SDS removal and loss of sample

[dimtypas]

Hello everyone,

I am a grad student in Leiden University Medical Center. I have set up a method for purifying ubiquitinated proteins and I am trying to combine it with MS but I am now stumbling on various technical difficulties. I have isolated ~3-4mg of ubi proteins, in a buffer that contains SDS, which I then trypsinised into peptides and desalted with a C18 cartridge. As I figured out afterwards SDS is not removed by C18 and as I learnt the hard way it's messing up both SCX and HILIC chromatography. Thus I bought the detergent removal columns by Pierce (4mL resin for samples up to 1mL) and I tried to clean my sample. To my surprise I pretty much lost all my peptides but not all SDS, although I followed manufacturer's instructions to the letter. I had few if any peptides at MS and when I measured protein concentration by Bradford I could barely see anything above background. Does anyone have any suggestion about how to remove SDS? Does precipitation with KCl work or do you also lose peptides there? Additionally does anyone have an idea about why SDS would mess up with SCX but also HILIC chromatography?
Any suggestions would be rather helpful and greatly appreciated!
Thanks a lot,
Dimitris

[HW Mueller]

Ultrafiltration is a possibility, you probably have to wash with a correct buffer several times. The Piece columns may also work if you find a suitable buffer?
Since detergents modify proteins one should expect differences in chromatography in their presence (as compared to their absence).

[jonmike.reed]

Try replacing SDS with SDC (sodium deoxycholate) in your buffer. This detergent can be be removed after digestion by addition of 1% formic acid and 2 volumes of ethyl acetate. Here, most of the detergent will precipitate, and the remainder will go into the ethyl acetate layer, though you may want to repeat the ethyl acetate step to remove all of the detergent. We have found that a single extraction isn't sufficient to remove all of the SDC.

More details on this are at: J Proteome Res. 2008 Feb;7(2):731-40.