I am trying to identify an unknown molecule/peptide that is secreted by a bacteria and has a particular activity in my cells. I have narrowed it down to something that is less than 3kD. I have tried using trypsin or proteinase K to determine if the unknown is a peptide as well as heat-treating the sample. I know that I can use HPLC to separate small molecules but I have some concern if the acetonitrile will have an effect on my activity. I am looking for some other suggestions to try and characterize the unknown in a quick and easy way.
Thanks!
Acetonitrile is normally used in peptide separations, so if your substance is a peptide, then attempting to purify it by reverse-phase HPLC in a water:acetontirile gradient with TFA ion pairing would be a sensible approach. You could also try ion-exchange chromatography.
You can avoid the effect of acetonitrile and remove the solvent just freez-drying your samples after the HPLC. Separate your samples as Adam suggested, subsequently freeze-dry them and resuspend them in water or what is best for your experiments, then use the fractions on your cells.
I think in the end I will have to run the samples on HPLC. We do have some concerns about acetonitrile affecting the the activity of what's in the fraction. But freeze-drying them is a good suggestion. Since HPLC can be a bit pricer we were hoping to find a quicker and cheaper way to narrow down what's in the conditioned media so we know which type of chromatography would be best to use.
You could try another chromatography. E.g. gel filtration or size exclusion
Otherwise, you could try a fractional precipitation of your samples by salting out and resuspend your precpitated fractions... but I don't know if this method is OK for the kind of molecues you are loocking for. Anyhow this is a quite cheap procedure, you could just try, if you prefer avoiding HPLC.
Good luck!!
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