I have not used any of the reagents you named. My first thought is to use chymotrypsin. Since it cuts predominantly at aromatic residues, it tends to produce larger peptides than trypsin.

Dear Fridtjof,

The peptide cutter predicts potential cleavage sites cleaved by proteases or chemicals in a given protein sequence. In order to digest proteins in cell lysates into large poly-peptides with 50 aa or more, I recommend chymotrypsin like Dr. Shapiro said.

You may also look at the following link

https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html

Regards,

cyanogen bromide cleaves only at methionine residues, and should work for you. It is very efficient and very precise. You can look this up on Wikipedia.

Thanks everyone for good answers! I will give these a try and let you know the results. If anyone else has experience, I would be very grateful if you share it.

Hi Fridtjof Lund-Johansen ,

You may consider digesting the cell lysate through Pulse proteolysis (Digestion for shorter time period) method using either Chymotrypsin or Trypsin enzyme.

Please see attached link

https://www.nature.com/articles/nmeth740

Your cell lysate will surely contain a huge number of different proteins, the majority of which will already be longer than the required 50aa. What is it you are trying to achieve?

Hi Geoff,

I know that a cell lysate contains a huge number of proteins and that most polypeptides are larger than 50aa. I want to achieve partial digestion into peptides between 5-30 kDa.

Saravanan's idea should work for a lot, maybe most, of the proteins, but I would make a very dilute solution of proteinase K and do a pilot (various times) digest, assessing the average fragment size using SDS-PAGE and coomassie staining. But that assumes I dont mind if each molecule of the same protein (in the lysate) is cleaved at a different position. But thats true of any partial digest.

What do you plan to do with this very heterogeneous mix of peptides?