Hi Masrure,

I can get my bacterial proteome (Bacillus) resolubilized in 0.2% Rapigest in 50 mM Amm. Biocarbonate.

Rapigest is an acid-labile surfactant. It doesn't interfere with trypsin digestion, and to get rid of it, you simply acidify the solution (1% TFA final concentration). This cleaves the rapigest, and the detergent part of the molecule pellets away, and whats left doesn't interfere with downstream applications. I do not have a reference for you b/c we did not publish this work, but I can say from personal experience it works!

Here is a link to rapigest:

http://www.waters.com/waters/partDetail.htm?partNumber=186001861

Hope this helps!

Val

Hi Masrure,

if urea works for you, I would resuspend the pellet in 8M urea with 50mM ABC buffer and dilute the solution prior digestion to e.g. 2M urea by 50mM ABC. Trypsin should work in these conditions. The presence of urea could introduce carbamylation at elevated temperatures (>37°C), some experienced this also at lower temperatures. I would try this approach and digest your sample at maximum of 37°C and check MS/MS data for the presence of primary amines carbamylation (variable lysine and peptide N-term carbamylation modifications). If you are going to do label-free quantitation this could be problem.

I would perform digestion in the same tube (i.e. in presence of the residual pellet if there will be any) because the digestion itself could also help to solubilize the proteins. Again the question could be whether this will be quantitative in your case...

You could also try totally different approach using cut-off filters - look for FASP and "high recovery-FASP". This approach is relatively robust to your lysis buffer and you do not have to use precipitation.

David

Hi Masrure,

I'm not sure if you really need to clean up the proteome before tryptic digestion. There are protocols around for direct whole cell tryptic digestion which are quite straight-forward. Basically, you can remove residual liquid of your samples and resuspend them in ammonium bicarbonate buffer and directly digest them with trypsin over night. Stop the reaction by lowering the pH using formic acid and clean up your peptides using "zip tip" pipetting (pipette tips with a small silica column inside).

This method is a standard procedure in our labs and also published, see Jehmlich et al. 2010, Jahn et al. 2013.

It is described for cytometrically sorted cells but should work for everything else as well.

Cheers, Micha

http://www.jiomics.com/index.php/jio/article/view/115

Thanks a lot guys.

Hope your valued suggestions will help me a lot. David, can you give me any reference where this type of tryptic digestion (in presence of 2M urea) had been performed? so that I can cite them. Will the carbamylation really affect the quantitation? If I follow this approach, will the data be publishable?