Sunday, October 9, 2016

Time to shake up the clinical proteomics paradigm?!?

This paper is from March. Not sure how I missed it, but I absolutely love it!  Its from Philipp E. Geyer et al., and comes out of some lab at Max Planck.

The gist here is this -- maybe we've had this whole clinical plasma proteomics thing a little backward. The way we do it:
1) Get the deepest possible plasma proteomics profile you possibly can on a few people with your disease.
2) Repeat on people without the disease. Heck, maybe add them together and do Super-SILAC or TMT or something. Either way -- spend a TON of time on just a few samples
3) Find something cool -- probably with bad statistics, cause you had an N = 2 or 10
4) Do a targeted analysis that is much more rapid on a bunch of people.

This paper isn't really revolutionary or anything, but it does shuffle things up a little, and I like the logic here. They work out an extremely rapid method starting with a pinprick of blood. Okay, actually, I am going to change the picture above and put in the summary picture cause its better at explaining it -- TADAA!  What they did is in the picture above!

The highlights here -- rapid, reproducible sample prep followed by EXTREMELY rapid nanoLC separation and analysis on a QE HF and then label free quan (with cumulative analysis at the MS1 level -- i.e., if a peptide retention time and isotopic profile matches within a tight parameter, the peptide doesn't necessarily need to be fragmented for MS/MS in every single clinical sample -- this is going to be a repeating theme coming up!)

Short gradient? Yeah! 15 minutes for the main elution stage!  Out of their cohort they identify and quantify 345 proteins.

345 proteins? That's terrible, right?!?  But this isn't the focus. The focus here is getting rapid reproducible reliable realistic confident(!!) proteomics on as many clinical samples as possible. Out of these they come up with 50 or so of the approved FDA clinical biomarkers. 50!! (or 49!)  For comparison sake, if they do some fractionation and run more traditional 100 minute gradients they only come up with about 15 more. Waaaaay more time and not that much yield.  What do you want out of those patient samples? Here they're saying -- 350 protein quantified in the time it would take you do ELISA for a couple. Rapid diagnostics and huge cohorts real fast!

This paper is really written with clinicians in mind. It talks about blood draw techniques and how they can determine (from the proteomics) whether it was not done correctly (excessive lysis)

I do have one criticism. And I hate to say it after such a nice study, but.....these authors go to an awful lot of work to build a super awesome canned method. If I was tasked with setting up a clinical proteomics diagnostics lab somewhere -- here is the complete template -- I'd put copies of this paper up on the wall everywhere and follow it to the letter. With the exception of the chromatography.... The authors use a custom prepared in house 40cm column. And from the CVs they get from the study and everything it is obvious they are really good at it. But I'm not. Want a really crappy nanoLC column prepared? Give me 3 hours and access to your bomb thing. I'll screw up a 10cm 15 out of 16 times. 40cm? Not a chance. I don't think I'm the only one that would have trouble making a good reproducible 40cm 75um column.  If this paper had used a commercially available QC'ed column from any manufacturer I think that it would be just a little bit better for being a canned template -- and I wouldn't even look -- I'd just order 100 of whatever they said from whoever they said to get it from and be running samples a week later.

Outside of that? Seriously, just a killer paper. Maybe the paradigm is wrong -- or it isn't the best solution for every study, whatever -- but this is the best study I've seen with a solid alternative.

Side note: No plasma protein depletions!! WoooHoooo!!!

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