Saturday, April 13, 2024

MAG-NET - 4k proteins from plasma with accurate quan without spending $4k/sample!


I just prepped and ran an absolute shipload of plasma samples last week using a lot of the best of today's technology. S-Trap 96-well plates so it's fast and reproducible. EvoTips and EvoSep one so they're clean and - again - ultra reproducible and the newest best diaPASEF on a system with all the updates so it's screaming fast on nice little windows. 

And......700-ish proteins identified. Which is just about the same f'ing numbers I'd get for plasma in 2011 prepping in a FASP filter and using a terrible old brown Eksigent nanoLC and running samples on an LTQ Orbitrap Velos system. Give or take a 100 protein groups. My memory has faded over the last 13 years. 

The point is that it's generally pretty easy to see those top few hundred proteins in plasma and it doesn't seem to matter all that much what instrument you use. You're still stuck at the top few hundred.

But we've been seeing people getting 4,000 proteins from plasma recently. Not the completely unproven to have any quantitative accuracy whatsoever aptamer based stuff, but with super smart sample prep and runing on the newest and best instruments in the world. 

The magic for LCMS based proteomics of plasma appears to have been solved. You just need ...$1,000 per sample and a TIMSTOF HT or an Orbitrap Astral...... and most of us have none of those things. 

What if you don't need them? How would that change EVERYTHING? 

You'll note that this isn't brand new. I wanted to buy a lot of ....some....reagents....before I shared this. I made the mistake with posting some labeled DIA reagents a while back and then had to wait 6 months to get the reagents myself. 

You might recognize some of these names as being the most annoying nerds about "quantitative accuracy" and "matrix effects" and a bunch of other annoying things in proteomics. You'll find their most annoying traits in full force in this manuscript. Translation - holy fuck this looks like the real fucking deal. 

The prep looks a little annoying, but it sure beats most of the ways we need to get past those 700 plasma proteins (deplete, run 74 offline fractions) and they conveniently automate this magnetic prep with a generally affordable KingFisher robot. All the scripts to automate it are already available and this Github is the fastest way to get you to the actual RAW data.

However, since this is a MAG-NET based method I don't see why you couldn't use other convenient things 

I don't have a KingFisher but who hasn't experimented with automation on an OpenTrons and has one in use or being used for pipette tip storage somewhere. Drop in one of these and I don't see why you couldn't do this whole prep. 

If I have an issue with this method/preprint at all, it is not from me. It is from criticisms from a super Promising method from a while back that I loved and the criticism that it got from others. It was called Promis-Quan and while it had a couple of assumptions that annoyed people, one was that it was mostly analyzing extracellular vescicles (EVs) and some people thought that was a different thing than plasma proteins. Just relaying what I remember. I guess you could worry that there is criticism that this is the same thing? Don't ask me, I think if it's being circulated in blood we probably want to know about it.

Anyway, that's enough on this. The 4 groups doing single cell proteomics that I'm not actively collaborating with are all waiting on reviewer 3's comments on their manuscripts and I should be tearing these things apart 😇 instead of writing more on this amazing study. Also, they did use nice mass spectrometers, just not the ones you'd expect to see for numbers this high! 

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