Attentions in Peptide Assay
  • 1.Is the peptide content equal as the peptide purity?

    After peptide synthesis, one of the important quality indicators is the purity of peptide. Among the quality indicators of peptide synthesis, there are often two indicators: peptide content and peptide purity ,these two are often easily confused.


    Peptide Content:The weight of freeze-dried peptide not only contains peptide we need, but also some non-peptide components, such as water, absorbed solvents, coordination ions and organic salt etc.The peptide content refers to the weight percentage of all kind of peptide in it.Its usually accounts for 50-80% of the total peptide weight, which is generally obtained by amino acid composition analysis or Nitrogen analysis.


    The purity of peptide refers to the content of target peptide detected by HPLC at 214nm (214nm is the absorption wavelength of peptide chain).Purity is defined as the percentage of components in a peptide sample that contain the correct sequence.A few other impurities include deletion sequences, truncated sequences which didn’t been removed in the process of purification and incomplete sequences produced by de-protection.

    2.What impurities are in peptides which not purified by HPLC?

    It mainly refers to crude peptide and desalting peptide.Here is a summary of peptide impurities:

    ImpuritiesCrude & desalting peptidePurified peptide (HPLC)
    Deletion sequences
    Truncated sequences
    Incomplete sequence
    Dithiothreitol (DTT)×
    Trifluoroacetic acid (TFA)√ (microscale)
    Acetic acid×
    Other peptide (by side reaction)√ (microscale)

    3. What are the factors that affect the stability of peptides?


    The factors affecting the stability of synthesized peptides include deamidation, oxidation, hydrolysis, disulfide bond , racemization, beta - elimination, aggregation, etc.The results show that the most common degradation products are deamidation products, oxidation products and hydrolysis products.


    Among all kinds of amino acids, Asparagine and Glutamine are easy to deamidate (especially under the condition of high pH and high temperature).Methionine, Cysteine, Histidine, Tryptophan and Tyrosine are the most easily oxidized and sensitive to light.Aspartic acid is easy to lead to the formation of peptide chain breaks, especially Asp-Pro and Asp-Gly peptide bonds.Because there are many unstable amino acid residues or peptide bonds in a peptide molecule, the possible degradation mechanism and degradation products of synthetic peptides are complex.


    4. Peptide Dissolution & Preservation

    If peptides are difficult to dissolve, it is incorrect to think that there is a problem in peptide synthesis.If it contains a high proportion of hydrophobic amino acids such as Leu, Val, IlE, Met, Phe and Trp, it is difficult for peptide to dissolve in aqueous solution. Whether these amino acids are purified or synthesized, there may be problems.


    The solution is: our synthesis experts will provide you with a series of advice on sequence optimization at the beginning of sequence design to fundamentally solve the problem of water solubility,or with the help of organic solvents.Our services include free solubility test to support your research.


    Peptides need to be stored in dark for a long time, and should be stored at - 20 and 4 centigrade for a short time. It can be transported at room temperature for a short time. Peptides are stable at - 20 centigrade, especially freeze-dried and stored in a desiccator, which can be placed at room temperature before they are exposed to air.This will reduce the impact of humidity, when it is not possible to freeze-drying, the best way is to store a small amount of working samples.


    For peptides containing Cys and Met or Trp, deoxidizing buffer is essential for their dissolution,for more suggestions about peptide dissolution and preservation,please click to know more

    5. What are the factors considered in the binding of peptides to other compounds or carriers?


    Some peptides need to be connected with drugs or compounds with specific functions sometimes, or to connect peptides to some carriers, which need to be linked by chemical bonds.Another option is to connect the thiol and maleimide in the pH 8 environment.