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.)
Sunday, September 20, 2026
Monday, September 14, 2026
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.
Not all of the tools that people made before will accept the parquet files, but this new tool is specifically made for it.
Sunday, September 13, 2026
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?
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
Friday, September 4, 2026
An elegant way to sample peptides and intact proteins from important stuff!
Oh shit. That's a snake in the picture!
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!
Thursday, September 3, 2026
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?
Wednesday, September 2, 2026
Tuesday, September 1, 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.
Monday, August 31, 2026
NMN + Sugar = Reduced hypobaric hypoxia!
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.
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.
Sunday, August 30, 2026
...iHUPO single cell initiative TIMSTOF Ultra2 methods don't seem to perform super well in my hands.....
I'm back at this great iHUPO single cell initiative paper again, I guess. Sorry if you aren't interested, but it's TIMSTOF Ultra2 vs Astral on standards and real single cells, so I'm interested.
Another thing that caught my interest was how very different the instrument method on the TTU2 is compared to the basically default instrument method we have ran for single cells for about 18 months.
Above you can see our method on the left and the method from the iHUPO paper on the right. The format is a little different since I had to cut the darned table out of a PDF. Which is always a joy.
But you'll see that we use 8 cycles and we run from something like 0.65 to 1.4 and the iHUPO method runs 5 cycles and from 0.65-1.3.
I feel like we tried a ton of different methods and ramp times and finally ended up with basically the default method plus 50% more ramp time worked better for everything in our hands. But we optimized on mouse hepatocytes last year, but those are kind of big and also kind of friendly (there are like 550 proteins that you can detect even if the instrument is vastly underperforming, because they make up about 90% of the total protein content. Compare that to human plasma where 1 protein makes up about 90% and you'll see what I mean).
We run a longer ramp time which gives us a full ramp at a speed of 6.37 Hz according to the software, so each precursor gets measured over 6 times/second, right?
The iHUPO method is faster and has smarter looking windows
I was super pumped to run something that looks so good on paper, and....it's kind of meh....
To be fair, this is just K562 standard at 200pg. BOOOOOORRRRING, but we get a steep drop off when we go from 40SPD to 80SPD on the EvoSep One. And I've never even tried 120SPD until now. Maybe with a 50% faster method it makes sense?
This is a terrible way to display data, but I've got like 15 free minutes on a Sunday night to type this up.
There are only 2 replicates of the 80SPD for each. EvoTips are expensive, yo. But we run the 80SPD standard method all the time. It's...like...our standard method.... and these results look normal.
At 80SPD the iHUPO method drops off nearly 4k precursors and 450 protein groups? Ouch.
I ran more of the remaining tips on the instrument in 120SPD because we don't have benchmarks for this and it's not ...as...bad....but ouch. It's still totally worse.
Obviously, we're using an EvoSep and they used a funny preformed gradient on a Vanquish Neo. And quantitative accuracy measures more than protein numbers.
BTW, this is just command line DIA-NN running at the end of each file. Nothing special. No MBR, no whatever.
But if you're looking at this paper and thinking WHOA, the Astral gets better data than the TIMSTOF, I'd take it with a grain of salt. This doesn't look like the best method to me, and instrument comparisons are dumb anyway.
Thursday, August 27, 2026
Did an Abird outperform a FAIMS Duo source in this new study?
I recently blogged about this new preprint and while going over it in more detail to see how we could improve our workflows I had to be impressed by one of the findings I didn't mention.
Two of the Asstral / Astral (spelled different by geography, I guess) systems had the FAIMS 2 Duo system and one of them didn't. Instead, it had the humble Abird system.
Abird has been around a long time and some labs use it on every system and other labs laugh about it seeming like a silly idea to reduce your background signal.
In a couple of places in this single cell study the one Astral equipped with Abird OUTPERFORMED the 2 Astrals with FAIMS. For some context, the one time I had access to FAIMS on an Orbitrap Exploris 480 the reason we didn't like it was that we couldn't generate enough nitrogen for the silly thing. To make up for it we rolled in big liquid N2 dewars which cost us about $250 every 3 days or so if we used the FAIMS. In one month we could have paid for an Abird.
I don't know what a Duo costs (I hear they often bundle it in with new systems almost for free, but if you want to add one to an existing system it can be in the HPLC price range), but if you're looking for better proteomics results at an ultra-low load level this seems like a low cost investment. (BTW, I don't know if it works on anything aside from Thermo systems). Excerpt from preprint.
Friday, August 21, 2026
Wait. How many proteins are in the nucleus?
https://www.proteinatlas.org/humanproteome/subcellular/nucleoplasm
So...I'm surprised by a whole lot of this. I'd personally expected that when we got around to doing single nuclei proteomics that there wouldn't be a lot there.
Something like
HISTONES
and all the AHNAKs
and that would be about it. Sure, some transcription factors, but they're low copy number at the best of times.
And there is a whole lot more going on there which doesn't make much sense to me at all, and that's okay.
So far we've seen two studies with single nuclei proteomics, one published and one that I'm sure will show up any day now.
In this one, Derks et al., used multiplexed DIA reagents on a TIMSTOF SCP and optimized out some carrier channel levels for the nuclei. Across the study I count around 1,800 protein groups in their data, however the actual protein IDs are missing from a lot of the processed data. I assume that's so they can do unbiased clustering downstream. SCP-Viz requires an identifier to perform clustering which makes it harder for me to reanalyze the data, but it's still a big number. Mun et al.,
Thursday, August 20, 2026
Discovery proteomics with O-Link reveals new HCC markers?
When I think of proximity extension assays (PEA) which is either famous because of O-Link - or is? O-Link, I think of purely using it for large population studies or for validating discoveries by high depth mass spectrometry based proteomics.
But...could you use it in place of mass spec proteomics? Because that is basically what this group did here.
Wednesday, August 19, 2026
Lyse your cells in 100% formic acid to get 40% more membrane proteins??
They start out by laser capture microdissecting a pretty big area of cells. Something like 500 micron x 500 in 15 micron cuts. And you stop wondering why the cut is that big when you see the digests are loaded on a Q Exactive Classic. Man, I love those old things, but proteomics has moved forward a good bit since 2012.
They seriously just put 5 microliters of 100% formic acid onto the slices and dry it off by speedvac and then LysC digest for 3 hours and then trypsin digest overnight. Big boost in membrane protein IDs over just DDM lysis alone.
Then they repeat it with smaller sections using an Astral. There might be two rounds of this to get to very small cuts. This ends up letting them see everyone's least favorite super important proteins, those awful SLC things. And it isn't a small list. They pick up several of them.
If you are also struggling with low input preps under-representing membrane proteins, this might be worth checking out. I suspect you probably have LCMS grade formic acid sitting around somewhere.
Tuesday, August 18, 2026
Multi-omics day on-site at Waters corporation!
Hey proteomics people! Did you know that Waters makes mass spectrometers? They totally do and, while I'm not at all sure how this happened, I got invited to drop in and meet scientists and see some talks and touch some instruments.
I'm pretty sure I got to visit because of some impressive numbers out of the P10 instrument which looks like a compelling piece of proteomics hardware. I'm trying to talk them into running some dumb stuff on one of them for me. The day featured a small group of people with very different mass spectrometry interests getting to wander around the demo labs and touch things and see the Cyclic MS thing (that I know Padula has and likes, but I wasn't really sure what it was) and lift the MALDI and DESI sources that you can just move from one instrument to another. Which, I can't remember anyone else doing, but they were just sitting there.
There were also fun customer talks from this guy https://info.liningtonlab.org/ at Simon Fraser.
Did you know there was an effort to compile all identifications of all natural products on earth? And maybe if you had all of them together you could look to see if evolution has biases toward certain chemical scaffolds - which, it absolutely looks like it does. Super ridiculously cool talk. He did admit that most of the natural product efforts have probably not been published, but they've got 37,000 of them all pulled together.
You should 100% check out the Natural Product Atlas here.
While trying to figure out my hand writing and misspelling of Eric's last name in my notes, I stumbled across this JASMS paper featuring open source software for integrating data from the ion mobility roller coaster.
One takeaway from the lab was that Waters instruments are a lot physically larger than I expected. The cyclic ion mobility system I saw was easily Astral size, but the new one is a benchtop. A very big benchtop. But they've clearly got their niches for these hardware. Moving native proteins and isomeric drugs around, purifying them by doing multipass IMS and then kicking out the ones they want for MS/MS? That's all stuff I saw people do and things I can't think of anything else that could do.
Monday, August 10, 2026
Wait. Are we doing this wrong? An argument for SUPER MASS SPECTROMETRY!
This is an interesting one.
What if it wasn't the most efficient to have proteomics mass spec instruments EVERYWHERE?
Rather, what if mass spectrometry proteomics was, instead, a fully centralized system where each sample could be measured comprehensively on multi-capacity hardware without all the compromises that come with solo 150kg - 400 kg vacuum chambers?
In the simulations presented here it seems to make a lot of sense!
The author makes the argument for splitting the ion beam from a single sample and routing it through to multiple mass analyzers simultaneously. Considering this from the company that can route ions around a lab with lenses that resember a garden hose. This is not an endorsement, but you might not know that the Spion is a thing, and it's crazy. https://www.tracematters.com/technology
Sunday, August 9, 2026
The biggest proteomic human atlas yet should reduce our reliance on transcripts to determine specificity!
2,800 human samples analyzed at incredible depth.
You have to be asking yourself that with a dataset this large and comprehensive, clearly we'll know for sure that the correlation is between transcript abundance from GTEX and this, right?
Uuuuummmm..... yeah, it's...the same....though that 0.105 from the pancreas is....special.... (this is from Supplemental Table 4). There is SO much data to dig through here.
I found out about the paper (how did I miss it??) when I stumbled onto this.
Friday, August 7, 2026
Bruker is boosting customer support and hiring.
Today I had the privilege of meeting with some fancy people from all over the place to discuss some concerns I have voiced and/or amplified about Bruker Daltronics support system. My concerns largely centered on what looked like a model I've seen before where an LCMS business is the cash cow (wtf is a cash cow?) for a big company to buy a lot of dumb shit. Who cares if a company uses their profits to buy other little companies? Not me, as long as that doesn't mean you lose all your field applications scientists or field service engineers for the stuff that you've already sold.
It is possible that I over-reacted. It would absolutely be the first time that has ever happened, however. As anyone who has ever worked with me can attest to 😇. But it's also possible that the mass spectrometry at the company isn't in charge of the whole thing and that sometimes there are corporate level shakeups where you have to do what you are told and deal with the fall-out.
Shortly after my ASMS possible over-reaction we had one of their top engineers on site followed by the amazing Dr. Josh Beri that most of us know who made sure our Ultra2 was back to pumping out world class data. To be fair I should have probably updated a really negative post yet again and I'll link it back to this one.
More importantly, I have independently verified that what I heard from said nice fancy people that they are actively building up the LCMS support network. At least one person I got used to emailing when we were stuck will be back in a couple of weeks and there are a pile of open field service jobs at least around the US. I've screenshotted some of those above. Which, given, the fact the economy in the US is rooooouuugh right now, sounds both very positive and hard to do. As an aside, the actual Bruker career page is a silly mess. Support jobs are distributed between the categories of Technical support and sales/support and you have to actually look at "ALL" to find the open applications jobs by manually going through. Interestingly it maxes out at 3 pages. So you might have to go through several iterations to find that there is a job posting on their website. I've seen worse.
I'm not sure I've seen any openings for field applications scientists while digging around, but there was a specific TIMSOmni Apps job on Linkedin that appears to be closed and possibly filled so I'm optimistic.
New people I've met from Bruker now have been legit as well. There are some serious key hires who are accomplished mass spectrometrists who are currently adjusting to their roles and willing to get on planes to talk to customers even if they're weird bloggers in small cities who were kindof a lot mean to them right when they started their jobs.
Summary - science business stuff is hard. My TIMSTOF is back up and generating really good data and I am a lot more confident and happy with my customer experience right now than I was a couple of months ago.
If you are wondering if there are people standing in my office making me type thiiiiiiiii HELP sdlkjdsfkjldasfljk980909098890098fjljdsflkjdsflksfjkfdaslkas;dlkjsdf lkhjasdfljksdf
That's a joke. I wrote this because I felt like it even though it was very very boring to do.
Thursday, August 6, 2026
Defining quality standards for single cell proteomics OR Astral vs TIMSTOF Ultra2
Okay. So what if you sent the same single cells out to a whole bunch of labs that agreed to send the data back?
Now....I am a little confused about the HPLC setup. It looks like they used a bunch of instruments but there is exactly one NanoLC method in the paper. I know people who like the Neo enough that they have their LC on one computer and their MS on another. So maybe that's what happened here?
It's a bunch of labs so you do get to see lab to lab variability on the same instruments as well.
Wednesday, August 5, 2026
NanoLC dead volume calculator since I can't find the MSBioworks one!
This is a nanoLC volume calculator inspired by the AMAZING (but dead?) MSBioWorks phone application I miss a lot and made with the frustrating assistance of some AI thing my university lets me use. It's honestly pretty good at color palettes, even if it's bad at math and confident/arrogant about it.
nanoLC System Volume
To use this, I've assumed some things about your flow path. You'll need to select how many columns and tubing sections. Let me know if I'm not considering modern nanoLC options, yo. It'll autofill some normalish stuff, so you'll need to fill in the inner diameter and length for each. It sums everything into total system volume live.
00
Set Up Your Flow Path
Sections 02 and 03 below build themselves to match — enter the inner diameter and length for each capillary as it appears.
Line weight and column shading scale with each segment's volume — thicker lines and denser fill hold more mobile phase.You'll have to believe me because I can't figure out why these fonts are OUT OF CONTROL
Total system volume
0.0 nL
01
Injection Loop
0.0 nL
02
Tubing Sections
0.0 nL total
03
Columns
0.0 nL total
04
Additional Dead Volume
0.0 nL total
—
Breakdown
Internal Dead Volume
Injection loop + pre-valve tubing
0.0 nL
0.0% of total
Operational Dead Volume (Probably the one you care about)
Valve → columns → MS inlet
0.0 nL
0.0% of total
| Component | Zone | Volume | Share | % |
|---|
Subscribe to:
Posts (Atom)