Can any one please help me to resolve this problem!
I was doing in-solution tryptic digestion of a sample with 100 ug of proteins. Sample was dissolved in Urea/Thiourea and for lowering the Urea concentration I added tris 100mM. Next day I added formic acid to stop the trypsin action but after adding the formic acid I observed that most of my samples formed a ppt!! Why this happens? Is this because of lowering the pH? Or does it mean that trypsin didn't worked and still enough proteins were there? Should I repeat the digestion?
The answer to your question about repeating the digestion depends on how much sample you have left and the cost of your MS run. I am somewhat puzzle as to why you added Urea/thiourea to your in-solution trypsin digestion. Also their is no obvious reason to stop the trypsinization, likely trypsin will stop itself by self digestion.
After the digestion, I usually zip tipped the sample with a C18 tip and apply to MS. Remember that MS is so sensitive and that you do not need 100 % digestion.
Hope this helps
Marc
Is the precipitate composed of protein-derived material, or some of the other stuff you put in there - urea, thiourea, Tris - precipitated by the formic acid? It would be better to avoid adding so much material that will later have to be removed. I would recommend treating with trypsin for a few hours, then boiling the sample to denature any large protein fragments, then adding more trypsin to complete the digestion. As Marc Ouellet pointed out, the trypsin will digest itself, so you don't need to inhibit it.
I agree totally with previous answers, but I'm curious to know where you got your method.
Dear it seems you have a problem with urea concentration. I guess I am replying late but I want to drag your attention that urea affect protein folding in short. From your statement I guess you destroyed it's native conformation. In future come across with such problem, the best hope is to do dialysis.