diaTracer - my first impressions (diaPASEF data!)

I've been generally confused about the whole "FragPipe + DIA" thing for a while. What's the DIA-NN node doing there....? Why wouldn't I just run DIA-NN...? (for examples.)

When a new way to process DIA data showed up last year, I had my hands full and just didn't get around to it. I've got SpectroNaut, BPS and automatic command line DIA-NN (academic version). Why would I need anything else? 

What if you are an academic and someone set you up a very nice interface where they'll just load you up anything that will run in Linux? And you don't want to type stuff into a command line? And you don't want to convert your Bruker files? And you don't want to pay anything for it? And you don't want to mess around with making spectral libraries yourself? 

Enter diaTracer! So I'm checking it out vs the other tools and - HOT DAMN THIS THING IS FAST! 

Maybe you can't read the box above but diaTracer took 5 minutes and then everything else took about 5 minutes. That's super fast for these files!  

Also! 

1) I didn't really have to learn anything. I already know how to use FragPipe. (I did have to download FragPipe 24 and then look very hard for the diaTracer button) 

2) I didn't even give this search a lot of resources. I pointed at 2 random single cells, gave it 18 threads (parallelization)  and I have a report in 10.2 minutes? On diaPASEF data? I can't convert these 2 files to .HTRMS to upload into SpectroNaut this fast!  

3) I'll have to compare the results (or make someone smart and meticulous do it.....) I'm just eyeballing the DIA-NN data and they seem similar. 

WHAT??? And here is one of the proteins that I'm looking for! Oh. This might be super cool. 

May Institute Essentials! In November! Get LCMS proteomics computations statistics fast!

 

Need THE crash course in doing shotgun proteomics statistics? Enrollment is open for the upcoming May Institute (I think that is what it is called. Not the month.) 

You can find out more and register here! 

Need to enrich some weird peptide (PTM)? Make your own stagetips!

A long long time ago when I was an (unlisted and unacknowledged on the website to this day) summer instructor at the Cold Spring Harbor Proteomics summer school a couple years in a row, we made some phospho-enrichment tips.

Then (2014-2016? Something like that) it seemed a little silly when there were good commercial ones. But...I also worked for a vendor (why I'm unlisted as a guest instructor) and I could just request someone to send me some tips when I needed them. Also....while they are out there for phospho- what if you wanted to enrich something super weird? 

The ultimate method for doing so! With all the part numbers and fantastic pictures and graphs and summaries of the chemistry you'll need to get started on the weird stuff! 

DIA-NN EasyFilter Workflow!

Do you (or your reviewers!) need more than a text file to prove you did some proteomics?

There are some nice DIA-NN tools out there now that make looking at these data at depth easier, and this is seems like a nice new entry.

Importantly, there was a point where DIA-NN started using something called a PARQUET file. Which is pronounced like this 1980s goodness, which I guess they still manufacture today...

This is the gold standard for people who like cooking to be unpredictable at different temperatures and to contain an extra dose of Calcium Sodium EDTA. Which is fine. I'm only putting it here because I kept saying "PARK-ETT" like a dummy. 

Not all of the tools that people made before will accept the parquet files, but this new tool is specifically made for it. 

Mag-Net gets a lot of proteins from plasma...but are they actually useful?

 

I have to admit I'm only just now starting to understand the differences between all these plasma proteomics thingamabobs.

Since we've got our hands full with more single cells than we can possibly run, I even missed the absolutely hilariously weird stuff going on with the owners at SEER.

Importantly: I desperately hope all of this summer's drama over ownership hasn't had too terrible of an impact on the great scientists at SEER who are doing great and important stuff. And if it has, and you see a CV from a SEER proteomics scientist, you should probably hire them immediately. There are some amazing people there.

Not familiar? Look it up, I'm a little scared to give you my summary of all the stuff I've read. Lawyers and accountants are expensive. I just deleted it, but you don't need my summary of investors said, CEO said. Though there are some pretty funny plots about how the company stock has performed vs the CEO's salary...the latter wouldn't be considered bad for an NBA player. 

But here is the thing that threw me off even though I think people have mentioned it.

Mag-Net appears to get big numbers because it's an EV (extracellular vesicles) enrichment technology. Which is cool and everything. If you're an EV researcher you should be stoked. This stuff looks super smart. But....I'm not an EV researcher.....so...like...do I care? 

I stole this picture above from a 2025 paper that I'm sorta flipping through because my brain will do ANYTHING ELSE when I'm on a deadline. 

Which...honestly...I guess is sorta the question about anyt of these technologies. If Aim 4 of basically every grant I'm a high part of is "and then we port this marker to the extremely limited list of FDA/CLIA LCMS instruments for making a LDT or IVD.  Can I afford to put a nanoparticle on the front end of it? My hope/guess/approach is that the problem is we can't do discovery, right? But if we go to the completely over the top pain in the butt that is validating an LCMS QQQ assay, then it will see stuff. Right? 

I really like the paper above, but it let some things click into my brain so I understand these different technologies a bit better.

Oh. I did actually post this. Since it's been up for a couple of days I guess I'll leave it.

And if you are going to do this EV stuff, this automated procedure from EvoSep looks extremely straight-forward.

https://www.evosep.com/wp-content/uploads/2023/09/Poster_Auto-Mag-Net-final_HUPO2023_web.pdf

An elegant way to sample peptides and intact proteins from important stuff!

Oh shit. That's a snake in the picture! 

Okay, so maybe this'll sound obvious, but it only seemed obvious to me after I skimmed this new paper. 

What if you just wanted to sample out the endogenous peptides before you did your intact protein stuff - or digestion for shotgun proteomics? 

They stage tipped out of the solution to enrich the small peptides, and then precipitated all the big stuff to do the rest. It's probably a solid assumption that most of the big proteins won't either go into the little stage tip, and the ones that do won't elute out under the same conditions that the endogenous peptides will! 

Smart, right? And then they can do all the venomomics! 

Let's build a high resolution digital microscope out of Legos!

 

My wife has a conference that she chairs or is president of or something important sounding very soon and I'm very concerned about the heat index. Holy fuck it has been hot in this mountain city. Kindergarten on zoom is rough, y'all. There is far too much dancing for this to occur in my office space around a bunch of serious looking virologists and pharmacologists.

Emergency backup ideas, like this one? 

The 5th annual Johns Hopkins Mass Spectrometry day 9/17/2026!

 

Tracking predator cues in marine prey!

 

I liked this paper from the very first sentence...

Abstract

Prey rely on chemical cues to detect predators, especially in coastal ecosystems where danger is concealed by turbid waters. Juvenile oysters (Crassostrea virginica) respond to blue crab (Callinectus sapidus) chemical cues by growing stronger shells, while mud crabs (Panopeus herbstii) reduce foraging to avoid detection. 

...because...what...? 

Did you know that? No, I did not. This is the paper, and this isn't what I expected to learn about at lunch today. I expected to be at a liver center seminar but it's so hot in Pittsburgh (a small city in the Appalachian mountains) to safely have school. I tell you what, the climate collapsing isn't going to be boring. Also, kindergarten remote learning is a lot of dancing.

Wait. Okay, how do you set up an experimental design for something like this? 

Oysters were settled onto 4.5 cm × 4.5 cm marble tiles and allowed to grow for 1 week in 1250 L mesocosms with flow-through seawater prior to experimentation. Oyster spat were then transferred to aquaria and repeatedly exposed to 1 mL mixtures of blue crab urine from crabs that subsisted on either an oyster or mud crab diet (n = 3 mixtures in block 1 and n = 9 mixtures in block 2 per predator diet), settled seawater (negative control; n = 3 mixtures in block 1 and n = 9 mixtures in block 2), or a positive control of predator water 

Ummm....okay, that makes sense, but it's sorta funny, right? 

Oh. Okay, I had all sorts of dumb ideas about how they'd ever get the molecules in the sea water. What they do instead is see what oysters make thicker cells in response to crab A or crab B's urine and then they save back some of the urine that they analyze. Then they can track down the molecule that the oysters respond to. They use NMR for the metabolomics. So - no proteomics here - but it is in Journal of Proteome Research so it counts. 

NMN + Sugar = Reduced hypobaric hypoxia!

Despite the weird repository used for the data in this new paper, it's still pretty cool in this context.

Imagine that you're trying to climb Everest and your goal is to have mice carry all your stuff. Maybe you can't afford a human to carry the stuff? Maybe you're just really mean to animals. 

Everything is going great, until about 1/3 of the way up you discover that these mice are getting really lazy. Do you have an oxygen tank for a mouse? NO? I think you're just doing this because you don't like animals. 

Okay, but I wouldn't type all of this because I don't have a solution, right? What if it's a solution you can get at a gas station?!? Check this out. 

Through extensive trial and error of the stuff you can absolutely get behind the counter at shady gas stations, this team came up with the solution. 

Nicotinamide mononucleotide (NMN) and sugar! To prove that this made the mice better at carrying stuff, they chopped a bunch of them up and then did a bunch of multiomics on the resulting tissue. 

Metabolomics and lipidomics were performed on an Exploris 120 and the Proteomics of just the tendons and ligaments were performed on an Orbitrap Eclipse. 

Something interesting is that they used a specific kit for extracting proteins from the ligaments and tendons. I don't know if it's any good, but it is interesting to me that such a thing (and a company I've never heard of) exists. You can find it here. 

The end findings are extended to humans as the mechanism and how to reduce hypobaric hypoxia in us as well. Given the ENORMOUS differences in how humans and mice respond to these conditions...

(for real, mice drop their body temperatures at high elevations, on purpose. Humans generally never change their body temperatures.... human do a funny thing with a word I can't remember where we jack our body pressure way up to compensate for extremely high altitudes, which is part of the reason we need time to compensate and recover at high altitudes. Here's a good link to a whole lot of reasons we have to be cautious interpreting rodent and humans in these kinds of experiments) 

... this could be a stretch, but I like a good over the counter solution to problems, so if anyone ever talks me into running a race in Colorado again I'll try pumping myself more of more NMN and sugar than I normally do just to find out, I guess.