SDS-removal by C18

[AdrianJJ]

Can anybody help me?!

Essentially my question is:

Can you use a C18 RP column to remove SDS from a (peptide) sample?

I have a small peptide (50 ng of 1 kDa) which is in 100 ul of buffer containing approximately 0.05 % SDS and 0.5 M Tris. I wish to clean up the peptide prior to MALDI-MS analysis.

I have been trying to clean the peptide using standard C18 disposable columns but I am concerned the SDS is not sufficiently being removed. I have been led to believe that SDS will irreversibly bind to the C18 phase and have been using a 50 mg C18 column which should have more than enough binding capacity for the peptide and the SDS.

I have acidified my sample with TFA and eluted from the column using a stepwise gradient of acetonitrile. I have checked and I am managing to essentially recover all my peptide from the column eluting with 40 % ACN. However, my peptide does not seem to want to fly in MALDI-MS (it normally does fly).

I am concerned I have not removed all the SDS from my sample. Will all the SDS bind to the C18 (assuming sufficient capacity) or will some come through? Will SDS also be eluted by ACN?

I am working on removing the detergent by other means before the C18 column but if anyone knows how the C18 is likely performing then I would be very grateful to hear!

We are also interested in buying a C18 RP column to perform seperation but are concerned the SDS/Triton X-100 will permanently damage the column.

Yours hopefully

Adrian.

[Uwe Neue]

No, SDS does not permanently stick to C18. It elutes in the same way as your peptide.

You know from your step gradient experiments, at which acetonitrile concentration the peptide is eluting. Do the same experiment for SDS. This will help you to finetune the separation between your peptide and the SDS.

Unfortunately, I do not have a clue how much acetonitrile is needed to elute SDS. Maybe somebody else has some info on this.

[Serg]

Hi,

I have not checked that myself, but there are some reports in the literature (PubMed PMID 15378713), that adding SDS can in fact improve MALDI. Also, this page may be of use: http://www.nestgrp.com/protocols/polylc/sds.shtml. They say that they use 70% ACN to elute SDS, which makes me wonder if you 40% ACN is enough, (considering possible strong peptide-SDS interaction)

Serg

[AdrianJJ]

Thanks for your help and advice.

You know from your step gradient experiments, at which acetonitrile concentration the peptide is eluting. Do the same experiment for SDS. This will help you to finetune the separation between your peptide and the SDS.

Thanks, good idea. I am looking into the DPH assay for SDS concentration though I am unsure if this works in ACN elution samples.

Also, this page may be of use: http://www.nestgrp.com/protocols/polylc/sds.shtml. They say that they use 70% ACN to elute SDS, which makes me wonder if you 40% ACN is enough, (considering possible strong peptide-SDS interaction)

The 70 % ACN elution of SDS as I read on that site is not from C18 but from their hydrophilic interaction guard columns. I do not know if the SDS will behave the same on a C18 column?

SDS improving MALDI-MS results is reported in that paper only over the CMC which I am pretty sure I am below. I guess I could try adding some SDS!

I am currently trying out the detergent removal spin columns from Pierce though it seems to like to hang on to my peptide. I will keep going!

Adrian.

[Kostas Petritis]

You can try the kit below, but I would be careful to make sure to ask what is the minimum amount of peptide/sample you need to process in order to get the high recoveries mentioned (i.e. >95% most of the time).


http://www.piercenet.com/products/brows ... id=keyword

[Vlad Orlovsky]

You can pass your sample through anion-exchange guard or SPE to trap SDS.

[AdrianJJ]

Thanks again everyone.

I am in the process of trying the Pierce SDS removal spin columns though my peptide (FAM-ADQNATA-NH2) seems to like to stick to the resin and I have to apply quite a large volumn of buffer to wash it through. Not ideal.

We did try a SDS removal SPE from Michrom though we lost the peptide. I am thinking that our fluorescent tag is causing the peptide to stick to these resins.

I am now fairly certain I am getting a lot of SDS eluting with the peptide from the C18 column. If anyone knows what % ACN SDS elutes it would be great.

Still testing these different ideas....

[XL]

SDS can be removed quite easily with 70% acetonitrile. It is cationic ion-pairing reagent that could be stuck on the silica based C18 column due to the strong ionic interaction between silanol groups on the silica surface and the quaternary amine functionality.

To move or trap SDS in the sample, you could use a reversed-phase/anion-exchange mixed-mode column in a small format, say 3x50-mm. Both reversed-phase retention and anion-exchange retention will help retain SDS. During separation, acidic condition (I assume the mobile phase contains TFA or formic acid with pH between 2 and 3) will keep the peptide neutral or cationic. At the result it can elute with solvent gradient.

Both Dionex and SIELC provide RP/IEX mixed-mode columns.
http://www.dionex.com/en-us/products/co ... 71737.html gives you some information on the Acclaim Mixed-Mode WAX-1 column that might work for your application.
You can find ordering information here: http://www.dionex.com/en-us/products/co ... 71737.html

[AdrianJJ]

It seems I am in a slightly lucky position.

You can precipitate SDS with K+ ions (KCl typically). When I add 1 M KCl to my samples the SDS precipitates but unlike most proteins/peptides, my peptide stays in solution. Therefore I have romoved the SDS problem and the remaining KCl in the solution should be washed away on a C18 column.

I think this is problems solved, though it would still be nice to know where on an ACN gradient SDS is removed from C18.

The RP/IEX columns are useful to know about though thanks XL.

Thanks to everyone for their help!