Science Buddies: "Ask an Expert"

I have posted my questions here a few times before and have gotten a lot of great help. I am still working on my project of sequencing a myoglobin protein. Well I managed to get my SDS page bands digested at a facility near me and mass spec'd + fingerprinted, and the results, while good, only resulted in 81% coverage of my entire sequence. Since they only do tryptic digests I'm faced with the task of doing an alternate digest myself to increase coverage. I've heard good things about chymotrypsin so I think I will use that.

Does anyone have a simple and good protocol for a chymotrypsin in-gel digest? I am worried I won't have access to the kind of equipment I may need such as speed-vacs, agitated water baths, and the like. I've also heard a little about immobilized chymotrypsin but not much. Any alternative suggestions are very welcome.

Thank you

http://i.imgur.com/BNJC5iZ.jpg

I've taken a picture of my results. As you can see, I am missing everything in the black text. The red lines represent where the trypsin cleaved.

The dots represent where chymotrypsin would cleave if I used it. The blue line respresent where Glu-C would cleave.

Does glu-c seem a better enzyme to use? I am worried that the mass spec databases won't have as many matches as chymotrypsin. Also would one digest with trypsin then chymotrypsin or just one or the other?

Lots of questions! Thanks all.

Hi there,

I am sorry you have not received a timely response. You clearly have an excellent project and well articulated questions.

Your questions are outside my area of expertise, but I think we do have experts on this forum who can answer them for you. We had a backlog of posts from the holidays, so I suggest you try reposting your questions (all together) as a new topic. We usually ask students to keep everything in the original thread, but I think a new thread will get the attention of the experts who are trying to find what has gone unanswered.

Best wishes,
Heather