Iodinated peptides spread on RP-HPLC. Why?

Mr. P.A. Sansom psansom at
Sat Jan 14 15:20:51 EST 1995

Iodinated peptides spread on RP-HPLC. Why?

I'm using synthetic peptides of varying sequences which I analyse by C18 reverse phase HPLC (TFA and acetonitrile gradients). UV absorbance at 206nm shows nice sharp individual peaks with no major contaminants.

Because they all contain tyrosine I've labelled them with 125-iodine using Iodobeads (from Pierce; immobilised chloramine T method) under conditions that should give me mono-iodinated tyrosine only. This is to facilitate quantitation.

When I run the labelled peptides on HPLC with 125-I detection (Berthold, inline after UV detector) I see very broad peaks and a lot of extra peaks. Analysis of labelled peptides by isoelectric focussing shows multiple pIs for a single peptide.

Why don't I get sharp HPLC peaks with the labelled peptides? Is it

(1) my equipment? (ie peak broadening is happening between the UV and the 125-I detector?) - I've checked as best I can and there doesn't seem to be any dead spaces.

(2) because the label is incorporated into such a small proportion of the peptide? I estimate 100pg is labelled per ug starting material, and my assay system thus uses 1-10pg labelled peptide per HPLC run. Bearing in mind that the iodine atom incorporated causes a *biiiiiggg* shift in retention time on reverse phase, it means that I'm hoping to get a well defined peak from a very small amount of material.

(3) my gradient protocol? I go from 5% to 45% acetonitrile over 20 minutes. Shallower gradients don't improve things, and take a lot longer! Would a step gradient be better? It's a lot of work to optimise it if I'm not going to see an improvement.

Why do I get multiple peaks? Is it

(1) my peptides? I'm new to this lark so I've got no criteria to judge the quality of the peptides by other than the synthesis report and the say-so of the maker. Although they appear clean, is it possible that the minor contaminants are taking up the iodine more effectively than the peptide of interest? If so, what's the best way of cleaning them up? Commercial suggestions welcome 8-) - must be quick though.

(2) my labelling protocol is not as good as I think it is, and I'm getting incorporation of 125-iodine into the histidine residues? I must say that the differences in retention time are not very dramatic, so I don't think I'm getting anything other than mono-iodotyrosine.

Also, does incorporation of iodine affect the pI of a peptide?

If anyone has any advice or suggestions I'd be grateful for them!

Thanks in advance

psansom at

Paul A Sansom                                  tel: 081-748-9966 x4208
Department of Biochemistry                     fax: 081-748-5090
The Kennedy Institute of Rheumatology
6 Bute Gardens
London W6 7DW

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