Hi Trung,

Post reduction with DTT and alkylation ieth IAA, you will need to dilute the Urea to about 1M before adding eg., Glu-C. After 4-8 hours, you can follow this with Trypsin at a concentration of 5ng/ul, overnight. I think that switching the order of the proteases should also work, i.e., use trypsin for 4-5 hours followed by Glu-C. I also suspect that this second approach (trypsin followed by Glu-C) may result in better efficiency since trypsin is known to be a very active and specific protease. The peptides you generate with Tryptic digest will then be digested further by the addition of Glu-C.

Good luck,

Hediye.

Hi Hediye,

Thank you so much for your suggestion. I actually will switch the digestion solution after Urea, IAA, and DTT to 50mM phosphate (p.H 7-8). I think I will try Trypsin and then Glu-C. I want to enrich O-GlcNAc proteins from 3mg cell lysate. Since they are quite low abundance, that's why I have to start with more material. This is cell culture, so that wouldn't be a problem for me. After enrichment from 3mg cell lysate, I suppose 15µg of Trypsin and 5µg of Glu-C would be sufficient!!!

Hi Trung,

I have not used 50mM phosphate so I cannot comment. You can also dilute the 8 Urea to 1 M Urea using 25mM NH4HCO3 which is ~pH 8.3. Trypsin works well in this buffer.

The Enzyme : Substrate you have suggested is 1:200 and that should also work.

Good luck,

Hediye.

It seems to work fine for me. Thank you very much

Won't the enzyme cleave another enzyme ? what if that creates more complex data ? Rather then increasing coverage. Moreover is there any protocols for double digestion ?