I swear, I thought this was on the blog somewhere and I can't find it.
If you're in Europe or other places, I guess, you can just disregard this entirely. Rumor is you have a variable or variation DIA window button. If you're in the US, there is a trademark thing and only one vendor(?) can give you a V on your instrument by default.
Huge shoutout here to the Slavov lab for posting RAW files in the plexDIA preprint back in 2021 or something because this way of setting it up is more efficient than how I was setting it up.
As you can see above - you need to set up multiple experiments. In this case I'm acquiring MS1s on my old Q Exactive in the dark and generally extremely damp Biophysics basement at The John.
A Q Exactive Classis is slow by today's standards, so I think you'll definitely see people who will drop the MS1, depending on what their software actually requires.
This may actually be exactly the Slavov lab method that I am showing here. It might be worth noting that plexDIA was using mTRAQ tags, so the m/z of the peptides are shifted up a bit by the mass of the tag - but I tell you what - this method will absolutely smoke a standard 20 AMU fixed with DIA method.
So the trick is to set your loop count to match the number of windows in your "Inclusion list" file.
This is what your inclusion list should look like - I strongly recommend going into this and going File --> Export --> .csv or .tab delineated (whatever makes you happy) then opening that format in Excel or something else. Then you can use quick equations to set you center mass and overlap. Here I think we're getting a 1Da overlap on each side of each window, but don't quote me on that.
I'm 91% sure that you can also have Skyline do this for you, but - yet again - my Skyline expert is off making a lot more money than me, so the infinite cycle will continue. My cycle is this - sometime in 2025 I suspect I will pay to have some super bright young person go hang out with Brendan MacLean and some other brilliant targeted mass spec people and she/he will come back and tell me how cool Mike MacCoss is and for the next couple of years I'll have a Skyline expert! Then we'll have loads of pretty plots and matrix matched curves and then disaster will strike - Asta-Zeneca or Merck or Moderna will offer that brilliant young person more money than I make. I'll be super happy for them and they'll leave and reviewer comments will come in where it's like "can you do this very simple Skyline thing and we'll accept your paper" and I'll say "...no. No, I can't. I'm way way too dumb to do anything in Skyline myself even though I have tried to sign every support document for the software since 2012 or something.
What was I typing about? Aha! VariableDIA windows!
Above you've got the first 25 windows that are 12.5Da and then the next batch is 25Da and the final is great big 62 Da or whatever.
Considering where most of your peptides are in overall m/z ratio, you might want to adjust this. For example, if you were just LysC digesting and you were pushing up the distribution of the m/z of your typical peptides by about 70%, you could see a 4th experiment where you were using wider windows in the lower m/z mass range, narrow in the middle and then bigger then really big. This specific bit of vaguely and probably not usefulness I just typed was inspired by the r/proteomics entry that inspired me to spend 33 minutes before my first meeting today typing this instead of getting a shower and what appears to be a desperately needed second coffee.
Since the Virginia Tech/Google $20M drama continues, my GoogleDrive that basically every link on this blog went to has been deleted. What would be helpful, I think would be if I put the actual .meth and these tables up in links somewhere. I'm now at 36 minutes somehow, so I can't do it now. If you need these, email me, my contact is over there somewhere ---->
and I'll try to get these up where you can direct download them later
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