Science?!? What?!?
PepMap C-18 is a really common LC material these days. After looking at the last week of runs on 3 different instruments I feel like they should slap a warning label in it. It should say something like
ALL OF THE PEPTIDES COME OFF AT LOW LOW LOW ORGANIC!
I've got better runs than this now but I haven't processed yet. 200ng of human digest lysate. In the top run you have a normal-ish ramping of 5-35% acetonitrile in 100 minutes. On a whole cell human lysate, I'm getting around 3,700 unique protein groups on OT-OT scans. The peaks are just bunched too closely in the middle to get much more.
On the second one -- no changes to the instrument method -- just using a 2 stage ramping (to 20% B!!) I'm over 4,500 protein groups. I'm picking up around 3,000 new PSMs just by compensating for the fact that PepMap is ditching all my peptides super early in the organic. Again, I've improved upon what is shown above, but it gives you a feel for where I'm heading with this!
What is the mobile phase composition you are using? Is it with or without trapping? Can you share the entire instrument method?
ReplyDeleteMr. CoolJ,
ReplyDeleteI'm glad to help. Please don't go insane, startin' a hurricane, or releasin' pain.
Shoot me an email: orsburn@vt.edu and I'll send you the full method.
hi Ben, thank you for this useful information. Even we have been using 5-35% acetonitrile over 100 minutes routinely. It was nice to go through this post of yours. What was the length of analytical column for the runs that you show here?
ReplyDeleteManish Kumar