THUNDER-PASEF - Get those real immunopeptides!

 

I need more time to spend on this, but I'm going to put it here anyway. 

What's this about? 

Based on comments I've received about what I write recently I'm going to be trying to provide what I think is important context in anything I review. Obviously, context is from my perspective and background

Okay, so HLA peptides are super annoying to analyze but they are very very important. These little peptides are on the surface of our cells and they are part of the system that tells our immune system DON'T EAT ME. It's all self-self recognition stuff, sort of. When that system isn't working perfectly it's bad. Cells that are diseased (like cancer cells) aren't being targeted and destroyed. When it is working too well, it is also bad (auto-immune problems). So it's important that we can categorize those cells on the surface. HLA Class II are easier. The peptides are bigger and easier to sequence. Not easy, per se, but easier than Class I.

Class I peptides are much smaller than the ideal targets for mass spectrometry. Also, as they are produced after proteosomal degradation, it is very unlikely that the amino acid on the end is a lysine or arginine. Which is the opposite of useful for mass spec based sequencing.

And - we've known for a long time that just running a standard proteomics method on HLA peptides is suboptimal.  Now that I'm an academic, I am wondering why I didn't publish that..... I probably should....

So...how would you go about doing this if you had a really nice 200 resolution ion mobility instrument on the front of a mediocre mass spectrometer? 

THUNDER! Na na na na na-na-na-na (caution: actually the video link. might be loud)

Okay, so from Figure 2 it doesn't look like Thunder makes a lot of sense, it looks more like you should just not use a TIMS octagon. Which.....in my experience has never ever been a good thing...

Get further into the paper when they model the octagons. It definitely improves things. 

Big takeaway here could be - YOU CAN USE MORE THAN 1 TIMS OCTAGON!  I did know this, but I didn't learn it until a few months ago. The company that had a 2023 revenue of almost $3 BILLION still hasn't found the money to make any instruction manuals for said instruments, but that's understandable, of course.

But the numbers here are solid. The gold standard for immunopeptidomics is around 50 milligrams because that's about the size of a normal biopsy punch sample. If you assume 30% of that is protein you're at 16mg or something. If you start with 100 million human cells, assuming a protein content of around 200 picograms/cell you're at 20-ish mg of material! 

If you work out HLA peptides from 100 million cells, you're at a level where you might actually help find a cell surface marker that could be used to inform an immunotherapeutic therapy - or - design a new personalized therapeutic to target and destroy that tumor. And this is where we've been trying to get to from a sensitivity perspective for a long time.