Hi All,
Does anyone have experience with isolating native peptides from mammalian cells? Can anyone suggest a good protocol. The protocol I have is "Thermal denaturation of the cells at 80degree in water". I wonder I will loose many hydrophobic peptides.
So to enhance the overall yield I want suggestions.
Thanks.
It is difficult to isolate endogenous peptides for MS/MS because almost anything you do will co-isolate metabolites that have similar charge and polarity.
A lot of recent papers on neuropeptide isolation are using a combination of ultrafiltration and size exclusion chromatography to isolate endogenous peptides.
First, you spin your lysate through a 10 kDa ultrafiltration device, which retains intact proteins and other large macromolecules like DNA. Then you take the flow through and apply it to a size-exclusion column that only lets small things
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