Immunopeptidomics on low cell numbers without crappy antibodies!

 

Big shoutout to Anthony for sending this preprint over! 

We spend a lot of time talking about how much specific antibodies can hurt science. We've been talking about it for as long as I've been able to have somewhat intelligent conversations about science. 

What hasn't gotten as much attention is how terrible "pan" antibodies are. I see papers all the time where they still do phospho-tyrosine enrichment with the 4G10 antibody- and I first used that thing 15+ years ago it wasn't very good then, and I ask at conferences all the time if it's improved and I don't think it has. 

You know what is way way worse than 4G10? The absolute pile of poo that is used to pull down the conserved regions of HLA (MHC) proteins. They are clearly biased toward specific epitopes, if you want them to work well you have to slowly rock the antibodies with 4 kilograms of cell lysate, and it helps if you do it a few times and the same procedure carried out by 2 people following identical protocols get very different results. (This last part I saw at a meeting in 2023 but if the presenting author has published those results -she was in pharma R&D - I don't see it on a quick search). 

CAN WE DO IMMUNOPEPTIDOMICS WITHOUT GARBAGE ANTIBODIES? 

No joke, this is worth taking a look at. 

Mild acid elution has been around forever. Heck, the best way to get a zillion "immunopeptides" identified is to use a not-so-mild elution that will break open the cells and release the endogenous internal peptides and then to turn off your peptide FDR --> boom! a zillion peptides. To be clear, I'm only recommending this strategy because there are at least a couple "CRO"s out there that will do something similar and charge you $10k/sample for completely and totally useless garbage results that hurt our field while making people I can't tell are unscrupulous, dumb, or both a whole lot of money. 

Actual mild acid elution, though, makes a lot of sense if you can fine tune it in. Now, there is some controversy out there about whether that has a bias. It makes sense that from the shape of these little presentation "binding" pockets that 3D structural stuff would weigh heavily in  on what pH conditions would cause an effective mishaping and release of the presented antigen. (Stole this image from here) 

It's actually helpful to this rant because MHC II (the ones that are way easier to analyze by LCMS because they're bigger) is generally thought to be much more resistant to acid antigen release stuff. Although you'll find people who will contest this one as well.

What was I typing about when I should have been working on a proposal on my desk....oh yeah! This paper! 

So these authors do a very standard crappy antibody bulk pulldown from your typical 800 billion cells or whatever and they look at it on a TIMSTOF Pro. Then they do some comparisons to their acid elution thing which is helpfully contained within a simple little tip that then goes right on the EvoTip (wooo!) to get down to the 50,000 cell and below they do go to a TIMSTOF SCP and run the nanoflow (Whisper) methods on the EvoSep. They stick to DDA with fine tuned polygon IMS/m/z windows for immunopeptides (rather than just no polygon, which some people use for endogenous peptides on the TIMSTOFs). They also compare this to the super small window DIA stuff that they recently developed. That was called something confusingly similar to diaPASEF methods by other groups, but I think they decided to call it Thunder, because if there is anything the proteomics field needs more than confusing new nomenclature is is absolutely and without a doubt the same method from the same group with different names. 

Seriously cool method and I'm absolutely going to be trying it myself this spring. Probably step by step, with the exception that I've got an Ultra2 arriving in 25 days!