native and oxidized protein..separation on Ion exchange chr?

[alicee!]

hii i m trying to separate a native and oxidized protein (could be a single methionine oxidation also).. on Ion exchange chromatography? u think its possible? any method suggestions? shud i do it by pH gradient or salt gradient? which works better for resolution for such close species? thankss!

[danko]

I think you need to explore the reversed phase possibility in this situation.
Oxidation will not alter the net charge of your protein. When it comes to polarity on the other hand, you can expect a huge change due to oxidation.
Otherwise (to answer your other question) the salt gradient is most feasible.

Best Regards

[alicee!]

i do have an rp method.. i m looking for a complementary method by IEC.. for oxidized as well as reduced species separation from the protein.. u suggest salt gradient? why is that better? oxidized wud have a different charge right? why do u say no?
thankss!

[danko]

Hi again,

oxidized wud have a different charge right?

No. Where would the charge come from?

u suggest salt gradient? why is that better?

Longer story, but the short version is: More rapid action/effect = narrower peaks = higher efficiency.

Best Regards

[tom jupille]

Even if the net charge is the same, you may still get separation via IEX if the conformation changes as a result of the oxidation (moves the charges relative to one another). The only way to know is to try.

A salt gradient would be my first choice. pH gradients in IEX are always tricky because the resin "titrates" the mobile phase, so the effective pH during the column is not necessarily what you put in. In addition, pH has its greatest effect on selectivity as you approach the isoelectric point of the protein, at which point things start to change *very* quickly.

[alicee!]

Even if the net charge is the same, you may still get separation via IEX if the conformation changes as a result of the oxidation (moves the charges relative to one another). The only way to know is to try.

A salt gradient would be my first choice. pH gradients in IEX are always tricky because the resin "titrates" the mobile phase, so the effective pH during the column is not necessarily what you put in. In addition, pH has its greatest effect on selectivity as you approach the isoelectric point of the protein, at which point things start to change *very* quickly.

agreed.. so the differences wud be due to structural differences in the protein due to the oxidation at particular sites, as you say. since met oxidation includes s=o bonding.. wud electronegativity of oxygen there.. provide some sort of a charge too? what say?

and how to work on the salt gradient.. i m new to IEC! i ran a 0-200mM salt gradient for 30 mins after equilibration. i ran the control/native and oxidized protein ( i m confirming oxidation by RP-HPLC).. i dint see any resolution.. how do i change the method now!? its' a strong cation exchanger.. protein pI around 6, buffer pH around 5.4.
thanks!!

[HW Mueller]

You are doing some heavy handed oxidation? One normally has -SH >>
-SS- . If you oxidize further it may indeed produce ionic species (the ionization of -SH should be outside of your pH range). But don´t confuse changes in polarity with formation of new ions. Polar-ion interactions are quite different from ion-ion interactions.
From what I have seen about electrophoresis and IX, it seems that protein separation is as much an art as science. That may mean that you have to "fiddle" around quite a bit. I would start with changing the pH (not a pH gradient).

[danko]

Alicee,
“strong cation exchangerâ€

[Kostas Petritis]

Hans,

I think you confuse cysteine with methionine. Methionine residue goes CH3-S, first step oxidation gives CH3-S=O (sulfixide), more oxidation gives CH3-S(=O)2 (sulfone). Conformation changes might or might not happen, SCX might or might not work, you just have to try it...

[alicee!]

Hans,

I think you confuse cysteine with methionine. Methionine residue goes CH3-S, first step oxidation gives CH3-S=O (sulfixide), more oxidation gives CH3-S(=O)2 (sulfone). Conformation changes might or might not happen, SCX might or might not work, you just have to try it...

yess.. i m refering to methionine oxidation.. so now i see that there is a very li'l chance that i may see resolution on IEX..
what are the other methods that cud b used for such impurity identification/separation..? thanks!

[danko]

A realistic option could be HIC – although fairly similar to RP.

Best Regards

[HW Mueller]

Kostas, thanks for reminding me that methionine has a -SCH3 not -SH. But I am still surprised that alicee! has to bother with oxidation, . . . it appears that this should be a much slower affair? It surely would be interesting to find out what sort of protein has a methionine, but no cysteine.
I just also wonder where the current limit of molecular weight lies for a successful MS analysis of this?

[Kostas Petritis]

Hans,

I would say that methionine oxidation is a very common modification. It happens quite a lot due to biology but it can even happen as a side to sample preparation...

About the MS question, it depends if you are using chromatography or just infusing but I would say from accurate mass you can identify proteins of about 30K from isotopic distribution and maybe up to 80K from charge state distribution. Nowadays there are also a lot of fragmentation techniques such as ECD and ETD that can fragment the proteins and give a lot c and z fragments leading to easier protein identification... The numbers I mention are approximate...

[HW Mueller]

That should be good enough to quantitate the incorporation of an oxygen?

Also, didn´t we discuss some chemical derivatization or chemical analysis of S-amino acids some time ago?

[Kostas Petritis]

It really depends on the number of proteins you want to do simultaneously. If you have only a dozen proteins, that is a very easy task with modern instrumentation and software, as you can always digest your proteins and analyze them by MS/MS. Most people when doing SEQUEST searches they include methionine oxidation as dynamic modification (i.e. it will look for both variations of unmodified and modified methionine). CID MS/MS fragmentation will give you enough fragments to determine with pretty high confidence these peptides, no derivatization is required...

In proteomics, we can typically identify about 2,000 peptides (with high degree of confidence) from a < 2 hours run for a global microbial tryptic digest with UPLC-ion-trap-MS-MS. Only 10-15% of the MS/MS spectra will give a confident ID...