Friday, February 8, 2019

Doing MHC/HLA peptidomics? Please stop if you aren't using charge based mass filters!!!

Peptidomics or MHC or HLA profiling is all the rage right now. And for good reason. By figuring these things out there are all sorts of potential drug targets, therapies, etc.,

I'm going to screw up the nomenclature here. When I say "HLA peptide" I mean the endogenous peptide that is held by the HLA protein on the cell surface as shown in the HLA picture near the bottom.

I've rambled about these things on this blog almost since the beginning, but I'd always looked at it from the data processing side until recently. Our group got a cool grant this year on it and we've looked at it in detail and even hired a great young scientist to specifically work on just this.

We've downloaded just about every HLA study from the literature we can --- and -- wow.... there is loads and loads of wasted cycle time. In fact, in a lot of the studies we have in hand, the majority of the ions selected for fragmentation can not be HLA peptides. Like the picture above (files were from a paper in a journal called Cell? or something). We're writing this up and have proof, but one of the other Senior Scientists and I talked about this and decided we needed to get these ideas out to people ASAP. This is too important to wait for months of peer review. Here is the summary :  If you run HLA peptides the same way as tryptic digests (and, especially, just adding +1 peptides) you are doing you and your samples a disservice.

There are really easy ways to improve this, btw. If you've got a Fusion system, feel free to stop reading this and go to this paper.

After processing like 12 data sets and running around screaming -- I was thrilled to find out that there was a recent HLA study where someone optimized the mass spec for the targets they were trying to identify. It's that one from the Elias lab, and I'm thrilled that we're all working on the same project. This is the highest number of HLA peptides ID'ed/unit time of any study we've analyzed so far. (Having a Lumos doesn't hurt, but the advanced instrument logic is the star).

Let's think about these peptides for a second. This image was put into the public domain by Pdeitiker and is on the wikipedia page.

These peptides are loosely(?) packed into a pocket on the surface of the cell. As far as anyone can tell they're held in this 3 dimensional thing and they have extremely tight size restraints.

If you look online for information about these things you'll get to visit some amazing websites. Bioinformaticians of various level of skill have been working really hard with 3D modeling, fancy sounding statistics things, and what appears to be the AOL webpage editor to make the amazingly inaccurate predictions of what peptides will be "presented" by the HLA proteins. However, if you look hard enough you can find huge spreadsheets of all the actually identified HLA peptides. (Ben: Add link when you remember)

I found this one spreadsheet that has 156,000 identified endogenous HLA class 1 peptides (again, nomenclature) in it -- and this is the distribution pie chart from it:

Over 122,000 of them are 9-mers. and the next 18% are 10-mers.

Yo. Don't fragment something that has an uncharged mass of 4,000 Da!!!! It ain't what you're looking for.

So -- how do you set this up, fancy pants?

With size and charge based filters.

This is how Elias lab set theirs up. If it's a +1 and it's 300 m/z. It isn't an HLA peptide. Don't fragment it!! If it's a +3 and 300 m/z, SURE!

If you're asking -- what about me and my blue collar Q Exactive.

You need 2 scan events. I swear, it totally pays off. Check out the figure at the top where a QE spent >60% of it's time fragmenting things that are smaller than 8 amino acids and bigger than 14.

This is what you can get with 2 scan events in a QE

You set one dd-MS2 event that is only allowed to fragment +1 peptides. That needs to be a high mass range. For example only scan from 700-1600 or something. Trigger only on the ions that are +1.

Then put in a second dd-MS2 can event that runs from 300 up to 800 or so. Then only allow +2 and +3 peptides to fragment.

I have pictures. I can't seem to find them.

I promise, though, that the time you lose in adding an extra MS1 scan isn't anywhere close to a loss of 60% of your cycle time because you took your normal tryptic peptide method and said to the QE "go ahead and fragment +1 peptides as well."

I'll also post the methods to   Yes, I hope that is still a real thing. I haven't responded to an email from anyone who has volunteeered to help the project in 4 months because I'm a terrible person and very very sleepy, but it's about to become my #1 priority again. Hopefully the volunteers are still onboard (I have the strangest suspicion that I'm tough to work with).

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