Synthetic Peptides: Their Uses PLUS Tips and Tricks for Peptide Synthesis

In this article, we will discuss some applications for synthetic peptides, as well as the basics of peptide synthesis, with a brief explanation of blocking reactive side chains so that only the carboxy and amino termini react in the presence of the catalyst.

We’ll also explore various design tricks for peptide synthesis, such as improving solubility, designing long peptides, and incorporating post-translational modifications.

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.¹

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!² 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,³ which is the size of some naturally occurring polypeptides like the insulin A and B chains.⁴

You can also link synthetic peptides through chemical ligation to chemically synthesize larger proteins. By assembling and joining partially protected peptides, Nishiuchi et al.⁵ 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 later on when we cover tips and tricks for peptide synthesis. 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.

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	Cons
Can incorporate some post-translational modifications like Tyr or Ser phosphorylation.	Difficult to create disulfide linkages.
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 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 it in a cellular system is worth it	May not be appropriate in applications where the secondary or tertiary structure is critical or in making larger bioactive proteins.
	Can be problematic for interactions that require post-translational modifications that are difficult to incorporate synthetically, such as glycosylation, for biological activity.

Uses of Synthetic Peptides

1. 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.⁶ The amino acid sequence of the antigen is critical, though. While this is an inexact science, there are free online tools⁷ 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!

2. Protein-Protein Interactions

In general, synthetic peptides are great for breaking down the basic elements of protein-protein interaction.⁸ 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.⁹

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.¹⁰ This would have been very difficult if not impossible using cellular systems and looking for the interactions of native or recombinant proteins.

3. Mass Spectrometry

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 makeup 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 peptides.

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.

4. ELIspot

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.

Designing Synthetic Peptides

When planning peptide synthesis, you should take certain physical properties into consideration.

Peptide Length

The first is peptide length. You can easily synthesize shorter peptides, from 5 to 10 amino acids long, and it is typically possible to synthesize a peptide of up to 50 amino acids. The exact size you need depends on your specific application, but when you start getting longer than 50 amino acids, you will have issues with purity and yield.

Peptide Solubility

The next consideration is solubility. The more hydrophobic residues you incorporate (e.g. isoleucine and phenylalanine), the less soluble your peptide will be in an aqueous buffer. A good rule of thumb is that every fifth amino acid should be charged.¹⁰

If you are mimicking a naturally occurring peptide, you might have to modify the sequence by substituting in a charged amino acid to increase solubility. Some trial and error will be required to get a mostly hydrophobic peptide into solution.

Undesirable Secondary Structure

Another interesting consideration is whether significant hydrogen bonding will occur within the peptide. Polar amino acids such as asparagine, glutamine, serine, and threonine can participate in hydrogen bonding with each other and with water. In naturally occurring proteins, these hydrogen bonds help to stabilize secondary structures such as turns, \(alpha\)-helices, and \(beta\)-sheets.

Even though your peptide may not be long enough for these secondary structures to form, a peptide composed mostly of hydrophilic amino acids could gel in an aqueous solvent.¹¹

If you cannot alter the amino acid composition of your peptide, consider modifying the solvent’s pH. Changing the pH can change the charge of polar side groups, thereby affecting their ability to form hydrogen bonds and secondary structures.

Oxidation of Sulfur-containing Amino Acids

Sulfur-containing amino acids, such as cysteine and methionine, can oxidize easily, creating problems during synthesis, and lead to unwanted disulfide bridges (in the case of cysteine). You may be able to substitute these residues for non-sulfur-containing residues such as serine or norleucine.

You could also consider adding reducing agents to your solvent to prevent the formation of disulfide bonds.

Table 2 sums up these various considerations for peptide synthesis.

Characteristic	Consideration
Length	Anything larger than 50 amino acids will decrease synthesis efficiency and purity of the final preparation.
Solubility	Numerous hydrophobic residues will make solubility in an aqueous solvent difficult. Incorporate charged residues if the peptide sequence contains numerous hydrophobic residues.
Secondary structure	Sequences that promote hydrogen bonding or runs of Gln, Ile, Leu, Phe, Thr, Tyr or Val can promote secondary structures. Modify the solvent pH or introduce conservative substitutions to avoid charged residues and the formation of ionic bonds that may stabilize unwanted secondary structure.
Oxidation	Met and Cys are easily oxidized and can lead to issues during synthesis. Substitute these with Ser and norleucine when possible.

How to Synthesize Long Peptides or Proteins

If you need to make a long peptide or even a whole protein, how do you go about it knowing that synthesis beyond 50 amino acids is impractical? One option is to synthesize smaller peptides and ligate them together via native chemical ligation (NCL).

NCL relies on chemoselective bond formation between a C-terminal thioester and an N-terminal cysteine. Basically, the first peptide ends with a thioester, and the second peptide beings with a cysteine. The two react in an aqueous solution, ligating the two peptides together to create a larger peptide.¹²

What if your peptide sequence lacks a cysteine? One thing you can do is desulfonate the ligated peptide yielding an alanine.

There are also synthetic thiol-derived amino acids that can be incorporated at the N-terminus of the second peptide. After ligation, desulfonation will yield a native polypeptide. For example, \(beta\)-thio-leucine can be used as the N-terminal amino acid in the second peptide. After ligation and desulfonation, you will be left with a native peptide joined by a leucine.

Combining synthetic peptides with NCL can be a powerful technique and lends itself to efficient protein production without the need for a recombinant expression system.

How to Introduce Post-Translational Modifications and Unnatural Amino Acids Through Peptide Synthesis

Can you add post-translational modifications to peptides? Yes, you can! A few you might think of right away are disulfide bridges, phosphorylation, and glycosylation. Let’s take a look at each one:

Disulfide Bridges

Creating disulfide bridges can be done in a single step or sequentially.¹³ With the former technique, you simply deprotect the side groups of all residues and then expose the peptide to an oxidant such as dimethylsulfoxide (DMSO).

While relatively straightforward, this approach is inefficient and depends on the ability of the peptide to fold into a native state so that the desired disulfide linkages are made. Without this ability, you could end up with multiple and undesired linkages.

In the sequential method, cysteine residues are protected with different protectants that can be differentially removed. You deprotect as desired, expose to an oxidant, and repeat. This limits the number of available cysteine side groups available for linkage and increases the chance that the desired secondary structure is created.

Phosphorylation

Adding phosphorylated sites is a bit easier. You could simply synthesize the peptide using phosphorylated residues, such as phosphoserine or phosphotyrosine.¹⁴

Another option is to synthesize an unphosphorylated peptide and treat it with a kinase. The choice would depend on whether the use of a pre-phosphorylated residue would be problematic during synthesis or whether the kinase is able to recognize the intended phosphorylation target within the synthetic peptide.

Glycosylation

There are two methods for glycosylating peptides—direct and convergent.¹⁵ In the direct method, a pre-glycosylated serine (O-linked glycosylation) is added into the growing synthetic chain. For the convergent method, which is used for N-linked glycosylation, the peptide chain is made and the glycosylamine moiety is conjugated to a free asparagine residue.

Unnatural Amino Acids

Unnatural amino acids (UAAs) are amino acids not among the 20 amino acids used by living cells in proteins. Some, such as citrulline, occur naturally but are not proteinogenic.

UAAs are typically used in applications such as photo-crosslinking and activation, fluorescent tagging, and protein engineering. If your research can benefit from these applications, UAAs can be incorporated into synthetic peptides in the same ways as the 20 proteinogenic amino acids.

Design Tools

If you are convinced that synthetic peptides are for you, here are some design tools to help you get started!

Tool	Company/Source	Comments
Peptide Synthesis and Proteotypic Peptide Analyzing Tool	Thermo Fisher Scientific	•Analyzes physio-chemical properties and estimates ease of synthesis
Peptide Library design tools	GenScript®	•Epitope mapping •Alanine scanning among other analyses
Peptide Library Design and Calculator Tool	MilliporeSigma	•Overlapping peptide scan •Alanine scanning among other analyses
Peptide Generator	Peptide 2.0 Inc	•Peptide design and epitope mapping

References

1. 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.
2. Merrifield, R.B., et al. Instrument for automated synthesis of peptides. Anal Chem., 38, 13 (1966). doi: 10.1021/ac50155a057
3. 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
4. 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
5. Lee, B.S., et al. Antibody production with synthetic peptides. Methods Mol Biol. 1474, (2016). doi:10.1007/978-1-4939-6352-2_2
6. Peptide Antigen Database. GenScript®. Accessed on 14 Aug 2020.
7. Peptide-Protein Interactions. LifeTein. Accessed on 14 Aug 2020.
8. 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.
9. 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
10. 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
11. Peptide Design. Thermo Fisher Scientific. Accessed on 03 Oct 2020.
12. Kulkarni, S., et al. Rapid and efficient protein synthesis through expansion of the native chemical ligation concept. Nat Rev Chem., 2, (2018). doi: 10.1038/s41570-018-0122
13. Yang, Y., et al. Two-step selective formation of three disulfide bridges in the synthesis of the C-terminal epidermal growth factor-like domain in human blood coagulation factor IX. Protein Sci., 3, 8 (1994). doi: 10.1002/pro.5560030813
14. Chen, Z., and Cole, P.A., Synthetic approaches to protein phosphorylation. Curr Opin Chem Biol., 28 (2015). doi: 10.1016/j.cbpa.2015.07.001
15. Moradi, S.V., et al. Glycosylation, an effective synthetic strategy to improve the bioavailability of therapeutic peptides. Chem Sci., 7, 4, (2016). doi: 10.1039/c5sc04392a.