SEC of multimers of small peptides

[zopala]

Dear Chromatographers,

I am trying to develop an SEC method which would be used as a sort of an orthogonal method only for detection and quantitation of multimer content of a small peptide - ~ 1500 MW - under development. The peptide has 3 di-sulfide linkages (6 cysteines) in the sequence. The peptide is known to form dimers, trimers and possibly other multimers. It is also highly soluble in water.

My objective is to develop, if possible, a mass spec -friendly SEC method which would be able to resolve and quantitate the various multimers to a descent extent.

Unlike RP-HPLC, normal phase or even chiral separations I was not able to find a standard template approach for SEC. Since I am a newbie to this particular technique I would appreciate any input on where to start for the above mentioned peptide.

Thanks.
Zopala

[DaveM]

Hello Zopala,

I think your main problem with distinguishing those species is finding a column that will resolve such small (for SEC) molecules. Maybe there's a big gap in my knowledge that needs filling, but I suspect you will struggle separating 1500Da from 3000Da from 4500Da and so on. Unless it is an unfolded species in which case it may run with a higher apparent Mw and it may not be such a problem.

Another problem will be finding a MS compatible mobile phase. Water would be good for that, but I'm sure you'd have problems with non-specific binding to the stationary phase, ie. exactly what you want to avoid. Adding in salt would improve that but lead to MS incompatibility.

The severity of both of the above depend on the relative hydrophobicity of the peptide. It's highly soluble in water but perhaps there are some hydrophobic residues on the outside that will interact with the column.

So I'd suggest your first port(s) of call would be the manufacturers, asking if they can supply a column that will do what you want, then develop the mobile phase from there.

DM

[Gerhard Kratz]

Hello Zopala,

SEC for such small peptides is very difficult. One possibility is a 4µm packing material based on Silica, but there are some hardware issues. Please visit the following website http://www.separations.eu.tosohbioscien ... Brochures/ and download the brochure B07L04A Size Exclusion with TSK-GEL SuperSW columns. Minimum MW is about 1000 Dalton, minimum MW difference to get a base line separation is 500 Dalton. Anyhow, if you think that this column will do the job ask the manufacturer for a test column free of charge. Good luck

[Andy Alpert]

Hi, Zopala -

With apologies to Gerhard, the problem with SEC of solutes this small is that even the fractionation range afforded by a 60-Å pore emcompasses molecular weights too high for this. The solution is the following:

1) Use a stationary phase with a thick, hydrophilic coating.
2) Break the hydrogen bonds between adjacent ligands in the coating by using a mobile phase containing a weak chaotrope. Most people use 50 mM formic acid. This makes the space between the ligands permeable. In effect, this amounts to a class of "soft" pores 15-20 Å across, just right for fractionating peptides all the way down to individual amino acids by size. You can read about this in a book chapter I wrote in 1999, per the following link: http://www.polylc.com/downloads/SEC_boo ... r_ver1.pdf
Our PolyHYDROXYETHYL A columns have been used for SEC of peptides below 2000 Da for ~ 15 years. There are papers in print on its use for determining the size of peptides in commercial food hydrolyzates and the size of peptides exiting the proteasome. The 200-Å version seems to be everyone's favorite.

[pelle-plutt]

You could also consider the Superdex Peptide PC 3.2/30 column from GE Healthcare. It's has a fractionation range of 100-7000 dalton molecular weight, which seems to fit your sample. Use a flow rate of 150 μl/min and sample volume of 2-25 μl and a mobile phase that suits your mass spec.
Note that you need a special holder for this column if you want to hook it up to anything else than the SMART system!

[Andy Alpert]

Well, if you take a look at that book chapter I posted the link to in the preceding e-mail, you'll see that Superdex Peptide and all other carbohydrate-based stationary phases have a significant liability: the crosslinking agent that confers their low mol. wt. fractionation range interacts with aromatic residues. Consequently, peptides with a lot of Phe, Trp and Tyr residue can elute later than Vt in the SEC mode. A homologous series of oligoPhe peptides elutes in the opposite order than you'd expect in SEC. In that book chapter I compare Phe with Phe-Phe and Trp with Trp-Trp just to demonstrate that they elute in the correct order with a suitable silica-based material and mobile phase.

[dorota]

I do not want to put you off but it will not be easy. I have a short experience with insulin which tends to form dimers and hexamers. First of all as Gerhard said SEC for such small peptides is difficult. Second thing is conformational stability of monomers and multimers under chromatographic conditions. Noncovalent bonds in multimers are rather weak. Your peptides can undergo interactions with a mobile phase and a stationary phase and dissociate or associate (or folding/unfolding). I even cannot imagine what will happen with them in an ion source. I thought about a MALS or DLS detector but probably a hydrodynamic radius of your peptides is too small.
Nevertheless, good luck! Let us know when you succeed.