Answers to common questions in peptide synthesis
In custom peptide synthesis, people often have a lot of questions, the following is about the peptide synthesis process of common questions to answer：
Q:How does Omizzur biotech conduct quality inspection of the synthetic peptides?
A: All synthetic peptides were analyzed by HPLC and MS, and corresponding test reports were provided. All peptides are purified by reversed-phase chromatography, and the molecular weight of the peptides is determined by mass spectrometry to determine whether the product is correct, and the MS test results can also show most of the major impurities. If necessary, net peptide content tests, such as amino acid analysis or elemental analysis, are also available. These methods can confirm the amino acid composition of peptides, and they can be used as complementary methods for peptide confirmation. All delivered peptides meet the purity requirements of the customer. Those peptides that do not meet the purity requirements are discarded. Of course, it can also be sent to customers if they need it.
Q: How to explain the P+Na and P+K peaks in MALDI(MS)?
A: Na and K peaks are often seen in MALDI, and sodium and potassium are derived from solvent water. Even distilled and deionized water can contain trace amounts of sodium and potassium ions that cannot be completely removed. They also ionize and bind to free carboxyl groups of peptides when they are subjected to mass spectrometry. Because there is no purification system to remove sodium and potassium ions from the water, it is sometimes inevitable that sodium and potassium peaks will appear in the MALDI MS map.
Q: Why is N-terminal acetylation and C-terminal amidation necessary?
A: These modifications can prevent the peptide from being degraded, and can also make the peptide mimic its original state of alpha amino and carboxyl groups in the parent protein.
Q: How to dissolve peptides in DMSO?
A: Dimethyl sulfoxide (DMSO) is a sulfur-containing organic compound with the molecular formula (CH3)2SO, which is a colorless and odorless transparent liquid at room temperature. DMSO is often used in cell banks as a cryoprotectant. During cell freezing, DMSO prevents the formation of intracellular/extracellular crystals at a working concentration of 10%. DMSO usually binds to salt or serum albumin.
Hydrophobic peptides can be easily dissolved in DMSO. However, DMSO can increase cell permeability, and if the peptide is dissolved in DMSO, it will have toxic effects on cells. High concentrations of DMSO should never be used in cell culture. At a concentration of 5% DMSO, the cell membrane can dissolve. Most cell lines can tolerate 0.5% DMSO, and a few can tolerate concentrations of 1% without showing severe cytotoxicity. However, primary cell culture is more sensitive to it. Therefore, if the dose/response curve is made with primary cells (feasibility), the concentration should be less than 0.1%.
For some very hydrophobic peptides, try to dissolve them first in a small amount of DMSO(30-50ul,100%), and then slowly (drop by drop) add them to a constantly agitated aqueous solution such as PBS or other desired buffer until the ideal concentration. If the peptide solution begins to become cloudy during the drip process, it indicates that the dissolution limit has been reached. In addition, ultrasound helps the polypeptide dissolve.
A concentration of 0.1%DMSO is safe for almost all cells.
The final concentration of DMSO, which is widely used in cell culture, is 0.5% and does not cause cytotoxicity.
Although 1%DMSO is not cytotoxic in some cells, we recommend 0.5%.
There are also 5% of cases in which DMSO is successfully applied to certain cells.
The final concentration is always maintained at 0.5%, but it can be dissolved in 100% DMSO at 200 times the high concentration when stored.
Q: What are some suggestions for the design of phosphorylated peptides?
A: As the length increases, the coupling efficiency gradually decreases from the phosphorylated amino acid. The synthesis direction is from the C end to the N end, it is recommended that the residues after the phosphorylated amino acid should not exceed 10, that is, the amino acid residues before the phosphorylated amino acid from the N end to the C end should not exceed 10.
Q: How to choose between peptide N-terminal and C-terminal modifications?
A: By default, the end of the peptide is the N-terminal free amino group and the C-terminal free carboxyl group. The sequence of the peptide often represents the sequence of the parent protein, in order to be closer to the parent protein, the end of the peptide often needs to be closed, that is, n-terminal acetylation, C-terminal amidation, this modification avoids the introduction of excess charge, but also makes it more able to prevent exonuclease action, so that the peptide is more stable.
Q: I need a cyclic peptide that contains a tryptophan, will it be oxidized?
A: The oxidation of tryptophan is a common phenomenon in peptide oxidation, and peptides are generally cyclized before purification. If the oxidation of tryptophan occurs, the retention time of the peptide on the HPLC column will change, so the oxidized peptide can be removed by purification. In addition, oxidized peptides can also be detected by MS.
Q: How do you determine if a peptide has formed a ring?
A: The Ellman reaction is generally used to detect whether the ring reaction is complete. If the Ellman test result is positive (yellow), the ring formation reaction is incomplete. If the test result is negative (not yellow), the ring reaction has been completed.
Q: Were peptides containing Cys reduced before shipment?
A: If the peptide is not found to have been oxidized, we generally do not reduce Cys. All polypeptides are purified and lyophilized from crude products at pH 2, which at least to some extent prevents the oxidation of Cys. Peptides containing Cys are generally purified at pH 2, unless there is a special reason to purify at pH6.8. If purification is performed at pH6.8, the purified product must be immediately treated with acid to prevent oxidation. In the final quality control step, for peptides containing Cys, if a substance with a molecular weight of (2P+H) is found on the MS map, it indicates that Cys has been oxidized to form dimers. If there is no problem with MS and HPLC, we will directly freeze-dry the shipment. It should be noted that peptides containing Cys undergo slow oxidation over time, and the degree of oxidation depends mainly on the peptide sequence and storage conditions.
The above are some common problems in peptide synthesis, hope to help you, more details please visit www.omizzur.com.