In yet another blog post to originate completely by accident, some friends (who would rather remain anonymous) and I were doing some ETD optimization the other day and found that our first run (set up quickly prior to lunch) had ETD reaction times and anion target values that were far too low. This resulted in ETD spectra that were dominated by the parent ion and a few charge reduced species. Most of them looked like this (PD view):
Crappy, right? From the all-important manual look through of our data, it was clear that we needed to jack up our anion target value, our reaction time, or both.
Since it was our only run, we went ahead and processed it anyway. One of the things I wanted to toy around with was the "non-fragment filter" in PD. This vaguely named node has a bunch of parameters specifically for working with ETD spectra, including removing the parent ion, charged reduced species, and even neutral loss (think water loss) charged reduced species, and even something called FT Overtones (if anyone knows what that its, please comment!)
So we ran the crappy ETD spectra through this anyway, and guess what?!?! the ETD spectra were all the sudden awesome. Not good. Awesome. The MS/MS spectra below is the EXACT SAME SPECTRA (4613) as the one above, just with all these useless ions cut out and the spectra rescaled.
BOOM! And this was a match. A very nice, high scoring match.
Now, this node, which I am from here on out referring to the "awesome ETD spectra" node (I'll try to get it changed in later versions) does come with a drawback that my friends were quick to point out while I was recovering from my blown mind. The drawback is that if you didn't actually look at your RAW data you could miss the fact that your reaction isn't optimized correctly. Great point, right? Ideally, we'd want our reaction tuned up correctly, then run the "awesome ETD spectra node". But once you get it tuned up, definitely definitely use this node to get great, high scoring matches out of your ETD spectra!
(this is my first ever blog comment, woo hoo!) just want to mention, since ETD is somewhat like HCD in that its sometimes (especially with higher charged stuff) easy to over-fragment (which results fragmentation of your product ions and generation of internal fragments), sometimes what looks good to us intuitively is not the best ETD spectra... I lean toward under-fragmentation with ETD...the charge reduced species can be misleading, very often once removed, you have some GORGEOUS primary product ions there. The only ions there are no awesome correction nodes for are internal ions, so under-fragmentation can truly be your friend with ETD! don't let those charge reduced species bother you... PD has them covered!
ReplyDeleteWeird that you post this, I kind of ignored this node till this week and loving it.
ReplyDeletePost processing Blog very nice blog
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