Peptide purifcation

Dr Engelbert Buxbaum engelbert_buxbaum at
Mon Apr 12 06:18:36 EST 2004

Mehmet Sen wrote:

> Hello
>    I have a peptide, which is found in three forms. Unphosphorylated,
> singly phosphorylated and doubly phosphorylated. 
>    What I need is the way of seperating (purifiying from each other)
> these peptides from each other. Size-exclusion and ion-exchange did
> not help at all. 

Ion exchange is unlikely to work, because the molecular weight
difference would be too low (20 aa is equivalent to a MW of about 2200
Da, each phosphate group adds 95 Da or 4.5%). 

However, ion exchange should work based on the additional negative
charges of the phosphate groups. Of course you would need an anion
exchanger (DEAE or similar). 

Idealy you would work at a pH where the unphosphorylated peptide no
longer has negative charges, it would pass in the flow through. The
single and double phosphorylated form should then elute in this order
with increasing salt concentration.

For preparative IEC you may wish to look at volatile salts like
diethylammonium bicarbonate.

Sucessful separation is posible only if the phosphorylation is stable
enough to survive the procedure.

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