrometer. Mass spectrometry complements traditional compositional and sequencing studies to provide a complete picture of the peptide, from assessing sample purity and sequence to post-translational modifications. Virtually every peptide chemist, at some point in his or her career, will need to synthesize peptides for some research or clinical purpose (The Scientist, Oct. 1, 1990, page 24). Early on, peptides were synthesized in a series of cumbersome and lengthy chemical steps, requiring quantities of acids and esters and coupling reagents, along with washes, precipitations, and filtration steps. The synthesis of peptides longer than even a dozen residues required considerable time and effort. The development of solid-phase peptide synthesis by Nobel Prize winner Robert Bruce Merrifield in the 1960s released researchers from this arduous task. To create a synthetic peptide, the first amino acid is attached via its carboxyl end (C-terminus) to an insoluble support resin. The exposed amino end (N-terminus) of the residue is activated, or "deprotected." The next amino acid is added along with a coupling reagent that activates the second amino acid so that it will react with the first, leading to the formation of a peptide bond between them. The resin is then washed to remove excess reagents. This process is continued cyclically until the peptide is complete. The finished product is then released from the resin. Synthesized peptides have many applications. For example, once the amino acid sequence of the antigenic region, or epitope, of a protein is known, scientists can synthesize a peptide of only that region, which can then be used to generate antibodies. Researchers have found that these peptides are capable of eliciting an immune response in much the same way as the original protein. Using only a single epitope may increase the specificity of the antibody, while decreasing cross-reactivity. Synthetic peptides can also be put to use as models in active-site studies in an effort to better understand the interactions that occur between proteins and other biological molecules. Being able to sequence and then synthesize peptides also gives researchers the opportunity to selectively alter the amino acid sequence, and then study the effect on the peptide's structure or biological function. Today's peptide-synthesis technology includes both manual and automated machines of varying price. Some, like the RaMPS system, marketed by Du Pont Co. of Wilmington, Del., are so simple that virtually anyone can successfully synthesize a peptide with it. However, in today's climate of dwindling grant support, purchasing a machine may be out of the question. A growing number of companies, such as the Rockville, Md.-based Synthecell Corp., offer custom-made peptides to researchers who only use a few peptides in a year. The ability to analyze, sequence, and synthesize peptides brought about a revolution in peptide chemistry, and is shaping the future of biology as well. As the study of the human genome progresses, recognition of the value of peptides as research, diagnostic, and therapeutic tools will surely lead researchers closer to a better understanding of the intricate process of life. Holly Ahern teaches cellular and molecular biology at the State University of New York, Albany.

Peptide Chemistry Penetrates A Growing Range Of Disciplines

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