Method for determining solubility of modified peptides from modified peptide sequences
How to determine the solubility of a modified peptide from a modified peptide sequence?
1. What are the impurities in the non- HPLC purified modified peptide ?
A: Modified peptides and non-modified peptide impurities in modified peptides of crude and desalted grades: some raw materials such as DTT , TFA, etc., which are post-treated such as non-full length modified peptides and modified peptides.
2. What are the impurities of the purified peptide purified by HPLC ?
A: There are still some impurities in the modified peptide purified by HPLC . The impurities are mainly short peptides and trace TFA .
3. How long is the modified peptide suitable ?
A: Modified peptide synthesis needs to consider the length, charge, hydrophilicity and other factors of the modified peptide. The longer the length, the lower the purity and yield of the crude synthetic product, the greater the difficulty of purification and the inability to synthesize. Of course, the sequence of the modified peptide functional region cannot be changed, but in order to modify the smooth synthesis of the peptide, it is sometimes necessary to add some auxiliary amino acids upstream and downstream of the function to improve the solubility and hydrophilicity of the modified peptide. If the modified peptide is too short, the synthesis may also have problems. The main problem is that the synthesized modified peptide has certain difficulty in the post-treatment process. The modified peptide below 5 peptide generally has a hydrophobic amino acid, otherwise the post-treatment is more difficult. Modified peptides below 15 amino acid residues generally yield satisfactory yields and yields.
4. How to determine the solubility of the modified peptide from the modified peptide sequence ?
(1) If a high proportion of highly hydrophobic amino acids such as Leu, Val, IIe, Met, Phe and Trp are contained in the modified peptide, the modified peptide is difficult to dissolve in the aqueous solution or is impossible to dissolve at all. These amino acids, whether purified or synthesized, can be problematic.
(2) In general, the ratio of hydrophobic amino acids is <50% , which cannot be continuous for 5 consecutive aa. Hydrophobic, charged amino acids ( positive charge K, R, H, N-terminus, negative charge D, E, C - Terminus ratio) reaches 20%, the modified peptide N or C can be increased if short-polar amino acids, the solubility can be improved.
5. Why are modified peptides containing Cys, Met, or Trp difficult to synthesize ?
A: Modified peptides containing Cys, Met , or Trp are difficult to synthesize and it is difficult to obtain high purity products. Mainly because these groups are unstable and easily oxidized. Special attention must be paid to the use and storage of these modified peptides to avoid repeated opening of the lid.
6. Why are the synthetic yields or purity of some modified peptides lower ?
Answer: There is a big difference between the synthesis of modified peptides and the synthesis of primers. There are few primers that cannot be synthesized, but modified peptides that cannot be synthesized often have. When the amino acids such as Val, Ile, Tyr, Phe, Trp, Leu, Gln, and Thr are adjacent or repeated, the modified peptide chain cannot be completely stretched and dissolved during the synthesis, and the synthesis efficiency is lowered. In the following cases, the synthesis efficiency and the purity of the product are relatively low, such as: repeat Pro, Ser-Ser , repeat Asp , 4 consecutive Gly, and the like.
7. How is the modified peptide purified ?
A: Purification of modified peptides generally uses reversed-phase columns ( eg C8 , C18, etc. ) , 214 nm . The buffer system is usually a solvent containing TFA , pH 2.0 . Buffer A is 0.1% TFA in ddH2O and Buffer B is 1% TFA/ACN/pH2.0 . Buffer A is dissolved before purification; if it is not well dissolved, it is dissolved in Buffer B and then diluted with Buffer A ; for hydrophobically modified peptides, it is sometimes necessary to add a small amount of Formic Acid or acetic acid.
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