Peptide Synthesis Introduction

Peptide synthesis is the chemical process of joining two or more amino acids together via peptide bonds. Typical amide bonds are formed by the condensation reaction of the carboxyl group of one amino acid to the amino group from the growing peptide chain. Chemical peptide synthesis occurs from the C- to the N-terminus direction, and it is a precise step-wise addition of one amino acid at a time. In solid-phase synthesis, the process begins by first anchoring to a resin polymer at the C-terminal end, leaving the N-terminal amine unit free to attach to another N-protected amino acid unit. This newly added amino acid will then undergo deprotection of the N-terminal amine thereby allowing the addition of another N-protected amino acid unit. The assembling continues by repeating this cycle of coupling, washing, and deprotection reactions. Once complete, the final peptide is cleaved from the anchoring resin and purified.

Synthetic Peptide Applications

Synthetic peptides can be used in a wide variety of applications and research areas including; disease biomarker identification, drug target development, cancer diagnosis and therapeutics, epitope mapping, antibody productions, protein functionality and characterizations, enzyme-substrate or receptor-substrate interactions, and vaccine design to name a few.

Peptide Synthesis FAQs

Below are some frequently asked questions about the Biomatik Peptide Synthesis service. If you have any other questions, please contact us at Peptide@biomatik.com and we will be happy to assist you.

1. Do you have customer testimonials and publications citing your peptide synthesis service?
Biomatik has been trusted among top scientists and researchers from around the world for quality products and custom services since 2002. To date, Biomatik has delivered 70,000+ custom made products to our customers worldwide. Learn why Biomatik has been trusted by thousands of researchers worldwide by viewing our customer testimonials, selected publications and 3,000+ publications citing our fine products and services at Google Scholar.

2. What is the typical turn-around time for peptide synthesis? How do you ship and how long does it take to ship to me?
Our typical turn-around time is 2-3 weeks for a standard purified peptide under 30 amino acids. The turn-around time varies depending on the peptide length, solubility and difficulty. All peptides will be shipped in lyophilized powder, at room temperature. It is overnight delivery within USA/Canada, and takes typically 3-4 days to reach researchers in other countries.

3. Which analytical data do you provide for peptides?
Each peptide comes with CoA, MS spectra, and HPLC chromatograms. See Sample QA Report.
We can provide additional analysis if required, e.g. Peptide content analysis, TFA content analysis, and KF test (Water content test).

4. How do I design custom peptide sequence for my applications?
The sequence, amino acid composition, and length of a peptide will influence whether correct assembly and purification are feasible. These factors also determine the solubility of the final product. Please refer to our Peptide Design Guideline on some factors in the design of a peptide sequence for synthesis.

5. Which purity is recommended for my application?
Please refer to our Recommended Peptide Purity Guideline for more details.

6. TFA salt form vs. Acetate or HCl salt form: Which form should I choose?
By default, all research peptides are synthesized in TFA salt form. For cell based assays or animal studies, you should consider having TFA salt removed by switching it to acetate or HCl salt form (with TFA <1% guarantee) to avoid abnormal responses. Depending on the budget, you may also want to consider higher purities (>98%) to get optimal results.

7. How long you can synthesize a peptide?
Biomatik can synthesize peptides up to 130aa. Unlike many peptide suppliers who are only comfortable in making peptides under 30 or 40aa, Biomatik has extensive experience in making peptides ranging from 40aa to 90aa. However, the success rate is getting lower when it comes to longer peptides, especially 100aa or longer. If you plan to synthesize a sequence of 130aa or longer, please contact us for custom protein expression and purification service.

8. What type of end terminal modification choice is appropriate?
By default, chemically synthesized peptides have free amine at N-terminal and free acid at C-terminal. N-terminal acetylation and C-terminal amidation are uncharged, which reduces the overall charge of a peptide so the solubility may decrease. However, the modifications are desirable since it imitates its natural structure. It increases the metabolic stability of peptides and their ability to resist enzymatic degradation by aminopeptidases, exopeptidases, and synthetases. This enhances their ability to enter cells, thus increasing the biological activity of a peptide.
We recommend the modifications for intracellular, in vivo assays and in vitro functional studies. The modified peptides can then be used as substrates in enzyme assays. Amidation not only enhances the activity of peptide hormones, it also prolongs the shelf life. The modifications can reduce the influence of charged C- or N-terminal during ELISA binding assays.

9. How to dissolve my peptide?
Peptide solubility information is included on the CoA if you have requested solubility test at the time of ordering, free of charge. If it is not available, you can follow a Peptide Handling Guideline.

10. How do I store my synthetic peptides?
Most lyophilized peptides will be stable at room temperature for 2-3 weeks. For long-term storage, you should store lyophilized peptides at -20°C. Repeated freeze-thaw cycles should be avoided. Allow to come to room temperature before opening. The shelf life of peptide solutions is limited; a peptide solution once prepared should be used as soon as possible. For more details, please refer to Peptide Handling Guideline.

11. What if some problems come up during the synthesis or purification process?
Each peptide has its specific characteristics. If some problems happen during the synthesis beyond our expectation, and we cannot deliver your peptide on time, we will inform you as soon as possible. By chance that we are not able to make the peptide, you will not be charged for any costs - which is our "No Peptide, No Charge" policy.

12. Is C-terminal labelling of Biotin (or FITC) possible?
Yes. C-terminal labelling of Biotin (or FITC) is done by addition of a Lysine residue at the C-terminus of a peptide, and Biotin (or FITC) is attached to the Lysine side chain via an amide bond. Lysine's positive charge is removed.

13. What is the appropriate peptide length for antibody production?
Generally, a 10-25 residue peptide is recommended. A longer peptide could have more epitopes, but could also have a greater chance of forming stable secondary structures which are not native forms. A shorter peptide (<10aa) is generally not good unless there are valid reasons for it, such as potential sequence homology with a related family member or other proteins. Biomatik also offers custom antibody production service, please contact us for more details or check out our Antibody Production Service page for information.

14. Should I consider adding a Cysteine in my peptide for carrier protein conjugation for antibody production?
Chemical conjugation using Cysteine offers a single point attachment provided there is just one Cys in the sequence (added or part of the native sequence). If your peptide does not have an existing Cys in the sequence, it is preferable to add Cys at the NH2 terminus if the peptide is internal or it represents the very C-terminus. This will keep the COOH free (non-conjugated) as it exists in the native protein. For peptides representing the very NH2- terminal sequences, Cys should be added at the C-terminus of the peptide. For internal peptides, Cys can be added at either end but it is easier to synthesize peptides containing a NH2-terminal Cysteine. Cysteine can also be used to couple peptides to Sepharose for affinity purification of antibodies. Amino or COOH-conjugation chemistries should be avoided as most peptides contain several NH2 and COOH groups available in a given peptide sequence which can result in forming multiple attachment points or peptide distortion.

15. What is a MAP?
MAPS or Multi-Antigenic Peptide is a branched peptide at which linear peptide chains are linked at their C-terminus via polylysine core, thereby increasing the size of whole molecule. This is done to eliminate the coupling of peptides to KLH. It seems that, however, conformation of peptides on MAP is less flexible, and antibodies obtained by MAP typically recognize target protein less often than by conventional KLH conjugation. In addition, there is no free peptide produced when making MAPS, making it difficult to remove polylysine core directed antibodies. Purification of MAPS by HPLC is difficult, and MAPS is provided without mass verification due do its heterogeneity and large molecular size.

16. Why does my KLH-conjugated peptide solution appear cloudy?
KLH or Keyhole Limpet Hemocyanin is a large aggregating protein (MW = 4x10⁵ – 1x10⁷). Because of its size and structure, its solubility in water is limited, causing a cloudy appearance. This shall not affect immunogenicity and the turbid solution can be used for immunizations.

17. What is the purity for the crude and desalted peptide? How do you purify the peptide? What are the impurities?
For short peptides with normal sequences under 15aa, it is generally 40-60% by HPLC for crude grade; 50-70% by HPLC for desalted grade. The longer the peptide, the lower the purity for crude or desalted. Peptides are generally purified by HPLC using water and acetonitrile gradient. Most impurities are fragments or deletion peptides, incompletely de-protected peptides, and residual salt and water.

18. Can you explain the M+Na and M+K mass peaks in MALDI spectra?
It is common to see Na (sodium) and K (potassium) adducts in the MALDI spectrum. The sodium and potassium come from the water used in the peptide solvents. Even distilled and deionized water has trace amounts of sodium and potassium ions, which can never be entirely removed. These become ionized during the MALDI mass spec process and bind to the free carboxyl groups of the peptide. Because there is no water purification system that will remove every single sodium or potassium ion from water, seeing the sodium and potassium adducts at times is common and unavoidable in MALDI mass spec. This is not an indication that the peptide is not pure, nor should it be confused with an incorrect molecular weight.

19. How about batch-to-batch consistency with custom made peptides?
Research grade custom peptides come with limited quality control (MS and HPLC analysis only). Therefore, the variation is expected from batch to batch in terms of the desired peptide content, salt content, water content, impurities, solubility, etc. Please note that such batch-to-batch variation shall not create an issue for most experiments.

However, if you are engaged in sensitive bioassays and you want to minimize batch-to-batch variation, you shall request for additional quality control and analysis (e.g., peptide content, salt content, water content, solubility), at an additional cost.

For example, if you are engaged in cell related assays, you shall request for TFA removal (<1%); If you must apply the same peptide concentration for your experiment from batch to batch, you shall require peptide content analysis; If you require high solubility or concentration (e.g. 5mg/ml) in a specific solvent from batch to batch, you shall ask for solubility control and testing. For the ultimate batch-to-batch consistency, you may need to consider GMP grade peptides.

20. Which peptide analytical services do you provide?
MS Analysis: By default, MS analytical data is included in each peptide delivery from Biomatik. Mass spectrometry (MS) is an analytical tool used for measuring the molecular mass of a sample.

HPLC Analysis: By default, HPLC analytical data is included in each peptide delivery from Biomatik. HPLC Purity is the amount of target peptide relative to the total amount of material that absorb at ~220 nm (the peptide bond absorbs) i.e. the desired target peptide and other fragment peptides. Peptide purity by HPLC does not take into account water and salts that are usually present in the sample, since water and salts do not absorb at ~220 nm. HPLC analytical data is included in each peptide delivery from Biomatik.

Net Peptide Content Analysis: Net Peptide Content Analysis can be requested at an additional cost. Net Peptide Content is the percentage of all peptides (the desired target peptide and other fragment peptides) present relative to everything (including the target peptide, other fragment peptides, salts and water) present in the lyophilized peptide powder. Salts and water do not contain nitrogen, thus the net peptide content can be determined by nitrogen elemental analysis. Along with HPLC Purity, Net Peptide Content can be used to measure the net weight of target peptide in the lyophilized peptide powder. This information is important when calculating the concentration of target peptide during sensitive experiments.

TFA Content Analysis: TFA Content Analysis can be requested at an additional cost. As a standard in the industry, all of our peptides are delivered in TFA salt. TFA content analysis can be performed if you would like to know the actual TFA salt content in the lyophilized peptide powder. If you are engaged in cell-based assays and animal studies, you shall consider having your peptide delivered in acetate or HCl salt.

KF Test (Water Content Analysis): TFA Content Analysis can be requested at an additional cost. Karl Fischer titration is a classic titration method to determine trace amounts of water in a sample.

Please contact us if you require a quality analysis which is not listed above.

21. How to determine net weight and molar amount of the desired target peptide?
Biomatik ships peptides according to the gross weight of lyophilized powder. The lyophilized powder includes impurities such as fragment peptides, salts and residual water.

Net weight of desired peptide: Gross Weight X Net Peptide Content (%) X HPLC Purity (%) Molar amount of desired peptide: Net Weight / Molecular Weight

An example: Peptide sample, gross weight 10 mg, with the following analytical data: HPLC Purity 98.5%, Net Peptide Content 86.0%. Mol. weight 1360.65 g/mol.

According to the above formulas:
Net Weight of desired peptide: 10 mg X 98.5% X 86.0% = 8.471 mg
Molar Amount of desired peptide: 8.471 mg / 1360.65 g/mol X 1000 = 6.226 µmol

The above value allows calculating concentrations, when dissolving the peptide.