Single cell SDS-PAGE!

 

Wait. SDS-PAGE has enough sensitivity for single cell proteomics?  Hmmmm.... It probably does....at least for high concentration band recognition...and it would provide some level of proteoform resolution. 

I'll be honest, I first thought "...that sounds slow and silly..." but the more I think about it the more I like having this around as a concept - or even a first pass.

They use the migration patterns for their output and then use statistics that can tell different single cancer cells apart by those patterns....

I don't know about the 3D imaging part, and it is a preprint, so grain of salt over your shoulder or whatever, but I'm definitely going to think about this on my commute. 

Single arabidopsis cell proteomics!

 

The preprint is also on this blog somewhere, but it's cool to see it out! 

Proteomic (and transcriptomic) map of 28 primary cell types!

 

Great new dataset alert! 

28 different primary cell types! What a treasure trove (is that a word? I feel like it's a word. Like valuable stuff you'd need to sort through?) 

Primary, in most cases means something like "we didn't get this from a cancer patient in 1958 and somehow it is still growing and mutating a century later". It can mean different things in different contexts, though. Sometimes it's cells that won't divide, but they will stick to plates and divide for a little while. Just bringing this up so you're cautious about use of the term around biologists and pathologists.

It does give the feel of maybe a pre-pandemic study that finally got prioritized for writing. Orbitrap Fusion 2 system, DDA, SCX or SAX fractionation involved. That doesn't mean it's bad, by any means. It means it's high resolution fractionated DDA data that took way more time to generate than if we ran it today on one of the fast DIA boxes. In fact, it means a dataset that could yield new findings in the future, you'll just have a lot more (and smaller) files to keep organized. 

PTM analysis was done using BOLT! Yeah! First paper I've seen with this cloud based search engine (that I'm very biased about due to like 5 papers I'm on about it, including the very first one) for a while. If you're not into advanced PTM analysis and worried about that, the data was also analyzed with MaxQuant and the results summaries are available in the Supplemental and on PRIDE as PXD062642. 

Global proteomic structural analysis IN LIVING CELLS!

 

Dropping this here so I can get back to it! 

The omics molecule extractor! What a fun and easy way to visualize aptamer data!

 

All, this is legitimately a very nice and very easy to use tool.

You can load metabolomics data or transcriptomics or even proteomics data into it! The test data is some previously published aptamer data, and it's really cool to look at. 

You should check it out. 

You can go right to the online tool here and start pushing buttons. 

I haven't analyzed a load of SomaScan data before, but I've got a couple datasets and the Omics extractor can simplify my pipelines for looking at it down to a single push button. 

Check this out! Lots of proteins detected at just about exactly the same amount in every single sample! In this case it is people with or without arthritis! Look at that precision!  If I didn't know better I'd think that aptamers bind to and offload from proteins but do not do so in a quantitative manner outside of an extremely narrow dynamic range. 

Cricket enriched pasta proteomics!

 

Google said I could use this image, and it's amazing. 

Seriously, though, this is new study is also really cool. 

Look, I have no idea if the deoxidation potential of crickets as a food additive has any scientific merit. I can't possibly know this, and legitimately have no interest. I am, however, aware that the global population is still expanding and the climate is collapsing and no one is going to do anything to stop either thing. Other food supplies are going to be necessary possibly within my lifetime? Definitely within my child's. And we're going to need to think about the allergenic implications of doing things like introducing 20% ground crickets into our spaghetti.

This paper focuses on the benefits of adding cricket protein, but - wow - do they do some cool stuff with some endogenous peptides in ultra-complex matrices. Just about what you'd do to look for allergenic peptides. The LCMS was an EvoSep and a TIMSTOF Flex. Maybe the inflammatory stuff is real as well. 

Super fast targeted proteomics on an ion trap for IBD monitoring!

 

Sometimes you already know your targets, but porting them to a targeted assay is BORING, and typically requires one of those crappy triple quads. One transition per peptide? Repetitive and boring and you basically get a true/false for each transition per scan. Is it real? Is it coeluting in this one patient? Good luck sorting that out! 

Could you use a super fast, crazy sensitive, but absolutely overpriced (come on, y'all, you've got the only ion trap, you could claim targeted proteomics market share) ion trap to get PRM (multiple transitions per target!) to biomarker studies? 

Maybe! Wait. Maybe if someone proved it could work to the QQQ people then you could maybe then try to corner the targeted market? 

This group went up to 300 SPD with almost 1,000 samples! 300SPD wasn't everything they hoped and dreamed, but the quan at 140 SPD looks great! That's still a 1,000 cohort study in a couple of days. Okay, it's several days, but still. I'm impressed. Now...if only the clinics could afford it..... 

Deplete out 99% of the dead cells to clean up your proteomic data!

 

Even if you're not doing single cell type studies, chances are you've got some percentage of your data that is being biased by nonviable / dead cells that may not represent what you're trying to study. We kept trying to do single cell on toxicity models and - if you're doing an IC50 study...50% of your cells are dead. So....do you care about the dead cells? And do the ones that are not dead, or possibly more resistant, represent your phenotype accurately?

What if you could easily deplete out the dead ones? Would that clean everything up? 

Sure looks like it in this case! Over a 55% increase in signal for the proteins this group cares about. AND they got a list of high abundance proteins that seem to drop off when they remove dead cells. New markers for excessive cell death? Sounds like that to me! 

How does COVID affect hamsters? Proteomics answers the big questions!

 

I had a short day last Friday because one of the kids that plays Paw Patrol with my kid had a positive Covid test. Wait. Is that still a thing? Yes it is. So the very first thing I wondered was "I wonder what the virus does to hamsters???"

Proteomics to the rescue! 

If you're not good at reading or a conservative or both, the two seem to correspond, I will clarify that I'm being facetious here. 

I legitimately think that some friends and I were among the first people in the US to have Covid. There was an international trip and one person had just flown in a very long way east from a meeting and we were so so so sick. But there weren't tests then, and who knows?  And who knows what other people fall in that category of "possibly had the virus, but we'll never actually know?" How do you do "changed by virus" studies on people who may have had it? Or may not? 

Apparently these poor little rodents express all the correct proteins to make them a good model of something that can get the virus, but probably hasn't. So the animal testing is justified by the fact it's probably impossible to build a good human cohort that hasn't been exposed. I hate animals models. Abhor. Loathe. But I also can't CRISPR the most abundant protein in a human's hippocampus either. 

Animal model...is...I'll grudgingly admit, the only way to do some things. 

Proteomics was done by DIA on an Orbitrap III (Eclipse), pooled samples were used to make a spectral library then DIA was used for quan. Phosphoproteomics was also performed and a pile of IHC and validation was also performed. Solid looking study. Easy to write a silly headline. A+. 

Proteomics of butterfly metamorphosis!

 

A little disappointed by this picture, but nothing else in this great new study! 

Fixed it! Wrong butterfly, but I don't care and neither do you! This is the whole genomics vs proteomics argument - 

same genome - different proteome! Where are the wing proteins in the early life cycle stages, Mr.Genome person? Checkmate. 

Let's go! 

The data was acquired on a Q Exactive Classic using nanoLC and DDA. Data was searched in Proteome Discoverer 1.4 (I guess they could have cited something for the software. I wonder where they'd find a reference for that?) and a RefSeq genome with 19k protein sequences (whoa!) was used for analysis. Okay, peptide mass tolerance is too high by about 20x for this instrument, but maybe they did that to increase the number of bad hits for the FDR. The method for estimating FDR isn't disclosed, so it might be anyone's guess. 

Oh. Weird. I think they spectral counted. In 2025 2026 on a Q Exactive which has onboard electronics to limit repetitive peptide sampling...? Interesting.... And they used a microarray normalization R package for differential quan....which...is.... whatever. 

They did pull data from a previous paper where they did transcriptomics on another pile of these butterflies, which is cool, and demonstrates that basically transcript abundance is a pretty uselessthing to do with your time. 

The data is up on ProteomeXchange if you wanted to do a reanalysis with a less eccentric data analysis pipeline as well! 

New ScienV biosketch format - ORCID won't assign? Possible reason/fix.

Happy 2026 US Researchers! 

Here is your friendly reminder that the NIH did have time to invent not one - but 2 (two!) new required Biosketch formats. Ignore the one from the summer. Starting January 2026 your BioSketch MUST be made through the ScienV portal at the National Library of Medicine. (Yes that still exists, it's just the Goddard Library that is being shut down and the books thrown out).

I assume the NIH came back from the longest shutdown in US government history as surprised by this as we are, so there is a glitch or two. 

If your ORCID won't connect (which will completely prevent you from saving and printing a BioSketch) you can check it this way.

Log completely out of your internet browser or use "IgconoYolo mode" 

...you'll find that in the upper right corner of your Doogle Chrome AI enabled browser....

Make a note on your blog to investigate your 28 compromised passwords and wonder if that has anything to do with the alert you received from Chase yesterday of them rejecting an attempted $228,000 purchase. Definitely investigate. I figured the Chase thing was Phishing...?

But THEN go to the National Library of Medicine https://account.ncbi.nlm.nih.gov/

And try logging in through your ORCID. In my case I found a totally new account registered to -

...which....is neither my email...nor a valid email format. I don't have a lot of publications or grants, but this NLM account was linked to zero. And I have more than zero.

I reported this to the capital letters of National Library Medicine dash support@nlm.nih.gov - which I'm sure they're very happy to have listed publicly on an open to access blog. 

Boom! Now my fictitious NLM account is gone and it can be registered to my ScienV portal -I think. 

A new pile of mass spectrometry patents!

 

Link to the original post (with patent links here). I tried opening it in a browser I'm not logged in to (into...? en to? e tu? something) and it worked! 

Add another ion funnel for absolute sensitivity!

 

This is probably a great idea, but it ABSOLUTELY has a funny parallel I haven't been able to stop thinking about.

Wow. Did I ever go down a rabbit hole. I was looking for a MadTV skit and then discovered that SNL did it first - in 1976! SNL did a 3 blade razor. 

MadTV stepped it up a little with 20 ion funnels! razor blades! (Youtube link)

Was I wrong yet again? Single cell phosphopeptide enrichment??

 

Okay, so even if this is possible after I've repeatedly said it probably isn't (yet?), I hope we can all look at this and agree that it looks like a complete pain in the butt to actually do. But...I don't like bulk phosphopeptide enrichment, so I have my biases. 

Now, you could probably argue immediately that 83 phosphopeptides from one HeLa cell isn't very much. And I'd also agree with that, but it's probably more than I could get without enrichment. Though....I might legitimately see if that is true on an Ultra2. We do have some cancer cells coming in....

Another piece in the p53 (TP53) puzzle uncovered by dose dependent activation!

 

Paper link if you don't want a history of p53 and why I love it. 

p53 (TP53 to some people for some reason) is super weird, it's also super important, the TP doesn't stand for the bathroom paper Americans are obsessed with for some reason. It stands for Tumor suPressor (I didn't come up with the name it was discovered by 3 or 4 researchers independently the year I was born). It's one of my favorite proteins, not only because my first postdoc was on it's very close friend p53 binding partner #1 (53BP1), but also because its the best example for why we can't trust transcript abundance.

Perturb a cell and measure the p53 transcript and measure (or estimate with rabbit blood based tools like western blots) the actual protein and they won't line up. p53 is almost entirely regulated by degradation, not transcription /translation. The latter processes are just too slow for such an important protein.

Imagine this though, this protein that has been studied as long as my old ass has been bumbling about this planet, and we still don't have it figured out. But (and I did not know this - and a bunch of other things in this paper- until I read it last night/this morning) we now have drugs that selectively activate it in a dose dependent manner. (They appear to do it upstream somehow). 

These authors leverage these compounds to tease out a mystery of why too much p53 will stop cells from proceeding through cell cycle. AND why too little p53 does the exact same thing! 

I won't ruin it for you, my kid just woke up, but it's a really cool paper to read and a great example of a well designed study. The TMT proteomics is also top notch (whassup Akos!) If I have a criticism (I really don't) its that the authors should have cited a paper for the proteomic data analysis software they used. 😜😇 Actually, the westerns do look a little overcropped. I bet the unaltered ones are in the supplemental but that's the reviewer's job to check, not mine. The microscopy is just amazing, though, particularly Figure 4 (in particularparticularity 4I). Great and inspiring study all around. 

Seasonal changes in the proteome of fox semen!

 

I saw foxes and plasma in the title and I automatically assumed I knew one of the authors. I skimmed through and found that there were multiple seasonal proteins altered and thought that seemed pretty unlikely without extensive nanoparticle depletion. Those top proteins in blood plasma don't really seem to change all that much. Then I realized we weren't talking about blood plasma. We are talking about another plasma! Aha. That's a thing! 

And it's a crab eating fox! I didn't know that was a thing either! 

The method section is interesting, particularly the animal conditioning, but also the mass spectrometry! Proteins were separated by 1D gel and then analyzed by nanoLC into a Waters QTOF, perhaps the second one this week I've typed about that I'm not super familiar with. Data analysis was performed using Mascot with 1Da MS1 and MS/MS tolerance, which seems like kind of a lot, but - again - I don't know this instrument. I've seen people use these tolerances for data from the Stellar in preprints and Mascot knows what it's doing. Sometimes big mass tolerances help the downstream FDR stuff work better. I do really like the figures and while this fox is not considered at high risk for extinction, the last survey was done in 2015! I definitely think that all life on this planet has a higher risk of extinction today than it did in 2015...for...reasons..... so I'm glad they found the resources to do a study a mammal I'd never heard of! 

Saved you a click, no real time targeting in this paper "iRT Assisted" paper

 

If you were thinking "cool, maybe someone made the great iRT triggering tools for Q Exactive available again!" maybe they have, but this isn't where they did it. 

This group did do targeted by standard PRM assay for the host-cell proteins that they are most worried about, and I've never seen this before. Generally host cell protein analysis is done by untargeted means - at least in the literature. The figures are nice, and they focus a lot of their reproducibility and ability to port these methods to other chromatography gradients, hypothetically. They also back up some of their results by ELISA and it's altogether not a bad study. 

How the reviewers let a helpful (not helpful! we have enough acronyms for methods! especially this one which is literally the node in the instrument interface) new acronym to get through to publication for someone following basically exactly verbatim the methods in the 2015 Skyline Tutorial for "how to do PRM", I do not know. Hopefully I can save you a click. 

Whoops. I forgot to put a direct link to the paper in. Too late now. 

Multi-instrument comparison of a proteomics process control pipeline!

 

How would your favorite mass spec do in a head to head in a process control pipeline? What's that? Something practical in manufacturing things, probably! Ask an AI, it will make some dumb shit up about it. Google's moron of an AI I can't seem to turn off just lied to me extensively about how many points different field goals in basketball count for. Unless I started the New Year with a stroke, I'm pretty sure I can't trust Doogle for anything now and I'm happy about this surprisingly inexpensive IOS migration in progress. 

What, was I typing about a paper? Yes! A preprint. This one! 

These authors prepared multiple combinations of two bacterium mixtures and used everyone's instruments. There's an Orbi, a TIMSTOF HT, a ZenoTOF, and even two Waters instruments. One of which I don't think I've ever even heard of. Either it's old or they called it something different in the UK, or I did have that stroke. Further evidence for the latter thing is that they somehow got Waters data into DIA-NN and MSStats! Except the Waters I don't know about, which was processed with Skyline. Sure, there are raw numbers here, but the cool part is that the authors focus on the pathways that they actually care about, and basically every instrument does really well in that regard, with the Vion being the clear outlier (on the bad side, but that might be Skyline 😜). 

The single cell histone PTM papers are both out!

I am making this post to boost the Altmetric score of my accepted paper. 

Sorry, it got desk rejected from the target journal and ended up in one I'd never heard of and I want ALL THE ACCESSES. 

But...if you want to see my paper with the editorial glitch by the journal which inserted figures from a completely unrelated paper about COVID, check this out. It's legitimately weird. Thanks to Dr. Slavov for pointing it out. 

For an actual blogpost about these papers check out this weird Springer blog. 

https://communities.springernature.com/posts/single-cell-histone-modifications-can-be-readily-quantified-in-single-cell-proteomic-datasets

If you want to see the targeted one where they derivatized the lysines(!!) with robots, check this one out. 

Volcano enzymes for proteomics!

I kept forgetting this was a thing. Apparently, I even met some of the authors, but I was with the Director of Mass Spectrometry Innovation at some fancy place in California, and since we only hang out at conferences we tend to end up out until morning and I may not always form long-term memories at conferences with mass spec nerds everywhere. Which is funny when I pitch a collaboration and I'm all like "nice to meet you" and they are like "...ummm....what......" 

This is the original paper! 

This is the big picture! (Click to expand, or go to the paper link above)

AND the enzymes are surprisingly affordable!