After detection of biologically active peptides with antimicrobial activity, we need to fully identify them and know if they are new.
I would write to Michael J. Conlon. He is a true expert on antimicrobial pepetides.
peptide mass finger printing is the most common method for characaterization of peptides. You can go for either MALDi-TOF /MS or ESI-TOF after isolating the peptide by 2D Gel Electrophoresis.... Then from the known databases available for the antimicrobial peptides you can actually identify that which groups or moieties are unique to your sequence and whether the isolated peptide is new or not..... Wishing you gud luck for your research work....
Dear Mohamed
The route for characterisation will depend on your focus and the origin and type of peptide. First, you need to isolate your peptide each peptide has it own challenges. The most reliable starting point would starting with salt (NaCl of ammonium acetate) and/or acid precipitation* and then using cationic exchange chromatography followed by reverse phase HPLC. *Acid precipitation helps to get rid of proteases/unwanted proteins and you can reconstitute your peptide fraction from the precipitate with 50% acetonitrile for freeze drying.Try and limit your contact of the peptides and extracts with plastic and membranes and AMPs tend to stick to such polymers. Work in detergent free glass ware which you can prepare by thorough washing with 70% ethanol followed my MilliQ or high purity water and pyrolising the glass ware (ask your local glass blower).
If you are working with gene-encode peptides, for example those from a vertebrate, insect or plant you will first need to isolate enough peptide for N-terminal sequencing or de novo MS sequencing. From the derived sequence you can search for the gene and derive a full sequence from the genetic data. You can then either clone and express the peptide or make a synthetic version of the peptide. Latter only works well for peptides less than about 30 residues without a too complex disulphide bonding pastern.
If you are working with microbial peptides, they could either be gene encoded and modified or be a secondary metabolite. With such a peptide you will have to isolate enough high purity peptide for analysis by both MS and NMR if you really want to elucidate the structure.
Once you have your purified peptide a know the primary structure. You can elucidate their secondary and tertiary structure via NMR and CD, as well as their structural behaviour in different solvent/lipid environments using fluorescence, CD and NMR. You can also look as their interaction with lipid model membranes and the range of their microbial targets, as well as their toxicity towards different eukaryotic cells. You can study their stability/activity in plasma, high salt media and biological fluids. Finally, depending on the peptide and future application, you can do a qualitative structure-activity-relationship study using libraries of analogues of your peptides. From these results the next step would be to select a lead peptide for further development and study.
Good luck with your research. Hope it helps.
Regards
Marina
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