SEC - mobile phase for sticky proteins

[lisaannstone]

Can anyone here suggest a good mobile phase for sticky proteins? I'm using a superdex 75 column and my peptide just doesnt like to be seen. Most solvents give too much background at the wavelength I'm using.

many thanks
Lisa

[danko]

You might like to tell us which mobile phase compositions you've tried and which wavelength do you detect at. Then we'll try to suggest something you can go forward with.

Best REgards

[lisaannstone]

sorry I wasnt very informative before...
I've used standard PBS and also 10mM ammonium acetate pH10, I dont see anything. I detect at 214, but have also tried 254nm. I cant see my standard markers at 254 which is a problem. I havent tried any solvents yet since DMSO, which is standard, gives a giant peak just where my protein should be. My protein is 4.5 kDa, quite small.
many thanks for any help
Lisa

[danko]

OK. Firstly, you should be aware of the fact that acetate absorbs immensely light/energy at 214 nm . Another fact is: No, or very few proteins absorb light at 254 nm, so it’s a kind of useless wavelength in a protein context. My suggestion is, to just change the detection wavelength to 280 nm, if you need to use acetate in your eluent.
Secondly, if you still don’t see your protein eluting, then it might precipitate out upon contact with the mobile phase. Maybe you see backpressure increase when injecting? If that is the problem, make sure the protein is soluble in the mobile phase – just mix a small portion of the protein solution with a small portion of the mobile phase and make sure the mixture is completely transparent.
Then maybe you’d report back and we’ll take it from there.

Best Regards

[lisaannstone]

thank you for helping out the newbie. I will try 280 nm and let you know how it goes )))

many thanks

Lisa

[lisaannstone]

still nothing,
Tested peptide solubility in buffer.
I used my standard peptides that we use for MW measurement with PBS and normally a good signal at 214 nm with 10ug each (alubmin, cytochrome c and insulin) and my peptide at a higher conc of 100 ug (there was a very small peak at an earlier time point than it should be). I'm now investigating some other standards at higher concentrations to see if I can see anything remotely at 280 nm. I have also changed the filter lots of time incase there was a build up of aggregates.
Keep you informed, any other suggestions welcome
Lisa

[danko]

Maybe I should’ve mentioned that proteins absorb approximately 14 times less at 280 nm compared to the absorption at 214 nm. So you’ll need to load more sample in order to see adequate peaks. You can start by injecting larger volumes.
Remember to set run time to at least 30 – 40 min. What are the column dimensions and the flow-rate by the way?

Best Regards

[tom jupille]

In addition to danko's good suggestions, let me add a few general comments:

1. Are you dealing with a peptide or a protein? Proteins are detectable at 280 nm only to the extent that they contain aromatic residues. If your peptide has no aromatic residues, you won't see it at 280, period. The peptide bond itself absorbs at low wavelenth.

2. Detectability issues aside, there is no general fix for "sticky" proteins, because they "stick" in different ways: ion-exchange, adsorption, and hydrophobic binding come to mind. Combinations of pH, ionic strength, and organic modifier that work well for some proteins (or peptides) work poorly for others.

[lisaannstone]

thanks again for all your help

The peptide I'm working with is beta amyloid (in various forms, working with the "easiest" - ha, to set up at the moment) its small, hydrophobic and has only one tyrosine residue. Working at higher concentrations than I am already would be problematic so need to maximise the sensitivity of detection.
I use a Superdex 75 column 10/300 set at 0.5 ml/min flow rate.
If anyone could recommend a good buffer for 214 nm detection that would be good. Although even in PBS there was some issues... just got going on this and the peeps before me were seeing DMSO as beta amyloid in PBS so their methods were obviously rubbish ) I've tried so many of the published methods and nothing seems to work.
Thanks for helping out the inexperienced
Lisa

[tom jupille]

A couple of additional questions/comments:

1. are you trying to maximize *mass* senstivity or *concentration* sensitivity? If it's the former, then a smaller column will give better results -- but you will be limited in injection volume and you will have to be very careful about extra-column volume. If the latter, then a smaller column won't help (less dilution but limited injection volume cancel each other out).

2. Are you trying to recover the peptide or merely to measure how much is there? If the latter, GFC may not be the best way to go (limited injection volume). Something like reversed-phase would allow you to inject much larger volumes.

3. Along the same lines, if it's a case of assay, you should be able to get much better sensitivity with LC-MS/MS (UV has its limits!) or possibly with post-column reaction + fluorescence.

[lisaannstone]

Its a little complicated what I'm doing, I need to have a good sensitivity for concentration as too high a concentration will increase aggregates formed on the column. 100 ug is the max I can inject. Also, even though I want to look at this peptide under non-aggregation conditions the methods I'm trying to develop will eventually be used to assess this peptide that has be pre-aggregated, crosslinked to fix the aggregates, then also run under non-aggregating conditions to assessed crosslinked peptides only. I want to separate the different forms to collect and test.
I hope I'm making sense here...
I'm now back to PBS just to check what I can and cant see before I investigate other solvents.
many thanks for the help

[danko]

Hi Lisa,

I think you’re trying to do the impossible. And it’s sort of ironic that your boss has delegated the task to a newbie (as you describe yourself). Maybe you should go to him/her and ask him/her to demonstrate what they are expecting you to accomplish.
The aggregates you are trying to chromatograph are bonded by hydrogen bonding and are either unstable, under certain conditions (typically alkali as well as under shear forces), or insoluble and thus impossible to chromatograph. I’ve seen some articles that describe chromatography of non-covalent (hydrogen bonded) aggregates/fibrils, but I have my reservations about that.
As for the detection; you can just go back to 214 nm by leaving out the acetate from the mobile phase. Try phosphate instead.

Best Regards

[Furry_dice]

Mny proteins that stick with PBS will elute beautifuly with 0.1M sodium nitrate.