Hi to all,
I have to perform a trypsin in-solution digestion of a protein sample.
I would like to avoid the use of SDS/Urea/GnCl. Is possible to perform the trypsin digestion of my proteing operating at high temperature and using a little percentage of acetonitrile in order to denaturate the protein and allow the trypsin to work?
I have a rougly 150KDa protein, so which concentration of acetonitrile and which temperature do you advise to me?
May I perform the digestion over-night?
Thank you
Hi Antonio,
Trypsin will probably allow the use of up to 10% AcN in the digestion buffer; it might even cleave the protein you're digesting slightly better. I wouldn't advise to use temperatures much higher than 37oC. It's no problem incubating the mixture overnight, although most of the digestion will be done in the first 4 to 6 hours of the reaction.
Another thing you could consider is performing a sequential endoproteinase LysC - trypsin digestion: LysC works in relatively high concentrations of urea, so after an initial digestion using LysC, you can dilute your sample and let trypsin have a go at the pre-cleaved sample, which should improve overall digestion efficiency.
Good luck!
Hi Antonio,
Trypsin will probably allow the use of up to 10% AcN in the digestion buffer; it might even cleave the protein you're digesting slightly better. I wouldn't advise to use temperatures much higher than 37oC. It's no problem incubating the mixture overnight, although most of the digestion will be done in the first 4 to 6 hours of the reaction.
Another thing you could consider is performing a sequential endoproteinase LysC - trypsin digestion: LysC works in relatively high concentrations of urea, so after an initial digestion using LysC, you can dilute your sample and let trypsin have a go at the pre-cleaved sample, which should improve overall digestion efficiency.
Good luck!
ok, thank you Eef. I'm wandering if only 10% Acetonitryle is enough to denaturate a 150 KDa protein (an IGG).
I already suspected that you were planning to digest an antibody. I assume that you perform a reduction of the disulfide bonds (and alkylation of the resulting free cysteines) prior to digestion?
In our hands the combined LysC-trypsin approach I described worked quite well. It is difficult to get complete and consistent digestion all the time, but that protocol improved the quality and efficiency of IgG digestion.
ok Thanks. unfortunatelly I can't use Any DTT or Alkylating agent, because I have to study the behaviour of cysteine in the presence of metals
This has worked for me.
Anal Chem. 2006 Jan 1;78(1):125-34.
Efficient and specific trypsin digestion of microgram to nanogram quantities of proteins in organic-aqueous solvent systems.
You can digest in Methanol up to 80% where trypsin is compatible, however I'm not sure if it can denature better than Guanidine or SDS. You also run into the trouble of SPE clean up prior to LC/MS analysis since you have such high organic (which I would recommend to avoid).
I used to use trypsin to digest a 400 kDa protein in 2 stages. After incubating with trypsin at room temperature for a few hours, I boiled the partially digested sample, then added more trypsin and incubated overnight at room temperature. No organic solvent, SDS, urea, or guanidine needed.
Thanks Adam,
Can you please tell me the concentration of trypsin you have used...
This is the method:
CaCl2 (5 mM) and tosylphenylalanyl chloromethyl-ketone-treated trypsin (Sigma) dissolved in 1 mM H2S04, (20 µg/ml) were added to the protein sample (1 mg/ml). The sample was incubated at 23oC at least 2 h, heated in boiling water for 30 s, and cooled. A second addition of trypsin was made, and the sample was incubated overnight at 23oC.
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