diaTracer vs SpectroNaut - same single cells!

 

I'll start with the dumbest analysis possible. That's probably all I have time for anyway.

I reprocessed a small population of large single human cells (TIMSTOF Ultra2 with EvoSep One 40SPD "Whisper Zoom") 41 cells that are EXTREMELY difficult to extract intact out of a cadaver.

No filters, no libraries, just the same library free approach, same FASTA database. Both came back with a little over 7,000 protein groups.

Big difference? On the same PC (an i9 desktop tower purchased last year with a ton of ram put in it before the prices went to the fucking moon) diaTracer wrapped up in about 4 hours from beginning to end. SpectroNaut was close to double that.

The reason this analysis is exciting and not depressing is how the respective softwares name protein groups and genes. SpectroNaut gives you your output as a "Protein Accession; Another protein accession" where multiple proteins could fall in that group. The way I have FragPipe 24 set up I just get a single entry.

I strongly suspect the overlap is better than this. 

Besides being fast, I went back and reprocessed both of these datasets looking for PTMs in these big single cells. And diaTracer found over 650 phosphorylation sites! SpectroNaut found ...fewer.... Are they real? I dunno! It would be super cool if they are! 

Obviously this is only cool if you're an academic who can use FragPipe, but - hot dog - I'm super excited that FragPipe can 1) process my diaPASEF single cell data and 2) it looks like it's at least very in line with software I've used to process thousands of files and have grown to trust over the years! 

Spatial transcriptomics is far more expensive than spatial proteomics (deep visual proteomics)!

 

Hey you! You have so many options today for doing spatial measurements. You really do. There are at least 10 different options for measuring gene products in different cells. I was at a couple of recent meetins where biologists were talking about how much these things cost, and it's impressive.

Direct quote from a core director "Each slide costs me $7,000 in reagents" for her favorite spatial transcriptomics technology. She owns all the hardware! 

I pulled some prices for internal at my University and - wow -

There are now ways to do up to 1,200 antibody probes for proteins in addition to single cell transcriptomics. This appears very new (Bruker's new acquisitions) and I've heard it is in line with the spatial transcriptomics. 

Let's break this down for deep visual proteomics (spatial proteomics by microdissection) again this is internal at my University.

For a 10x Visium 2 tissue 11 mm capture for $10,900 you get 50um pixels with 100um center-to-center (according to their website) and adding on proteins is an extra $3,900. Let's call it $15k for 2 images with 100 micron resolution.

This is whole transcript sequencing in most cases so they expect 18,000 gene products theoretically measured across the slide and it looks like 35 protein maximum right now per panel if you add that on. 

For $11,000 if you cut out 100um spots by LCMS we could (internal) do between 200 and 400 of those spots with proteomics (you'd have to find someone with an LCM to do it - I know a guy) and unless I'm doing this wrong, 220 100 micron spots (center to center) would be more than one 11 mm capture. 

A 50 micron pixel would be somewhere in the 8 cell range so we'd expect a proteomic depth of at least 7,000 proteins per "pixel" not "over the total study" or hypothetical.  That's solid depth and could easily be mined for PTMs, etc.,

And....we'd deliver a fully processed visual report in a GUI so the data could be fully mined by the end user.....

Compare that to the Xenium at 5,000 genes and $23,500! Holy shit. Xenium might be far higher resolution, I'm not sure, but if there is a takeaway here -

Spatial proteomics (or deep visual proteomics, if you prefer) is WAY WAY less expensive than spatial transcriptomics.

And....at the end of a deep visual proteomics experiment you don't have to do a western blot to see if any of those gene products actually....you know.....are real at the protein level. Which, depending in how you count the cutoffs, about half of them are actually real. 

Maybe this is a rant, but holy shit, I do love spatial transcriptomics and their pretty slides. But when I see 4 slides in a talk now I know they spent $60k - $100k on their n=1 data. And then they did IHC or western blots after. It seems like a misuse of resources. 

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!