What Are Synthetic Peptides?
Synthetic peptides are chemically synthesized small polymers of amino acids. You can think of synthetic peptides as being to proteins what oligos are to DNA. The chemistry used to synthesize a peptide bond between two amino acids has been known for 100 years, and the first small proteins were chemically synthesized in the 1950s and 1960s. 
In brief, the synthesis reaction consists of joining the carboxyl group of an amino acid to the amino group of the previous amino acid in the peptide chain. Various reactive groups on the side chains and termini must be chemically protected to prevent undesired reactions from occurring.
Now, peptides are easily synthesized on a very large scale, and you can just place an order online and receive them days later!  In this article, we will explore the applications of synthetic peptides as well as their pros and cons.
Synthetic Peptides vs. Recombinant Proteins
Is it possible to make an entire protein synthetically? The size limit for synthetic peptides is about 20 to 50 amino acids,  which is the size of some naturally occurring polypeptides like the insulin A and B chains. 
You can also link synthetic peptides through chemical ligation to chemically synthesize larger proteins. By assembling and joining partially protected peptides, Nishiuchi et al.  synthesized the 238-residue precursor for green fluorescent protein (GFP) and found that the fluorescence profile in solution was identical to that of recombinant GFP. But we’ll get into more detail on this in Part 2. For now, know that it is possible to chemically synthesize an entire protein, it is just not that easy, and it is not always advantageous.
It is worth mentioning in vitro translation systems (IVT). Here, you use crude cell extracts (usually wheat germ or rabbit reticulocytes) along with mRNA, amino acids, and salts to create peptides in a test tube instead of in live cells. IVT provides the benefits of using biological components to make a polypeptide without having to worry about growing cells. This approach is ideal for producing proteins that may be toxic to the host cell. For some small-scale applications where you need to make a peptide exceeding 100 amino acids quickly, IVT may be the way to go.
So why use a synthetic peptide over a recombinant or IVT-generated protein?
Why Use Synthetic Peptides?
For one, you can make them quickly, and you do not have to worry about cell culture systems or downstream purification like you would with recombinant proteins. If you need to look at an SH3 binding motif or many of them, you can get a ton synthesized very quickly.
Second, you can isolate different elements of proteins like binding sites or kinase substrates. With full-length proteins, the presence of additional factors, domains, or motifs can confound this type of experiment. However, if an interaction is not simple, peptides may come up short.
And third, given the ease of making synthetic peptides in large quantities, you can easily do high-throughput experiments, like measuring binding kinetics or looking at libraries of peptides. Purified proteins require a great deal of preparation to generate enough material for measuring kinetics, and you are limited in scope to a handful of protein analytes at a time.
Pros and Cons of Synthetic Peptides
Here are some pros and cons of synthetic peptides:
- Can be cost-effective: For applications that only require a peptide 20–50 amino acids long, chemical synthesis is the way to go. There are vendors who can do this for you rapidly. The time savings alone, compared with cloning the gene and expressing in a cellular system is worth it.
- Can incorporate some post-translational modifications like Tyr or Ser phosphorylation.
- High purity and precision: You control exactly how the peptide is composed and do not need to worry about the complexity of a biological matrix. Furthermore, you do not need to add an affinity tag for purification, which can impact the native function of the peptide.
- Limited to the size you can create (20–50 amino acids).
- Can be problematic for interactions that require post-translational modifications that are difficult to incorporate synthetically, such as glycosylation, for biological activity.
- Difficult to create disulfide linkages.
- May not be appropriate in applications where the secondary or tertiary structure is critical or in making larger bioactive proteins.
Uses of Synthetic Peptides
Generating Custom Antibodies
Synthetic peptides can be used as antigens to generate custom antibodies. When coupled to a carrier protein, the peptide can stimulate a host humoral immune response and generate both monoclonal and polyclonal antibodies. This approach allows you to control the epitope much more easily than when using the whole native protein.  The amino acid sequence of the antigen is critical, though. While this is an inexact science, there are free online tools  to help you select the sequence most likely to elicit an immune response.
Peptide-based antibodies are ideal for use in Western blots. If you include a phosphorylated amino acid in the peptide, you could generate an antibody that specifically detects the phosphorylated protein. Couple this with an antibody raised against the same peptide containing the non-phosphorylated residue(s), and you can stain your Western blot for both the phosphorylated and non-phosphorylated forms of the protein. This approach is widely used in cell signaling work where you are interested in probing protein kinase and phosphatase interactions. And you won’t have to worry about cleaning up P-32!
In general, synthetic peptides are great for breaking down the basic elements of protein-protein interaction.  Biotinylated peptides can be immobilized on streptavidin-coupled beads and used to pull down proteins that interact with the peptide. This is used in epigenetics research where peptides corresponding to histone tails with and without post-translational modifications are incubated with nuclear extracts. Associated proteins can be analyzed via SDS-PAGE. 
Synthetic peptides are also ideal for methods like NMR or fluorescence anisotropy, where a smaller size is a key asset. Finally, another application of peptides is as substrates for enzymatic reactions. Sugiyama et al. (2019) used peptide libraries and purified kinases to identify direct kinase substrates.  This would have been very difficult if not impossible using cellular systems and looking for the interactions of native or recombinant proteins.
Synthetic peptides can be used as standards in mass spectrometry to aid in quantitation and identification. When a protein is digested and analyzed by mass spec, it can be very difficult to quantify the resulting peptides because the mass spec peak depends on the peptides’ chemical make-up in addition to concentration.
For example, if you are interested in quantifying the levels of a given post-translational modification of your protein, you can spike in an isotopically labeled peptide with that modification at a known concentration. You now have an internal standard that allows you to quantify the levels of native peptide.
Isotopically labeled synthetic peptides can also help you identify peaks in an MS/MS spectrum since they will produce identical patterns in the MS/MS spectrum as native peptides, simply offset by the difference in molecular weight based on the isotopes used.
One additional application of synthetic peptides that is germane to current events is in enzyme-linked immunosorbent spot (ELISPOT) assays, which is being used quite a bit these days to test host responses to vaccines against SARS-CoV-2.  T cells are harvested from patients and challenged in culture using peptides corresponding to the SARS-CoV-2 proteins used in the vaccine. If the cultured cells release interferon-gamma (IFN-\gamma), this suggests that the vaccine can stimulate cellular immunity in the patient. Being able to produce specific peptides allows you to home in on the exact antigen.
Do you have experience making or using synthetic peptides? Leave a comment below. Also, stay tuned for part 2, where we will discuss how to design peptides and the use of nonnative amino acids.
- Merrifield, R. B. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society, 85 (1963). doi:10.1021/ja00897a025.
- Synthetic Peptides. Monoclonal Antibody Core. Accessed on 14 Aug 2020.
- Merrifield, R.B., et al. Instrument for automated synthesis of peptides. Anal Chem., 38, 13 (1966). doi: 10.1021/ac50155a057
- Goeddel, D.V., Kleid, D.G., Bolivar, F., et al. Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci USA., 76, 1 (1979). doi: 10.1073/pnas.76.1.106
- Nishiuchi, Y., et al. Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence.?Proc Natl Acad Sci USA., 95, 23, (1998). doi:10.1073/pnas.95.23.13549
- Lee, B.S., et al. Antibody production with synthetic peptides. Methods Mol Biol. 1474, (2016). doi:10.1007/978-1-4939-6352-2_2
- Peptide Antigen Database. GenScript®. Accessed on 14 Aug 2020.
- Peptide-Protein Interactions. LifeTein. Accessed on 14 Aug 2020.
- Roos, A.K. and Wysocka, J. Peptide pull-down (PPD) assay for identification and characterization of histone PTM Effectors (PROT46). (2009). Accessed 14 Aug 2020.
- Sugiyama, N., et al. Large-scale discovery of substrates of the human kinome. Sci Rep., 9:10503 (2019). doi:10.1038/s41598-019-46385-4
- Zhu, F-C., et al. Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet, 396, 10249 (2020). doi:10.1016/S0140-6736(20)31605-6