Proteomics Show 102 - Dr. Jan Mulder and BRAINS

 

Lazy post day! Reviewer comments (....some a little past the due date...sorry....) on ....I shouldn't type how many papers....it'll make me a little stressed out...... on my desktop.....

I am legitimately loving recording this new season. Thank you US HUPO and these amazing guests we have lined up. Did you know a brain...just....unfolds....? I did not know this. With the podcast actually now racking up more listens than this blog, I should probably advertise this on the other thing.... But this took 6 minutes and most of that was trying to decide on a gif. 

Why do immunopeptidomics anyway? And what comes after?

 

Do you wonder why we don't just do immunopeptidomics by genomics technologies? Besides the obvious fact that it's impossible? Or just wonder what happens after you've spent a really long time working on the crappiest peptides you've ever tried to fragment?  

Then this is the review for you (and you)! 

I'm convinced! Illumina Protein Prep might be a game changer!

Brazenly borrowed from this whitepaper. 

If I have a super power as a person or a scientist, it is that I'm very okay with being wrong. It helps that it happens all the time and the fact that I have friends and a domestic partner who are way way way smarter than me. I'm used to be the dumbest person in the room and I can just discover that I'm wrong.

And boy - was I wrong about this new Illumina Protein Prep thing. 

I thought it was just a repackaging of SomaScan, a product that has had the strangest propensity for avoiding the very simple experiment that would make me stop making fun of it. After a decade I was starting to think that 1) They were doing it just to get on my nerves or 2) They had done it - and aptamer off binding could not be used to estimate a protein concentration in a complex mixture in any meaningful way (translation - it doesn't work). 

But Illumina has been killing it for years and years! We have petabytes full of Illumina short read sequencing data all over the world. Sure, you could argue they missed the long read sequencing bandwagon and that is a little weird. But a behemoth of an organization like that has the money and the people to avoid becoming complacent.

So when Illumina acquired whatever SomaScan had changed their name to that month, you had to think "wait. maybe there IS something to it!" 

And here I sit while turning a TOF after a power outage that caused me to miss the last day of a conference. Embarrassed and corrected.

The A problem with aptamers is that they are only linear within an EXTREMELY narrow linear dynamic range. If sample A has x target and sample B has 2x target, you can basically see that difference. If sample C has 10x target, you're probably okay, but you're at the end of the dynamic range. If sample D has 1,000,000,0000x more protein, you get about the same value as sample C. More on that and other problems with aptamers here. 

This new product is so much more than the original product it was based on - because after you have your aptamer readout you NOW do NGS sequencing on tags on those aptamers. And then you do the quantification off of the NGS readout! By counting the reads! And we all know that there is no better way of doing quantification than counting things. And if there is, it's probably counting an indirect measurement of an indirect measurement. Wait. Didn't we do something like that before? 

Okay, but that doesn't fix the linear dynamic range issue of the original measurement. But now you've got rock solid absolutely amazing quan on those narrow measurements, right? 

And this is where I change my mind about this whole thing! 

This group took a good hard look at precision and accuracy in a pile of different ways to do RNASeq, with a special emphasis on low input techniques like scSeq and scNSeq, but lots of work on the bulk as well.

The CVs ARE AMAZING.

Less than 1! Across the board! Okay, fancy mass spec people, tell me how many times that you've reported a CV <1 across an entire dataset. I'd love to say that I only report out proteins with less than 10%, but we use a 20% CV cutoff.

Oh...fuuuuuuuuuuuuuuuck..... they mean CV%, right? Not CV 1 = 100%??

Oh. So...a CV of 1 is a CV% of 100%. Right. So I'm going to puke. Hey! And the new TIMSTOF water pumps reset their temperature after a power outage. That's cool. So..I have more time since I have to set my water cooler temperature to the temp written on it in sharpie (25C) and I assume wait for this thing to re-equilibrate...

Okay, so maybe we need to look at these numbers a little more. 

It's hard to see but there is a red line which is a CV of 0.1 or  CV% of 10. As you might notice. They don't often get very close to those numbers. Now, we could argue this is cheating. The maximum number of cells analyzed in each study was used to generate a pseudobulk metric. So this is averaging thousands or tens of thousands of cells. What we need is - yeah! 

This paper - 

Which features a super duper method for improving RNASeq reproducibility in measurements! 

And - ACROSS A GENE they get to 

Around 22 to 24 CV%. Ouch. 

This is where it gets way weirder. My TOF is finally back so I need to go do work, but do you think they're attaching a huge gene to each aptamer? Or do you think they're attaching a single short oligo? I'm no expert, but I suspect it's the former and this is like global proteomics CV% on a single peptide compared to across a protein. The numbers get better when you've got a higher sequence coverage.

I'll be honest, I started out this post as an April Fool's joke, but it turned out that I learned a lot. 

I'm not going to change the title, though. I think that this product will change the game and I don't think it's going to be in a great way. On paper this product looks like it will still not be able return quantitative protein values, and it looks like when it does, the variability in metrics will be worse than the product it is based on due to the difficulty in reproducing the output data consistently. 

We'll see, though. If you are using this product, or have access to it and you want to do the easy and obvious experiment to show me I'm wrong and this works, please reach out. In the meantime I'll still tell every conference audience and every classroom I'm in front of that there is zero evidence that this stuff can quantify a protein. 

On site at ABRF 2026!

 

This is the first time ABRF has happened in a city that I live in! Man, once upon a time there were so many proteomics people that we had competing initiatives. We were standardizing methods and standardizing standards and comparing software and it was all a lot of fun and it all kind of stopped. But you know what you can talk about anywhere you want to? 

Single Cell Proteomics! Dr. Kyle Swovick, photo taken from the front row, organized a session and, for a small conference with 3 competing sessions at a meeting with sessions with riveting titles like "asset management" I'd call it a hit. Definitely better attendance than last year's session I organized that was called "Yo, Justin Walley and I are totally going to do SCP in core facilities, hold our beers". This was sort of a victory lap for me because we now have paying customers in our core at Pitt for SCP and the first data delivered received this response, that I don't mind at all.

Kyle's core at Rochester also does it! It's a real thing! Kyle talked about what a pain in the butt it was to reproduce methods from the literature that largely forgot important details and I called the authors of a preprint that said "5,000 proteins per cell" in the title "a bunch of assholes" on video for a second year in a row! 

And we had great questions and interactions.

Was that all the proteomics? Nope! It sure wasn't! 

Someone who isn't an ABRF member nominated John Yates for the lifetime achievement in BioAnalysis award, and they were like "wait. didn't we give that to him 15 years ago? Holy shit. Why haven't we given him this award already??" So they did.

And I got to be like 4 rows back for John's history of protein mass spectrometry. Super funny with xeroxes of papers (younger scientists, we used to go to the library and find papers in actual journals and photocopy them) and a lot of self-effacing humor and reflection. And cuttings from papers of the past that seem either predictive or ridiculous in the context of today. Or both. 

Super fun thing I was very glad to catch.

The rest of ABRF? Hmmm....there are some cool new LCM solutions I'm investigating, and some cell sorters. And a great convention center! 

Maybe I'll write more about it when it's over. 

As an aside - 

Conference travel in the US is really really hard right now. Last weekend at BWI TSA screening was taking over 5 (FIVE) hours. I've flown out of BWI easily 300 times in my life. It's a small airport. 30 minutes is a big deal for TSA there. I agreed to speak at an awesome university a 7.5 hour drive from my house. And I don't think I can get there faster in a plane right now, but it'll cost me about the same in gasoline. ...yaaay...

Transfer single cell peptides all over the place with NO peptide loss at all!

 

4 people have sent me this new preprint and I've had to go "...oh...it's in my draft's folder..." but the power went out in my building while I was at a conference and it's sort of a mess and I'm grumpy, so Imma post this. 

So...sometimes I see these papers where someone does something like - 

1) Get better results than anyone in the world has ever gotten with that instrument (or at all) 

2) And when you do that...if you don't make the data publicly available, I have to think....

Let's do background first! 

THE number one challenge in single cell proteomics (or any low input stuff) is informatics, probably, but right there with it is just protein and peptide loss through plain old surface adhesion stuff.

If you put a 200 picograms of peptide diluted in a 4 microliter droplet in a 384 well plate you'll lose about 30 picograms of that material to that plate surface (say..125uL well volume). It's just gone and DDM will help, you you aren't getting that back. Pick that up in a DDM coated pipette tip and you'll lose another 20 picograms. If you take that droplet and you add enough liquid to it to allow it to effectively vortex it and then thoroughly vortex it  - you'll lose a whole lot more! Peptides stick to plastics.

Even when we're dealing with hepatocytes at 400 picogram of protein (single nuclei) we're probably not getting 200 picogram to the instrument because with our workflow. Loss loss loss. And you can buy a more sensitive instrument for $1M or you can cut your losses by 30% and get the same thing.

In this study - this group transferred a single cell peptide load over and over again! Like 4 times! (four, 四, cuatro) and then - got more proteins per cell than anyone has ever reported on that same instrument with nanopots (single transfer, but exceptionally low loss during prep) or with single well digest, pickup, load, on that same instrument. They just pipetted the cells from here to there and used magnetic nanoparticles and then desalted and then transferred it again and then still got huge numbers. That's a big big claim and you generally expect big evidence to support something like that. But...when there isn't any....it's hard to say or think nice things. 

The Proteomics Show is back! Check out chapter one of "All the Parts!"

 

In this US HUPO sponsored season (thank you!) we're being serious scientists who are asking the big questions of bigtime experts of specific tissues and organs. Neely kicks it off in this week's episode with "...What...IS...hair...?" And one we just recorded features a guest who speaks to our level when referencing the activity of adrenaline as - and I quote - "Yo, get down there and dilate, we gotta go!" 

US HUPO is letting us do a big unsupervised season and so far, I'm happy to say I'm enjoying it. Listen to Episode 101 with Glendon Parker, wherever you get podcast things.

How do commercial procedures impact the blood plasma proteome!

Oh. This. Is. Sick.

Matt Foster and I were JUST talking about this at US HUPO. Sometimes blood gets frozen in transit, y'all. And sometimes it probably sits around on some phlebotomist's cart for a whole day and then gets frozen. 

The last paper I could find on this weird stream of consciousness website was almost 10 years old. Wait. How long have I been doing this? That seemed like a recent paper... Whatever. It was before all these fancy nanoparticle thingamabobs showed up.

Actually, I'm procrastinating - here is a rant. I'm convinced that absolutely no one understands how these nanoparticles work. For real. Y'all will give me the "protein corona" like that explains anything at all. 

That's what it said in the first Steve Carr paper and I swear they just made that term up in Boston and everyone just repeats it. Also - how do you copy a GIF on a MacBook? Why is this thing so fast and why are so many things impossible to do on it? 

Okay - but that paper! The paper takes a look at the practical real life concerns in blood plasma proteomics! Like travel time and storage time and freeze thaws! And - obviously old fashioned and new fashioned blood plasma proteomics methods. But everyone has done the latter, even if they can't agree. This one looks at the interesting stuff too! 

Non-reducing proteomics opens up a whole new pile of PTMs under stress!

I don't have my notebook on me but someone who was on The Proteomics Show dropped a knowledge bomb on us about cysteines a couple of years ago. She said something like only 12% of cysteines are involved in those disulfide bridges we're all so worried about. Might have been a he and might have been 2 or 40%. 

So...when this PI is in the lab, he commonly skips the alkylation and reduction steps. Not just because he doesn't know where the reagents are, but also because I spent a couple of years studying drugs that bind cysteines. True story, about 1/4 of the pharmacology faculty at Pitt seem to study similar drugs. 

This group shows that it might be a good idea even beyond cysteine alkylating compounds! 

For real, what are all those other cysteines doing? Just being sulfury? Sulfur isn't the most abundant thing and it only gets incorporated as a nutrient by plants through a painstaking process that starts with a slow oxidation by bacteria. Seems like a lot of work for something that just does a couple of things, right? Evolution is too cheap for many single use materials. 

Super cool paper and definitely worth thinking about when you can't find that DTT thing on your first pass through the -20C? Chemical cabinet? I honestly don't know. 

Trap column optimization for single cell or sub-nanogram proteomics!

 

Okay, so this might be more interesting to me right this second than it normally would be, but I'm very glad to have this group's notes and findings! 

There is a paywall on this, but if you were a little sleuthy you might find an earlier version of it that isn't quite as polished that is not. 

Proteomics of organoids IN OUTER SPACE!!

Y'all know what organoids are, right? 

Some people got together like 20-ish years ago and were like "yo, I wonder if it actually makes sense that all these human cells are growing in 2 dimensions...?" For real, like, they don't grow like that inside a human being, outside of perhaps Lady Cassandra....

So they make cells grow in little balls instead. Still...not...like normal...but if you give people a dose of a drug and measure a response and you work out that same dose for cells growing in 2 dimensions vs growing in the little balls of cells (organoids) the latter is closer to the human.

SO.

What if. 

You (not you, but some NASA affiliated people, unless that IS you. Then you, obviously!)

PUT ORGANOID IN OUTER SPACE???

 (They did single organoid, so I assume it's singular when referencing them?) Sounds funnier, at the very least. 

We send way dumber shit than that to outer space all the time. A ketamine addict who runs a racist social media platform keeps failing to get rockets into space that probably just have pictures he colored of himself inside them. Who knows? Certainly not ol' Space Ketamine Karen.

What was I typing abou...THIS STUDY THAT I SAW PREVIEWED BY ALINE AT US HUPO! 

To make this even more spacy! They ran the organoids on - you guessed it - an Orbitral Trap tandem Time of Flight Mass Spectrometer that we know as the Asstral!