Thursday, March 8, 2018

EncyclopeDIA -- Gas phase fractionation + chromatogram libraries!

I have no intention of waking up enough to get this paper right. Primarily because I've got a huge and awesome day ahead in the lab....

My sleepy readthrough of this new study was something like this.

" this...?"
"Oh..maybe...come on, brain...maybe...?..."
"Nope. No idea what I just read. At all. Did they give a monkey ritalin and then let it dance on a keyboard? These aren't words! Why is everything capitalized wrong places.?!?...Is this the weird SpongeBob mocking meme..?...Where is my coffee?"
" is really smart...oh no...I should probably try this...cRAP...I should try this...oh no...that means I'd have to explain it to someone...but it's probably smart enough to be worth it...."
"I need more coffee or whatever they have for breakfast up there..."

What I've got (I'm gonna try. No promises):

Gas phase fractionation is one of those things that we've looked at for years -- it will obviously work -- but, in the end, it always comes off as kind of disappointing considering how much instrument time it takes so we never really do it. I fell for it for the 11th time less than a year ago! 

DIA is really powerful but the sensitivity sucks and background noise makes it one of those things that you hate to show your collaborators. "Yes, your peptide is these 5 fragments. No, ignore the other 175 fragments. They're supposed to be there. QUIT LOOKING AT THEM! (Why did I show you this...)"

And lots of chromatographic centric people -- PNNL comes to mind for some reason -- have been telling us for years that we need to work on the chromatography and it needs to be an important factor in our peptide ID and quan.

What if the gas phase fractionation DIA was used in conjunction with fantastic retention times to build the libraries for your DIA analysis? Would it then justify the time? Is this the chromatographic library? This is where I'm unclear....)

What I do get is the gas phase fractionation reruns in 100 Da windows with narrow isolation DIA (4 Da?) with the speed of the Q Exactive HF allows the authors to get really deep identification and they assign the identification with some fancy math things to a specific retention time (and I think this is the chromatographic library?) and then they can go back and match their "normal" DIA experiments.

Critical point here -- it relies so much on retention times that the authors are clear to point out if they use a home packed column this probably won't work.

Unrelated reminder:

 What do you get out of all of this? What about DIA that is actually more sensitive than DDA? What about more IDs? Like 50% more IDs than equivalent DDA experiment!!

Major bonus? You can show your collaborators a chromatographic peak from run to run and not a DIA fragmentation window (don't ever show anybody else a DIA fragmentation window!)

And (


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