Friday, September 10, 2021

Carrier channel math on Orbitraps! Something proteomics people agree on!

There is so much important foundational work being done in single cell proteomics right now! I know several people who are very interested in starting to do this once "it's all done" and this is the stuff we need to get through. 

1) has some of the prettiest plots I've ever seen for proteomics data 
2) started in a completely different and really smart way to work out carrier channel ratio compression (using swapping TMT126 chanel for TMT0 to get some sense of ground truth.)

And what's really impressive is how consistent every study that has looked at using amplification via (SCoPE-MS or BOOST or whatever terminology this all lands on in the end) has been in the end. Now, this is the most conservative estimate to date, but everything has been pretty much in the same ballpark. There is a single order of magnitude between the lowest estimate and the highest, we're somewhere between 20 and 200).

Always worth considering here, and I apologize if I'm starting to sounds like a broken record (whatever that means) but every study so far has been on an Orbitrap, with only one group using the bigger D30 Orbitrap and everything else using the D20. Not to oversimplify the physics involved, but it's not just coincidental that the larger Orbitrap measurements have biased toward higher carrier. 

We've known all along that while the Orbitrap is one of  the single most awesome things that has ever been invented, the instrascan linear dynamic range is NOT why you buy one. You want big instrascan dynamic range that's linear? Get a QQQ. Everything else falls in the middle. 

Back to the paper: 

Another really good paper I'll be referencing all the time has done the carrier ratio suppression math and just to flag why I'm so impressed by them getting to the same conclusions: 

We're a 20+ year old field and not one single one of us can digest a sample the same way. Proof in point? 

Paper earlier this year? 

1:50 trypsin with overnight digestion at room temperature

10mM DTT for 30 min at 56C

55mM IAA for 45 min at RT

Some other changes: 

In Claudia's new paper we see something slightly different (surprise)

1:100 trypsin overnight at 37C 

50mM DTT for 30 min at 37C

40mM IAA at room temp for 30 min

Does this change anything? Probably not, but if 2 proteomics people from different labs land on the same conclusions about anything it's really impressive. 4 or 5 studies coming up with very similar conclusions? Maybe this current generation of proteomics trainees will fix the rest of it! (I'm super pumped they both used the same alkylating agent.) 

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