Extract nuclei from fixed tissues - optimized protocols!

 

More nuclei extraction proteomics! This is for bulk analysis and this team went all out in testing different procedures for the best yield. Super cool stuff! 

"SomaSignal tests: The next step in the evolution of multiplexed proteomics..."

 

..... (come on Ben, type something nice you can do it....got one!) The illustrations in this new advertisement review for aptamer based proteomics are very nice!

...and....some peer reviewers were fine with letting this thing through, so there must be merit in it somewhere. 

Unfortunately, I was skimming it and saw section headings like this and didn't want to throw up on my keyboard, so I'll leave it here for others to find and read. 

Every author on this review, btw, appears to be an employee of SomaLogic. 

Reminder that misleading mouse studies waste medical resources

I'm great Mr. Mouse, thank you for asking. So rather than share any annoyance about what I read over my lunch, we're going to remind people about these 10 year old findings! 

Look, there are places for animal model data. There really are. Core metabolism is just like ours with few changes. However, if you're using mice as a neurological model and testing something that impacts higher level functioning in humans - using mice, which absolutely do not have the pathways in question - you're just being silly. I linked the editorial, because no one has to read much past the fact that 0 ALS drugs that worked in mice had any level of efficacy in humans.

Rapid one-pot workflow for phosphoproteomics prep (and DDM coating procedure)!

 

One issue with phosphoproteomics enrichment methods is the large required sample input. There are other issues, such as the mod is intrinsically transient by evolutionary design, but this group totally tackled the first one. 

I figured that anything sounding this complicated would require me to wait until a commercial kit was available, but the set up sounds surprisingly (shockingly?) simple. The paper is open access, so you can get that part list yourself if you are interested.

Another reason to check out this paper is that they coat their low-bind plastic in n-Dodecyl-Beta-Maltoside (DDM) and show that helps a lot. If you're interested in just reducing your surface loss binding stuff but possibly also worried about the longitudinal effects of a recently described detergent blasting into your mass spectrometer, here is a process to coat and discard the active solution! 

41,000 (forty-one THOUSAND) human plasma proteomics samples!

 

41,000 plasma proteomic samples?

Clinical data linking 218 different diseases across these people? 

Yes, this was O-link explore, so just under 3,000 proteins probed (which, reminder, does NOT mean measured, it means they were theoretically detectable because there are probes for the protein).

This is still exciting.

LESS exciting.....

Single NUCLEI proteomics (and identification of cancer mutations in single cells!)

 

I'll start with the figure above because it's early in the paper. Super exciting to me personally because of my personal research interests. These peptides are hard to identify in bulk. This group is doing them in single human cells.

How? They're building libraries for the software that we like to run library free the old fashioned way. They're capturing the specifics of their instrument as well as the unique characteristics of how peptides at super low intensity/concentration tend to behave. So this is all label free proteomics by data independent acquisition used to resolve both human mutations AND PTMs in single human cells. Wow, right? 

THEN this group broke through to the next level of what single cell proteomics (SCP) needs to do. THEY ANALYZED SINGLE NUCLEI. For context, if you look at what a large percentage of "single cell seq / scSeq" is actually analyzing, it is actually nuclei harvested from fixed tissues. The nucleus is pretty tough and it can often be separated from materials where the rest of the cell won't be recoverable.

Things like fixed tissue. And we have LOTS of that.

They only analyzed around 100 single nuclei here, but I honestly thought we'd see nuclei 5 years from now. When I first saw these results around the end of 2023, I couldn't believe it. It should be noted that we've recently saw a new preprint that did a few thousand single nuclei, so I was waaaay off in my estimates. Super super cool new paper. 

Struggling with protein acylation? You should try Acyl(S-) Trapping!

 

If you are a rational human being and you want to stay one, you should probably just forget that proteins reversibly acylate all the time. Most notably, acylations tend to occur to drive intracellular spatial stuff. Like your protein gets a terrible acylation on it that helps it migrate to the membrane where it now has activity while it's tethered there. 

Typically to measure these awful things you start with a huge input then do some enrichment and cleave off your enrichment tag or something. Other times you over-express your protein in a system where the acylation is forced to occur but it has no biological function. Force that KRAS to go to the E.coli membrane expressed at completely nonphysiologically relevant concentrations, because that will help you characterize those mods while learning absolutely nothing else about what that system does. 

OR you can and should do this? 

This clever use of both a modified suspension trap and what/when/how you add isobaric tags allowed this group to characterize protein acylation in a complex system starting from as little as 20 ug of protein. It's a super cool new approach to get at very releavant protein modifications that are very very tough to do otherwise. 100% recommended. 

ARE WE THERE YET?!? What does single cell proteomics need to do next?

 

Just leaving this here before I run out the door! Great insight (perspective, even?) from one of our most forward thinking protein informatics groups. Cool that we can do this stuff, but is it read to help people yet? Why not? Totally worth a read. (Open access)

Proteomic analysis of a super promising new active RAS inhibiting drug!

Y'all, KRAS small molecule drugs are sort of my jam and I have exactly zero guesses of how this drug could possibly work. My calendar is packed today, though, and I'm going to drop this here without looking it up.

Here is the idea, though: KRAS by itself is generally not bad. The problem is that the active sites get mutated and then the stupid new version of the protein stays active all the time. As KRAS and it's cousins NRAS and HRAS sit on top of pro-proliferation pathways, having them active all the time is a terrible idea.

This new drug, currently RMS-7977 (the name generally changes if it has success in human trials, you can guess by the designation that it's probably not there yet, so a healthy grain of skepticism should be involved here for this exact molecule) appears to only inhibit the active sites. 

Imagine a KRASG12C mutation where you've got a cysteine very actively holding onto GTP so that it is activating those proliferation pathways. You've got too much active stuff around. This drug doesn't block the GTP site, it blocks the activity of the active GTP bound protein. 

What's surprising is that there is a very different shape for a G12D mutated pocket and a G12V or G12R, etc., but this thing doesn't care! 

What's funny is that they did some awesome proteomics and it didn't really make the paper. TMT proteomics and phosphoproteomics on an Exploris 480 using the turbo TMT mode. 

If you're as interested as I am, the files are here. There appears to be a second repository, but there is a typo in the paper so I can't find it without some digging. 

Really really cool stuff. And some of the early small molecule RAS inhibitors aren't really doing well in the clinic at all right now, despite being approved for use. However, the molecules based on those are rapidly evolving and each one is better than the last. Even if this one doesn't go forward, the fact that you can inhibit a bunch of deleterious mutations with a single drug is a super promising development!! 

Save the date! ABRF 2025 - Las Vegas March 23-26!

 

Today we'll talk about why you have the coolest person in your field head your academic associations. 

I'm of course talking about -- 

THE Dr. Sue Weintraub  -who may have had nothing at all to do with the fact that the Association of Biomolecular Research Facilities is meeting in 2025 in Las Vegas, but I'm going to pretend that I have inside knowledge that it was entirely her idea. 

Thanks Sue! You can find out more and register here! 

I haven't been to an ABRF meeting in a few years. I did a talk on single cell proteomics in 2022, maybe, because I haven't been in a core facility in a few years. I'm a huge fan of the organization and the conference. 

If you are an early career researcher (ECR) and you are working on building up your credibility and exposure ABRF can be an amazing thing to be part of. Look, we already know who is going to be headlining ASMS next year, right? Same dudes that founded the whole thing in the 1960s. ABRF heavily promotes ECRs because it is great for everyone. Core labs sometimes don't get to do a lot of method development because they're too busy applying established methods for the hundreds of customers necessary to keep their lights on. You come in from you academic lab with the techniques you developed and you can get a chance to present those new methods to the lab pros who may have a very different angle than you've considered. Someone with 20 years of applying proteomics techniques can be an unbelievable untapped resource for new insights and ABRF is where you get to hang out with them.

I've never gambled in my life but I've gotten to climb in the Red Rock Canyon just outside of Vegas.... that might still be my profile picture for this blog.....

Obviously you go for the science, but it never hurts when there is fun stuff to do before it starts or a day or two after all the science has wrapped up.