Dear Tobias,

Two ways to go at least. Elute your proteins from the beads with Laemli buffer (SDS, DTT,..and boiling. Load on a SDS-gel and cut out all visible bands or even all the gel (don't forget to add negative controls). This is what I call salami proteomics but could give you good results.

Another gel free approach is the FASP method developed by Matthias Mann group at Max Planck. Gives good yield and it's an in solution digest.

Don't forget that because of the high amount of antibodies you will have to go through LC-MS (nano if possible) to find the needle in the haystack.

Good luck

Dear Tobias,

You may try both ways, sometimes they give complementary results. But I would not be afraid much of loosing peptides in in-gel digests, especially if using trypsin.

Best,

Yakov

Dear Tobias, we routinely use the SDS-PAGE/nLC/MS/MS approach described by Didier, cut the lane into 23 bands irrespective of staining with Coomassie, and are very satisfied with results. The SDS-PAGE also works as a clean-up step prior to mass spec, provides additional fractionation so higher degree of separation, plus a reality check through the Coomassie staining. Yes you will lose some peptides during the extraction from the gel, but then the same is true for e.g. FASP where I have not seen credible studies on recovery so far. If anybody else is aware of publications where FASP recovery was thoroughly evaluated - I would also be interested!

Dear Tobias, somethng useful to understand recovery from gel-based studies that came up on a different forum: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291619/pdf/v011p00074.pdf

I think both protocols are working well. In my case, I always prefer in-gel digestion, due to its another practical advantage - you are sure your protein is highly pure. And I have never observed any significant losses.