Friday, March 15, 2024

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. 

Thursday, March 14, 2024

I missed the 20th US Human Proteomics Organization meeting!

 


I missed the 20TH MEETING OF THE US HUMAN PROTEOMICS ORGANIZATION thanks to my immune system being a slacker. The committee I chair, the US HUPO Virtual Media Outreach Committee (most of the group is above) sent me pictures like this. 

I just found this and I'm brazenly stealing it -- 


Is this the same meeting that we'd been trying to interview all the invited speakers for? Yes. Well...not  all of them, THE Proteomics Show has to fit into space around our jobs and families and things, but we got a bunch of them. 

The same meeting that the spent months working on so we could have a cool proteomics video competition? Yes. Same one. Super bummed to miss it. 

The winning video was a parody video called "The Real Scientists of Beverly Hills" and came from Cedar Sinai. The concept makes me laugh every time I think about it, even though I didn't see it. 

Because proteomics is now (FINALLY) a thing the outside world thinks about once in a while - legit news outlets sent legit reporters. Not joking. Here is a link! 


The 60 second lightening talks at US HUPO are, of course, legendary. Apparently there was some beatboxing. If you've never seen these rapid fire talks, you're missing out. Again - sooooo bummed I wasn't there. 

THE Proteomics Show still happened, just with a legitimate upgrade of one of the hosts! (Whoa. RASR lab website is seriously cool....) 


I have every intention of listening to this one (it will be the first one) when it goes live. Maybe today? 

Big shoutout to the award winners this year. I am ultra bummed to have missed 


Kelleher's Don Hunt award! 


Parag's Computational Award winning talk! 


Jenny van Eyk's always inspirational talks on why we NEED PROTEOMICS IN THE CLINIC. YESTERDAY. 


And the Cotter Early Investigator Award winning talk by Ying Zhu

I like good award winning talks as much as anyone, but posters are my favorite part of any conference. That's where you get to meet the smart young people who are actually in the trenches running the instruments and pushing the limits of what we can do with any proteomics technology. CRITICALLY ULTRA BUMMED. Next time.

If you haven't heard - US HUPO 2025 is PHILADELPHIA!!

Also - have you ever been to a conference that was really disorganized? Like you can't figure out where things are going on at each point in time or stuff starts too early or too late? You know why this doesn't happen at this one? US HUPO has 


From left to right (ignore Pratik, Lydia, Jennifer and Brianna, ignore Ben) from Conference Solutions. And they make all the things work! 

Wednesday, March 13, 2024

Separate single cells, extract metabolites and ionize them in one go!

 

I'm going to bore (boor? bour?) anyone unlucky enough to visit this awful blog because this study is on:

Single cells

AND 

Metabolomics

This is a 3D printed ionization source that allows you to put in a population of cells. Then the cells have to go in single file because they are being pushed down something too small for them to bunch up in.

While they are being pushed through the metabolites are extracted out of them!

THEN the metabolites are directly ionized into an Orbitrap (looks like a Classic!)

And...no joke, it actually looks like it works.

If you're following along in the statistics world, someone recently realized that there are problems with some of the advanced statistics used in single cells. We'll ignore it right now 'cause the dust may settle one day, but according to these plots, they can tell different cancer cells apart using this crazy source!


Tuesday, March 12, 2024

FUNCTIONAL spatial proteomics is live!



In a very early entry for paper of the year....


Quick overview - one of the goals of any sort of bioenergy production has been to figure out how the actual fuck microorganisms break down cellulose into usable energy sources without literally catching it on fire. If you can release the base sugar molecules trapped in WOOD you could do easy things like convert that to ethanol or butanol or other handy things. 

The problem is that these things are not happening in a homogeneous way. There are typically diverse populations of organisms widely dispersed throughout the material that are doing these things. 

How do you decide where to actually focus your efforts? 

How do you know that where you are analyzing is actually where the cool stuff is happening? 

Do what this groundbreaking work at PNNL is doing! You first slice your material and you use mass spectrometry imaging (they're using MALDI-FITCR I think) to locate the breakdown molecules that you are interested in.

THEN you take that same material off and you cut tiny tiny tiny tiny sections out of the area where those molecules came from. They're using NanoPots / MicroPots and then doing proteomics / metaproteomics. 

So now they know what enzymes are directly linked to the small spatial area where the lignocellulose breakdown products are coming from! Killer, right? Okay, but get this, why can't you just use this exact same system and apply it to other biological materials? We got a glimpse of what is coming down the pipeline from PNNL when we interviewed Dr. Burnum-Johnson on THE Proteomics Show earlier this year. There is something coming that is definitely worth setting a google scholar alert on this PI's name. 

Monday, March 11, 2024

Single particle analysis enabled by 25 second transient times!



Okay....y'all. Are you ready for ABSOLUTE SINGLE MOLECULE SENSITIVITY?

Are you willing to accept 2 SCANS/MINUTE to get it?

No?!?!? 

Why? Oh. You have multiple studies you'd like to complete before you retire? 

Well.....crap....here it is anyway! 


Look, there isn't enough helium left on earth for unimportant things like cooling large magnetic drivn FTMS systems which...could...well.... do this...ummm....same thing....?...at higher resolution and 50x faster....? We need the remaining helium on earth for MRI scanners, which are actually important. Now....how the "Everything costs $1" store my kid loves can seemingly fill seven thousand Helium balloons a day...for $1....is still a complete and total mystery to me. 

Obviously, measuring a "single-ion" of anything is really really cool and not everyone's first motivation is how many chromatographic peaks across a scan they can get. Honestly, the most impressive part is probably the fact that "singe ion" didn't degrade in 25 seconds while making 400 zillion rotations around the inside of that little tiny vacuum chamber! 

Saturday, March 9, 2024

Need more surfactants for proteomics? Here is a big table!

 


Okay, well, that looks grainy and useless, but if you are one of those "low sample input" people, you might also be looking for surfactant ideas! 

We've all clustered around DDM because (so far, knock on wood) it doesn't seem to destroy everything. Longitudinal data would be nice and those of us using it to coat a lot of things will probably have some of that soon.

BUT THERE ARE MORE! And you can read about them in MCP here!


They seem to have one they like better than DDM, but they have to remove it from the peptides afterward, which....is less immediately interesting to me personally....

but I sure am glad to have a whole table of things to try! 

Monday, March 4, 2024

My alma mater ended a deal with Google - blog functionality impacted!

 

So.... short story is that Google and Virginia Tech had a long standing deal to work together. However, Google wants money for things these days and my alma mater was like - ummm....we'll go to the amazing people at Microsoft if we have to pay money.... cause.... outlook is clearly almost 1% as good as Gmail....

So....I need to migrate 140GB of data by tomorrow night to somewhere.....

Many blog links leading to my Google Drive are already down (ugh). This includes any historic methods on www.LCMSmethods.org that haven't been migrated successfully to protocols.io

I'm unclear what happens to the other 138GB of pictures and .gifs that are linked to this site. LMAO. DO YOU KNOW HOW LONG IT TOOK ME TO DOWNLOAD 138 GB OF GIFS?

Sunday, March 3, 2024

Veneer -- identify the real cell surface proteins!

 


Is that a cell surface protein? Oh yeah, sure it is, someone in 2003 BLAST'ed the genome sequence for it and found a yeast cell surface protein with 12% sequence homology....

And....that may honestly summarize some percentage of "cell surface proteins" we have on our lists. Legitimately somewhat informed guesswork based on organisms separated in evolution over 1.5 billion years. Or something.

Of course people have built off of these guesses by raising antibodies and verifying by microscopy, etc., but we have never had an easy way to rapidly annotate mammalian proteins as truly being on the cell surface.

UNTIL NOW.  Introducing Veneer!


There is a lot to unpack in this great new resource. The background of the resource was constructred from data from over 4,000 separate publications! 

"There are many different ways to enrich cell surface proteomes, I bet this only thinks about the one this group uses"



Shown here are the Veneer processed results from 4 separate enrichment methods! 

It doesn't take much imagination to think what Veneer could do for the biology community, right? I've never made an antibody before but my impression of it is that it is 1) expensive 2) takes forever and 3) requires you to go all vampire on 700 bunny rabbits, 11 camels and possibly 1.5 cheetahs. If you need to make a cell surface antibody and that protein really isn't a cell surface protein OR you are raising the antibody to a a section of that protein that is not actually the part ON THE CELL SURFACE. That's a lot of bunny / camel /cheetah blood for ....absolutely.....nothing......  

Vaneer doesn't have to be perfect to have a major effect on efficiency. Anything would help drastically but they obviously put loads of thought into this amazing new resource. If you're swamped for time, you don't even have to read this paper. Check out the logic summarized in Figures 2 and 4. 

Or just download Veneer from this Github and run it (might be a minor UTF-8 issue for Macs) now!

Saturday, March 2, 2024

The long overdue midiaPASEF post!


Almost a year ago I tried to figure out "all of these diaPASEF methods and what they do" and one of the most innovative ones didn't make the list. Maybe I posted the preprint link, but I honestly couldn't figure out what the Albert Heck they were even typing about. 

Today, thanks to the fact everyone in my house has been sick for an entire month I got to sit through a talk by Stefan Tenzer that is posted on YouTube, but you can only get the link for it by registering here. 

My motivation was something called VistaScan that is now available on some/most(?) TIMSTOFs if you have TIMSControl 5 or higher. VistaScan is related to midiaPASEF, but on a limited scale. 

My real motivation was trying to make VistaScan do something I want it to do that I can now say with absolute certainty that midiaPASEF can not do, nor will it ever be able to do it. Instead of being bitter about an hour of my life I will never ever get back (the video is shorter than that I had to stop and think about things and back up several times), I'm legitimately really happy to understand the concept. 

Like the other diaPASEF methods, this one leverages the very handy properties of being able to discriminate peptides by retention time, ion mobility and accurate mass. What sets this one apart is that the quadrupole (the fastest part) is moving in overlapping windows. Obviously, not new stuff, and this method was inspired by MacCoss lab's multiplexed PRM stuff that is so smart that no one aside from the authors have ever been smart enough to use it themselves. 

While on the surface you can look at the midiaPASEF posters and technical notes from the vendor and think - "okay, they're doing tiny window DIA while leveraging ion mobility" (okay, that's what I thought). It's not quite that. It is still using relatively large mass isolation windows, but because they're overlapping and the data acquisition is very very fast, they can deconvolute the data (again, similar to multiplexing PRMs). 

The trick, then, is on the software side, where the data needs to be modeled over the sliding windows. If, for example, you see your precursor fall off as the quad slides past a certain point, then you know the upper mass range of that precursor. Where this gets really smart is that they also have to track the fragment ions the same way. 

Your diaPASEF window is still loaded with fragments from everything within the 12 Th (or 16 Th, I forget) window, but by modeling the fragments that disappear as you tick the isolation window up or down, you can start to extract the fragments that correspond with that exact precursor.

You get the added benefit on top that you are altering the TIMS isolation as well. Where this becomes contrary to my motivations was that I was simply hoping to do the 2Da window overlapping DIA stuff that I was doing for SCoPE-MS on the ZenoTOF with an extra dimension - ion mobility.

But if I'm tracking reporter ions which are present in every MS2 scan, then these can't be modeled. It is definitely possible, however, to use the complementary ion pairs, but to process those data you've either got to have Thermo .RAW or have access to that super mysterious data processing server that has those capabilities. Neither of which match here. 

Doesn't work for my application, but it's still super cool. Where I have a disconnect is the VistaScan thing which appears to be a limited version of midiaPASEF that only works for some applications. I'm hoping to test some things on it (just got a 5.0 upgrade) and I suspect that we can generate the data but not process much of it. We'll see though!