I want to measure my peptide samples on mass spectrometry, therefore I want to avoid any detergent in the lysis buffer.
I can recommend the FASP protocol (http://www.ncbi.nlm.nih.gov/pubmed/19377485). You can perform your cell lysis with SDS. The SDS is removed on the filter by washing with 8M urea. After performing the trypsin digestion, peptides will flow through the filter when spinning. Afterwards you have to "desalt" your peptides with C18 solid phase extraction (i.e. Agilent SPEC PT C18AR (#A57219)).
Important: You have to use a flat bottom filter with 30K (or 10K). I used these:
http://www.sartorius.com/en/product/product-detail/vn01h21/
I tested the FASP protocol and an in-solution protocol and got better results with FASP for TiO2 enrichment afterwards. However, you can also use 8M urea in your lysis buffer. But you have to reduce urea concentration to < 1M urea when performing tryptic digestion.
Are you looking for peptides or proteins? Majority of peptides should be soluble in water or at worst, 1M urea.
Otherwise it depends on the protein you want to solubilise. Membrane proteins won't solubilise for instance. But you are right in avoiding surfactants.
You won't solubilise everything but then no single method really does. If you break the cells open in 8M Urea at pH 8+ (choose a buffer that won't interfere with TiO2 binding), reduce and alkylate and then centrifuge out the insoluble material. Dilute the supernatant to 1M urea in whatever pH 8 buffer you chose and add trypsin in a 1:50-100 ratio (enzyme:protein). I would then clean up the peptide by solid phase extraction (I like OASIS HLB for this but any C18 will do) and then add the TiO2 beads.
With the insoluble material, you could add trypsin in 1M urea and cut off whatever accessible peptides are there, leaving the transmembrane sections and bits where there is no accessible lysine or arginine. The transmembrane parts would not likely have phosphorylation.
Absolutely. You might jest need to "persuade" it back into solution with a sonicator bath or probe as the urea precipitates.
I can recommend the FASP protocol (http://www.ncbi.nlm.nih.gov/pubmed/19377485). You can perform your cell lysis with SDS. The SDS is removed on the filter by washing with 8M urea. After performing the trypsin digestion, peptides will flow through the filter when spinning. Afterwards you have to "desalt" your peptides with C18 solid phase extraction (i.e. Agilent SPEC PT C18AR (#A57219)).
Important: You have to use a flat bottom filter with 30K (or 10K). I used these:
http://www.sartorius.com/en/product/product-detail/vn01h21/
I tested the FASP protocol and an in-solution protocol and got better results with FASP for TiO2 enrichment afterwards. However, you can also use 8M urea in your lysis buffer. But you have to reduce urea concentration to < 1M urea when performing tryptic digestion.
As Stephan pointed out FASP is one method. You can also use acid cleavable detergents like Rapigest (via Waters) or Sodiumdeoxycholate. Upon solubilization and digest the detergent are precipitated by adding acids like formic acid or TFA and can be right away used for C18 purification prior to MS. We use it a lot and works fine. Here a recent paper from Ole Jensens lab where they compare insolution digests with FASP and found that both insolution digest and FASP using Deoxycholate works best for the conditions compared.
http://www.ncbi.nlm.nih.gov/pubmed/23792921
Most of the times the problem with the "solubilization" comes not from the 8M Urea solution not being able to dissolve most cellular proteins, but from problems the actual, physical lysis of the cell resulting in the solution becoming very viscous due to the release of cellular DNA.
One can use 8M solution for efficient cell lysys and sample preparation by applying the solution over a cell pellet (or protein precipitate) and briefly sonicating using a probe sonicator. Several bursts of 3sec with several seconds pause in between would results in complete lysis of the cell pellet / dissolving of the protein pellet without the solution heating too much and inducing urea breakdown and protein carbamylation. Followed by centrifugation to clear the solution of undissolved material one can use the supernatant for analysis.
Same with 6M Guanidinium chloride.
Yes, 8 M Urea lysis buffer is better option to avoid the use of detergents for cell lysis.
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