Multiplexed DIA on the Q Exactive

Okay, I've put this off long enough.
The final (so far!) method for quantification on the Q Exactive.  Multiplexed DIA!

You can start this fun monologue on QE quan methods here.

This is what you get when the crazy smart people at the MacCoss lab take on new technology.  Now, here is my disclaimer:  I've never ran mutliplexed DIA (which I'll refer to as MSX-DIA) so I completely have to go from what I've been told and from the papers and presentations I've read (and brazenly stolen from).

Unlike the rest of this series, Skyline takes the top of the post over a photoshopped image of the Q Exactive.  That's because you can't MSX-DIA without Skyline.  You need it to set up the experiment and you need it to process the data.

I've already talked about DIA in part 4, so pick up this monologue from here if thats what you came here for.

The MacCoss lab took a step back from our simple DIA experiment and our 20 Da stepping windows (sometimes called swaths) and thought that the specificity could be improved.  So they took the window and broke it into little tiny random chunks.

In this way, you can still cover the entire mass range because you've multiplexed random 4 Da windows together.  Imagine that we've now taken my biggest objection to DIA (only knowledge of your peptide MS1 scan +/-20 Da and you ditched it.  Thats what they did.  Now, since we are multiplexing we are still getting fragment ions from loads of different precursors, but we've now randomized them.

Looking at the data directly doesn't do you a lot of good.

You need to de-multiplex it first.  Whats that?  How does it work?  You've got me.  Skyline can do it though.  And if you doubt what Skyline can do, you probably haven't used it.

In the end, though, I know what you end up with.  DIA without as much of the MS1 specificity argument.  Since you are digging into 4Da windows (similar to a SIM) you get more dynamic range than you do out of your 20 Da windows.  And from the data that has been published (in some pretty impressive journals) it appears to be reproducible and accepted.  All good things!

And setting it up is easy (again, I haven't done it) but newer versions of Skyline have this nifty tab in it:

Multiplexed acquisition?  Yep!  And it outputs your "inclusion list" for the QE that provides your target windows.

And this is where I stop.
For more information, there is this sweet PPT from the MacCoss lab. (Warning: Direct download begins on clicking)

Thanks to Yue for explaining this to me and for the screenshots I stole from a talk she gave.