Poor RNA to protein correlations are an artifact of poor proteomics data?

 

I was making slides for a class lecture and went down a long and windy rabbit hole on what we now know about the discrepancies between RNA and protein regulation. I landed on this one from 2022, and while it may seem like I'm rage baiting....I think it should go here anyway..

Despite the title of this blog post we aren't firmly blamed for all the errors. Some error does exist in the mRNA measurements, but it's pretty clear that the disagreement in protein measurements between different studies is something that is worth thinking about. 

Acceleromater correlations to the UK Biobank proteome data!

 

Wooo! Okay, if your reading this in a first world country this won't apply all that much to you. In my country you can now spend $20k USD per year on private health insurance for yourself and if you actually need it someone will be very financially motivated to deny you coverage for pre-exisiting conditions.

What if you could wear an accelerometer (I'm sure my wristwatch has one) and it could predict you might have a pile of different diseases? 

BOOM - pre-existing condition. 100% profit for the most profitable scam in my whole country! 

Science fiction? Or science fact? 

A bunch of people in the UK Biobank agreed to wear an accelerometer for a couple weeks as part of their contribution! And this group remined those data against the O-link proteomics data and the clinical data they could access from these patients.

You accelerate poorly? Dramatic increase in a ton of different diseases! 

Moral of the story? 

Do you need DIA-NN QC? Do you also need retro visualization choices?

 

Okay, we all need more ways to look at the quality of our data, particularly before we send it out to collaborators who may do who-knows-what with it! 

Only one QC tool out there gives you retro visualization options! And it's this one!

https://dia-nn-qc.streamlit.app/

Load a DIA-NN file and choose 80s terminal or 90s webpage or just the boring regular thing. Who says you can't have a creative background while you're making sure you've got the correct number of scans/peak? Not me! 

MuPPE - Serial enrichment of the phospho- and glycoproteome!

 

What a great month for method names already! Introducing the... 

...sequential single pot digestion and then sequential enrichment of the phospho- and glyco- proteome! 

I'm not entirely sure what all the advantages are of the Muppet method. The authors make it seem very streamlined, and I'm guessing that you can get away with less sample and sample loss by keeping things in the tubes, but early in they have to spend a lot of time diluting urea down to functional levels. If you want a lot of the details on how this is performed you'll need to go to page 28 in the Supplemental Info PDF. There you will find that an Orbitrap 480 was used for all analysis with DIA for the peptides and phosphopeptides and DDA for the glycopeptides. So it is still 3 different injections per sample. I am always happy to see something like this, in any paper even if it's on Supplemental page 32. 

I also find this a little concerning

...in Jonathan Pevsner's book (which you can get on Ebay for $12 in first or second edition), he warns that smiling volcano plots can be either a lack of data points, excessive presence/absense, or over-normalization. Since I think they've got a solid pile of data here, it does make me concerned that the data has bene over-normalized. Though...they used Bionic and specify a rather small n-glycopeptide library was used, so it could be the other two. Smiling plots just make me nervous. When I have one I generally find out I did something silly upstream. 

Otherwise this seems like an interesting method, particularly if you're not always doing phosphoproteomics or glycoproteomics and you have to do them. I don't see any reason why you couldn't digest the peptides with a more traditional approach and then put those peptides into this workflow around step 2 or so. 

Target PTMs in single cells with ShtMtPro!

 

YES! Okay, so this is may finally be the smart solution to something we tried (and probably just about everyone else) with the SCoPE-MS/ScoPE2 workflows.

If you have a "carrier" "boost" "basil" or "oregonO" channel, why couldn't you load that thing with phospho-enriched samples (for example) instead of 200 cells or a a diluted perfectly digested pooled sample? The reason appears to be that your coisolated peptide (or junk) background ends up leading to a preposterous number of false discoveries. Remember that in these workflows your complete and total evidence for that peptide being there in single cells is just your single reporter ion. Since most PTM modified peptides are already in a suppressed region of signal to noise - and you only get one measurement of that phosphopeptide - you're already in trouble. (Wait. Is that too many dashes? Don't need y'all thinking some AI wrote this thing. Meh, I'll fix that in a minute). Throw in the contamination of your reporter ion signal with the isotopic impurities and now you've got tons of phosphopeptides and they may not really make sense at all. 

Ready to fix that? I sure am! Except...I don't have this hardware.... hmmm.... okay, but let's do it anyway! Introducing 2026's early entry for best method name......

ShtMtPro!

It's SureQuant with 

Super Heavy Tandem! (Sht) Mass Tags! (Mt) Professional (Pro) version! OMG. 

(Mandatory)

Okay, so the AMAZING name should not, in any way, distract you from how good these data are. Compare the number of PTMs you can pick up using this workflow vs DDA? ShtMtPro crushes it. Even vs PRM, ShtMtPro squeezes out a narrow victory! 

Intelligent - on the fly - targeting of chemically modified peptides IN SINGLE CELLS? Multiplexing so it's super fast?? Incredible idea that I bet no no one tried at all to talk another vendor into for 3 straight years. If you are thinking something dumb like "I can't do single cell proteomics, I just have this old Tribrid..." this is the second paper on this blog this week that should put you on the right track. If, however, someone offered you $75 and a pack of big red for that old Tribrid, I would happily give you twice that for it!

Edit: Okay, apparently they used an Exploris, which I was not aware could do the SureQuant thing. I thought it was a Tribrid exclusive workflow. Good news! There are a bunch of Explorises around!