Hello everyone, I'm working with predicted antimicrobial peptide using machine learning. Recently, we use an antimicrobial peptide from DRAMP which had been verified to have antimicrobial activity. We synthesized the peptide exactly as what provided in DRAMP. so we did test using disc diffusion assay but no activity. is there any possible issue like it must be tested in microdilution method or due to the synthesis parameter? Thanks in advance for the help and idea.
Does the natural peptide have any post-translational modification? Could be your synthetic peptide is forming aggregates so is not being absorbed?
Hi Robert, thanks for the reply. based on the database, there were no modification. there are reports that they synthesised the peptides without modification and show an activity. So, we are worried that the peptides were not being prepared properly before the assay. Aggregation also impossible as it was easily soluble before assay. Have anyone experience, using a control AMP to their target but showed no activity?
Dear Fareed Sairi Did you characterize the peptide, both with bioinformatics and experimental? I mean:
-By using for example Heliquest (https://heliquest.ipmc.cnrs.fr ) you get a proper idea about the peptide (its hydrophobicity, net charge, etc.). See for classification for example:
Article New User-Friendly Approach to Obtain an Eisenberg Plot and I...
Article Identification and in silico analysis of helical lipid bindi...
-Before checking in your assay, I think it is good to check the peptide in some way. If for example bioinformatics predicts membrane activity you can check quickly by using a monolayer technique or depending on what you have available in the lab check by circular dichroism (with and without lipids), see for example:
Article Anionic phospholipids are essential for α-helix formation of...
If this seems fine, then do realise that sometimes a pretty insoluble peptide (as described in the link above) that requires something like DMSO or TFE is all of a sudden reasonably soluble in aqua dest. as described here:
Chapter Characterization of helical structures in gelatin networks a...
However, in your medium there will be a buffer containing salt (and a certain pH) and the ionic strength in the medium can cause aggregation (this can be checked with the peptide and the use of for example circular dichroism).
Obviously, something might have gone wrong with the synthesis, or the prediction is simply wrong, and the peptide has no antimicrobial activity but a lot can be checked before one have to draw this conclusion.
Best regards.
PS. The last point can be checked quite easily. Solubilise the peptide in distilled water and add concentrate salt solutions and either follow it visual or use an absorption spectrometer to see turbidity (or not).
Hi Everyone, thanks for all the suggestion and help. The peptide that we choose was known to be soluble in water. The supplier also provide detail QC and suitable solvent to dissolve the powder. to Rob Keller, we did characterize in silico prior to the synthesis and we only chose the one that is soluble in water. we will try to check the turbidity, a great and easy way to check them. i think we just add them with the media and check the turbidity. if they become insoluble in media, then I know the culprit.
Thanks again
Hi Everyone, just some update. we got some results when using the broth dilution. I think disc/diffusion method might not be suitable for AMP as it might get trapped/bound to the disc or agar. that is my initial speculation.
Fareed
There are several possible reasons for the lack of antimicrobial activity in your synthesized peptide from DRAMP:
1. Synthesis parameters: Ensure that the peptide was synthesized correctly, including the accurate amino acid sequence, purity, and post-synthesis modifications.
2. Assay method: Consider using alternative assays, such as microdilution, to assess the peptide's antimicrobial activity.
3. Sensitivity of the test organism: Verify that the test organism used in the assay is susceptible to the peptide's activity.
4. Peptide modifications: Replicate any necessary modifications or environmental conditions used in the original study.
5. Peptide conformation: Assess the three-dimensional structure of the synthesized peptide to ensure it matches the native peptide.
6. Experimental variability: Minimize experimental error by using appropriate controls, replicating experiments, and using validated techniques.
Carefully evaluate these factors and consider modifications to your experimental approach to troubleshoot the lack of antimicrobial activity.
good luck,
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