Endoproteinase GluC has no Cysteines and therefore isn’t affected by DTT. 4 M urea will probably denature both Endoproteinase GluC as well as your substrate protein. Any denaturing should be done with a brief heat treatment, such as 95°C for 1 min. If 4 M urea is necessary, add it and then dilute 10-100 fold before digestion. We have never used either nitrosoguanidine or thiourea with Endoproteinase GluC. We would expect some low level of either may be tolerated by the Endoproteinase GluC but we’re not certain as they are also chaotropic agents. We have used both methanol and acetonitrile in digests at a level of 10% organic. Acetonitrile works better than methanol in our hands. At 20% organic, the results were more varied with improved digestion for some proteins and poorer digestion for others.