Hi,
I am looking forward for the extraction method that can extract most of the proteins from the serum for LC-MS analysis. I have many proteins of almost all the molecular weights for full proteome discovery. Some of the literature suggest that use of organic solvents such as TCA, methanol, and acetone etc. for protein extraction followed by depletion of highly abundant proteins. Few papers only immunodeplete the serum and solubilize the proteins using 8M urea with protease inhibitor. Which of the methods are best? Does this method interfere with in-solution or in-gel digestion further?
Thank you for your time!!
Best Regards,
Veni.
Hi Veni,
Read this article that suggests different strategies for excretion of proteins from different samples.
https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-strategies-for-protein-purification.pdf&ved=2ahUKEwj1yez2y5fsAhVU6XMBHZkXBDwQFjALegQIBRAB&usg=AOvVaw1dA12JQ3iSo-kqpHNkFy6M
I have prepared samples after TCA-Acetone preciptation for LC/MS analysis. As long as you boil properly to remove the acetone from the final white preciptate, it shouldn't interfere with downstream processing. Make sure to resuspend the preciptate in a slightly alkaline pH (I go for 8.5). If you are using Trypsin purchased from NEB, it'll remain active upto pH 9.
For total proteome study, in-solution digestion is your best bet.
Thanks Praveesh... Do you perform TCA-Acetone precipitation before immunodepletion or after immunodepletion? Which solution do you use for re-suspension of protein pellets?
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