I'm planning on analyzing phosphopeptides from tissue samples.
I was told to extract protein from tissue using either 8M urea buffer with 0.1% NP-40, 1 mM sodium vanadate and protease inhibitor + phosphatase inhibitor.
Is there a reason why I cannot use RIPA with SDS for phosphoproteomic analysis?
I will be using pierce TiO2 phosphopeptide extraction and enrichment kit, and use C18 desalting tips in between.
Hi Sulgi,
RIPA buffer is perfectly alright for protein extraction, but it shouldn't be there when you do your TiO2 enrichment.I would add other phosphatase inhibitors just in case, though.You can't(!) go straight from proteome extract to C18 desalting! and you have to digest the sample first...
no - any reverse phase attempt will lead to adsorbence of part of the detergents onto the column and turning it into a soap chromatography column with very unwanted results. Moreover, Trypsin won't work in 2% SDS and 8M Urea. you have to buffer exchange before you can digest. FASP is strongly recommended and talking to your local mass spectrometry facility is essential as they should have very strong experience with this type of sample prep process.
Hi Sulgi,
If you use RIPA buffer, as already pointed out, you will not be able to perform efficient in-solution digest and peptide clean up.
However, if you have to use RIPA buffer, you can proceed to SDS-PAGE and therefore remove any detergents from the protein. After SDS-PAGE, you can then perform in-gel digest on the protein and extract the peptides for TiO2 enrichment.
Alternatively, you can solubilize the protein in 8M Urea, dilute the Urea to ~ 1M Urea and carry out in-solution digest with trypsin. This workflow will result in peptides that can be purified and subjected to TiO2 enrichment.
Good luck,
Hediye.
Thank you Hediye,
As you suggested, I plan to use 8M lysis buffer with 0.1% NP-40. Then dilute 8M urea down to 1-2M by adding Tri-HCl for in-solution digestion with trypsin. I will also desalt after digestion with C18 tips before TiO2 enrichment.
Out of curiousity (and I think I should know this before my experiment), why is high concentration of urea not compatible with trypsin digestion?
Hi Sulgi,
Here is a very good methods publication where the authors tried the effect of chaotropic agents and detergents on solution digestion of proteins:
Best,
Hediye
Hi Sulgi -
again, desalting straight out of a detergent (NP-40 in this case) will lead to loss of sample.
good luck!
David.
Hi Sulgi,
David is correct - you should avoid NP-40. 9M urea is a great solubilizing agent and you do not need any detergents for the proteolysis. Just use 9M urea around pH 8.2 to solubilize the protein. After reduction and alkylation, dilute the urea down to about 2 M and carry out the trypsin digest. After the digest, desalt the peptides using Stage tips or Zip tips.
Best,
Hediye.
Thank you David and Hediye,
I will try 8-9M urea without NP-40 for lysis buffer.
So, here's another question. If 9M urea is enough for preolysis, why do some people use detergents in urea buffer? Maybe they are trying to isolate surface membrane proteins which is harder to get out?
Thank you very much
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