Get 70 Hz on an Orbitrap! At...3,000 resolution....

 

For anyone out there who wants an Orbitrap to go super duper fast and are okay with getting less mass resolution than a Q-Trap in 2002 do I ever have an advance for you! 

Introducing the FASTEST ORBITRAP EVER! All you have to do is ....give up....everything else....

...and run phase constraint! 

Okay - so I'm obviously just being a jerk. I got in all sorts of trouble a while back for ...running some old instruments at whatever resolution I felt like and then putting funny stickers on them because I  wanted faster scan speeds. 

I won't draw attention to those posts -lawyers are expensive - but when we ran the instruments at higher scan rates we had two major problems

1) We weren't getting anywhere near enough signal for most molecules

2) The mass accuracy went out the window. Funny to say now because I was super appalled by 20-30ppm mass accuracy, and that's before I started buying TOFs...where that's pretty good...

What this group does is a bunch of magic to fix problem #1. By balancing accumulation times to get more they show they can get enough signal - EVEN at 8ms fill times to get good MS/MS spectra. Impressively, they get a solid number of IDs on an Exploris 480 running 5 - 8 minute active gradients. They get the best coverage at a whopping 500ng of peptides on those 8 minute gradients. Fortunately, you can clean that quadrupole yourself!

Now...how's the mass accuracy...? Better than an Ion Trap! 

An honest description of a new type of in-cell enrichable lysine crosslinker!

 

Crosslinking proteomics is not really my thing. I've done it a few times over the years and I've personally had success with in vitro experiments with a few proteins at a time (metric of success being happy collaborator). 

I really like the tone of this manuscript which introduces a new crosslinker. From the very beginning the tone feels like "crosslinking mass spectrometry is still hard. And this is a new way of doing it. It's STILL hard, and could be improved, so here are ALL of our notes."

It feels very honest and open all the way through - from how to make it through how it compares to the other stuff -through 3 different mechanisms of enriching these crosslinked peptides.

If I had to do this I don't think there could be anything more helpful than - the exact masses observed for the fragments AND their delta masses. And they're all helpfully provided directly in the text! 

I feel like this is a great example that maybe we don't have to completely oversell every scientific advancement that goes very counter- to what we often read (or may be expected to write?) Cool science. Better writing. 100% recommended. 

Staining ancient collagens from fossils?

 

Honestly really surprised this worked, but the controls are seem to make sense. They take some current bones and de-mineralize them as their method development samples and/or controls. Turns out they can still stain some things (mostly collagens) from the insides of materials like rib cross sections and maybe inside of other dense materials like teeth? 

A study that is clearly way outside of my content knowledge, but encouraging that enough protein is around to do things with! 

Story that I thought was going to be real bad - turned out to be a great one!

 

Fun story (now!) 

Wednesday morning we had a super early recording for Season 7 of the Proteomics Show (thank you US HUPO!) it's called "Alt Proteomics" because "People who measure proteins in weird ways" was my suggestion and I swear everyone just ignored me. 

Actually - this just posted - it's a pre-ramble to the Season. Ben and I are getting WAY out of our comfort zones and asking smart people really dumb questions. Like (not joking) "so...is your 'lab' just a bunch of computer terminals into a super computer...?"

My AMAZING collaborators start calling. On Friday my "lab" is going outside of cancer cell lines and we have real human samples for single cell proteomics for the first time - and...it's not Friday..... I'm not technically a millenial (too old) but I embrace the sentiment that PHONE CALL = BAD STUFF 

And I was right. The cells were ready 2.5 days early. It was go now or have nothing.

My first hire here at Pitt was on his 7th day. I hadn't sent him the SCP protocol yet. We didn't have plates ready. I hadn't powered on our sorter in about a month, so he hadn't even seen it. Our cell counter was so new that I'd never even used it (you count nanoparticles in solution, and I'd done that). AND we had a Bruker field apps scientist on site doing instrument training.

Fortunately our on-site field Apps Scientist was Dr. Josh Beri. 

Yeah. This guy!  A Michael fucking Bereman (inside joke, I still miss that dude and the world was a cooler place when he was here) and Muddiman lab product. 

Our corporation's Field Apps Scientist jumped right into the trenches with us. He hadn't used my weird cell analyzer or our single cell sorter, but he obviously knows his shit, right? So we had an extra set of hands to analyze and count cells, buffer exchange them (not the term FACs people use), recount and reanalyze them, bust up clumps (real human cells are sticky, it's almost like they don't want to be a homogenous solution(? weird) isolate them, lyse them, digest them and run them.

And check this out! Now - it is a long held tradition in proteomics that you only show the best possible data, and I scrolled down to show injections 21-25 for that reason. And some of these cells are over 25um in diameter. Not small.  

This is Whisper40SPD Zoom on a Bruker 15cm column with 10um ZDV junction on the TIMSTOF Ultra2 (not AIP...yet...). There is a solid chance that cell 21 is a doublet or was going through mitosis, but the point here is that on Wednesday morning I thought I'd lost weeks of setup discussions and invaluable samples. And thanks a long couple of days of help from my vendor(??) I've got the best single cell data I've ever personally seen from myself or anyone else coming off my instrument right this second.

I sent a thank you out to every email address at Bruker that I had and they're going to send Josh back out so he can finish the interrupted training when my postdoc in onboard in a couple of weeks. 

Edit 7/1/2025 - This is part of the response I received from Dr. Michael Krawitzky at Bruker

"Glad we could help. Again we are trying to transform Bruker applications to become a trusted advisor in the lab and unofficial lab member."

(Possibly Josh's boss? Yo, maybe this is just some lip service, but I tell you what, when you convince people to drop this kind of money on an instrument it's sure a nice thing to feel like you aren't alone while trying to get evidence that they made the right investment in you and your program!)

Spatial proteomics takes on atherosclerosis!

 

Gotta move fast, our cells are dying way too fast (I wish this was a cool new chemotherapeutic....) and we've got to move up a single cell sort by a day or two.

However, I will take a break from making fun of Illumina acquiring my single least favorite proteomics technology to post this super cool new preprint! 

Check out this sweet bonus video of them cutting perfect squares with a laser out out of these tissues! 

SpectroNaut 20 PTMProbing! Can you finally find PTMs in DIA data?

 

Check out the above (click to expand) - Yes, that is the bizarre nomenclature used by our friends at SpectroNaut (PG.normal word you'd expect AND the funnest-to-parse [ mf'ing brackets ] AND a space after the second bracket ]  at the beginning of each sample name)! 

And in the "EG.PrecursorID" which roughly possibly translates to "peptide sequence with the addition of an underscore at the beginning of the peptide sequence" 

THERE ARE A BUNCH OF PTMS!! 

Wait! What? No one wants to talk about this, but 

YOU CAN'T IDENTIFY PTM MODIFIED PEPTIDES WITH LIBRARY FREE SEARCHES! Sure, there are buttons there, but you won't find shit.

Until now?

SpectroNaut 20 has a new pulldown thing called

"PTM Probing!" If this reminds you of an episode of South Park you might be old. 

You can find it here and it's pre-populated with a whole bunch of PTMs. 

Now...in my first check....I seem to have found only PTMs on keratins, but there are probably a lot of PTMs on keratins. I dunno.

Are they real? I also don't know yet, but it's absolutely a step in the right direction. 

Unless none of them are real and it's a pile of silliness, obviously.

So...what we should do is take some cancer cells and dump something on it that causes a crapload of PTMs. Then we should run some files DDA and run them with FragPipe and MetaMorpheus and then run some injections with DIA here and see how much they agree and disagree. And then we should call it something based on a late 1980s early 1990s actor based wrestling thing. 

Illumina bought SomaScan - using real money. That could buy useful things....

 

Okay. So. That rolodex of people who are interested in doing proteomics but don't want to investigate the technologies that are available that have evidence that they work? 

And...honestly....having competing technologies out there will keep O-Link from setting the prices of doing population scale projects at whatever they want to until LCMS proteomics works to throw off the reputations and limitations of the past. 

So....those things have value.... 

So...it's not the most expensive purchase in biotech history. Is it the one that right this second I feel most strongly negative about? Yes. 

Look - maybe Illumina will use their vast reach and capabilities to prove once and for all that a Somamer can provide a meaningful QUANTITATIVE measurement of a protein in a complex mixture. I have been waiting for one (1) single experiment to show that is the case... since I first heard of this in 2017? I think I'll change my mind just as fast as I did on the O-Link technology when evidence came out that O-Link protein readouts have value. 

Estimate the protein content of over 1,000 human cell types!

 

This came up when some old friends were visiting my lab this week. 

Where do I get these estimates for how much protein concentration should be in each single human cell? 

A couple of places, but the best organized place to start is the amazing 

Human Cell Tree Map - https://humancelltreemap.mis.mpg.de/

Is is the topic of this recent PNAS paper. 

Now - they did this the hard way. They took tissue from people counted the cells and weighed them. For real. It was an amazing amount of work. Supplemental File 3 is an amazing resource for getting these numbers. They didn't just come frome one white guy either. Cells were counted and measured from MANY donors.

Now - according to Harvard BioNumbers  - about 20-30% of each human cell is protein. 

Quick test of the data. In the screenshot above I have went to Male/Digestive System/Liver/Hepatocytes. And it gives me a range from 3.17 e9 to 1.49 e8 for the cells.

If it is a Sunday morning and my brain hasn't fired up quite yet. I go to Google convert and I have it convert 

So the low range for a hepatocyte is 3,170 pg and 20% of that is something like 640 pg of protein. 30% is something like 900pg of protein. Since I've got hepatocytes from patients under multiple conditions coming in this Friday, I'm pretty up to date on this. 

And this paper from a few years ago compared different human liver cell lines/types using deep proteomics and the Proteomics Ruler. 

And they calculated something like 700pg for a male human hepatocyte, so we're in the right range-ish.

There are clearly HUGE error bars here, right? But at least it points you in the right direction if someone comes in and says "can you do this cell?" And you can do a quick look and say...."an eosinophil doesn't sound like any fun at all!" 

Quantitative performance of O-Link with spike-ins in human plasma!

 

There is a lot to love in this short new preprint! 

Let's count them!

1) This uses a NIST reference material and uses a very standard analytical approach to tell if one of these "next gen" proteomics things actually has quantitative accuracy. 

As an aside - this is one way to approach the easy and obvious SomaScam experiment that I've been searching for and pleading for - for nearly a decade. 

The authors spiked in a some proteins into plasma and then used the O-Link assay and made sure that -in a complex matrices like a human plasma pool (wonders and wonders!!) the numbers make sense. 

2) And this is a big one - I have these data on my laptop right now. Because....

-the data is publicly available! On a proteomics data repository! This monumental accomplishment was performed using the PRIDE repository. You, too, can have these data right now! Click here! 

It's clear these authors use R/Tidy or some other format where they're comfortable with data in matrices rather than lists. If you're a list person, you'll have to pivot the lists. 

That doesn't seem like such a big deal, does it? PRIDE can accept these data and I bet they'll help you if you're challenged. This is absolutely critical as reviewers (and especially this one) is close to 0.00% tolerance right now with the "next gen" people. 

Important aside here that should get it's own post.

PRIDE could use your help. If you use the best proteomics data repository out there for free all the time, of if you just like science, please take 5 minutes and let PRIDE's funders know how important it is! 

You can fill out this survey here! 

3) O-Link appears to perform admirably in this test! What? Yes. This is really worth thinking about. The best I can tell this version of the technology measures a little over 1,000 proteins in plasma - and the spike ins work!! If you're on the fence between one of these technologies and the others - you should absolutely check this out. 

METAMORPHEUS SUPPORTS TIMSTOF DATA!!!!

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAHHHH!!!

One of the biggest challenges in adopting a new piece of hardware is that you might lose access to software that you've used for years and are very comfortable using. Losing MetaMorpheus by adopting TIMSTOFs hurt. For real. And this isn't a dig on other great software out there - I just know how to use MM and how to read the data and how to make MM do what I want it to do. I have whole downstream pipelines for HLA peptide analysis that uses MM scoring output as the input. 

This week when I thought I was seeing artifacts in a collaborators Orbitrap Eclipse data - boom - broke out MM - and found some weird peptide mods I've never even heard of before. 

If you need my pitch for MM this is it - it can look through your data for dozens or hundreds of PTMs and technical artifacts - and it can provide relative quantification data for them. If there is something out there that can point out ....ummm....that's a lot of iron adducts on your peptides...what's up in this batch?? This is the software.

And now I can use it again!?!?!?  

You can get MM here! 

You can fix cells and then ship them out for single cell proteomics! Here are the best ways!

This isn't the first study of it's type but this digs into more variations of how to fix cells. 

Something everyone doing single cell proteomics (SCP) has struggled with is HOW TO GET THE FREAKING CELLS TO THE MASS SPEC LAB.

At ASMS, I detailed my adventures preparing EvoTips and driving them across the completely insane and poorly maintained Pennsylvania Turnpike. In case you were wondering - I have far better luck keeping the tips at a stable temperature and being on wet ice seemed to be a detriment to the tips. n=1.

Meh. I was typing about something....THIS!

Okay, so there are obviously reasons why you might not want to put formaldehyde or something on your cells, but if there aren't, this may open the door up to all sorts of new collaborators! 

TesorAI Chat - An AI (genAI) designed for downstream proteomics data generation!

 

TesorAI has been on the blog somewhere before. As an academic I very much approve of the fact I can now process up to 500 Thermo RAW files/month! 

Edit/corrections - when I first starting using TesorAI it was only Thermo RAW file compatible. I'm punishing it right now and this is clearly no longer the case. 

The link above will take you to a high speed 45 second video demonstrating what you can now do with TesorAI processed data. You can tell a freaking chat bot to make your data into heatmaps and then just tell it to change the colors or add a heatmap - AND IT WILL DO IT. So so so impressed. Now....how does it do with 2 million human mutations as input sequences? 

Check it all out here. https://www.tesorai.com/

moPepGen - Find noncanonical peptides with graph based algorithms!

 

This new tool may only be exciting for a small group of people - but holy cow, yo - this is a just visually stunning. Maybe I'm just used to the fact that all LCMS proteomics figures generally come off the software looking like crap, but graph based algorithms seem like a great idea. 

You will need variant input sequences from transcript level data. And proteomic data. And someone who can use python - and can install a docker. All that magic stuff is here. 

Regeneron and Geisinger announce 200,000 patient proteomics study!

 

Watch out UK Biobank - Pennsylvania is coming for you with 2 HUGE proteomics initiatives!

Geisinger Health is well known for monitoring the genomics of my ...neighbors who prefer to use horse drawn carriages over motorized vehicles. Due to the limited speed of ...horses....people don't end up too far from home so it's easy to track geneology over time. Geisinger does other things, but that's what they're well known for. And Regeneron has the deep proteomics know how to pull off a 200,000 patient cohort!! Press release is here. 

Big DIA software advances - peptidomics and PTMs are the clear focus!

 I just upgraded to the fancy new SpectroNaut 20 and was flipping through all the things! 

Here are the release notes (in screenshot form, which is by far, the most convenient way to share text!) 

And I'm having the guys at the HPC upgrade FragPipe Academic and DIA-NN Academic to the newest ones for me as well (I don't have permission) and in the latter case (DIA-NN Academic) there are a lot of shared concepts!

In case it needs to be typed, I'm not accusing anyone of sharing notes. These teams are very user centric and we are all probably complaining about the exact same things.

Why can I find more PTMs in my non-enriched DDA data? 

Why is DDA so much better for peptidomics? 

Why isn't this faster? 

And here we are, at a great time to test some cool stuff out! 

Symposium on proteomics in the life sciences at the Broad! Registration open!

 

Wow. Sort of short notice, but this looks like an amazing use of a few days in Boston/Cambridge. This wasn't on my radar for the summer. The program is a little vague, but the organizers are some of the best scientists in proteomics and have picked extremely relevant topics. Definitely something to think about! 

Edit: I found the list of speakers as well. I was just on the wrong page. Geez. Check it out, the week is stacked. Here are the main themes. 

TMT in FragPipe with TMTIntegrator!

 I gotta move fast, but for those of you on the TMT32plex or 35plex or whatever it is, I think you'll really like this screenshot! 

How? What? I don't know! Check it out here! 

Peptides bistability in ion mobility might explain a lot of things!

 

Okay - somewhere on this blog a few years ago is a post that is called something like "what the hell is ion mobility"? And I'm continually learning - or trying to - and might have just given 3 talks doing proteomics with ion mobility devices. I might still only partially know what it is and I certainly couldn't build you one.

However, I'm occasionally surprised by measured versus predicted ion mobilities. And I'm often troubled by how ion mobility clouds are altered by things like isobaric tags. And for the former problem....this new preprint is really really interesting.

Why couldn't a peptide have multiple ways to be pulled through a gas gradient? I mean...it seems like you could probably flip 50% of your peptide population A one direction and 50% the other and they'd have different "shapes" - and it's not like the peptide bond is a crystal lattice. (Lettuce? Now I'm hungry). It seems like a curiosity until you see how much their IMS predictions improve when the model allows the entry of 2 stable IMS readings for each peptide....

ASMS2025! Press report 1 - Proteomics Hardware Advances!

 

Guess who finally got the ASMS PRESS PASS! Now....would....it have been an easier conference to have given 3 separate talks and not had a bunch of press responsibilities? Yes. it would have been. 

Waters saved me a bunch of time by inviting me to their conference but then not letting me in, so I used that to sit in my room for just a few and stare at the wall in the dark to recenter, and I sincerely appreciated that time. I don't think they had anything in proteomics to show off. I just went to be polite.  

On the proteomics side the big 3 had big hardware drops.

SCIEX dropped the 8600 which could possibly maybe put them up there as having the most sensitive high resolution system in the world? Maybe? Those numbers are crazy. Let's see where they go. And the proprietary quad sliding stuff on top of it makes it a really interesting and competitive bit of hardware. This launch was, of course, overshadowed to some degree by a very public disagreement with a popular open software development team. I'm not paying attention, but people being open minded about demo'ing different big proteomics solutions seemed to be paying very close attention to it. GenomeWeb dropping a story on it during the conference couldn't have been the coolest thing for their marketing people to deal with. 

Thermo had the Astral 2 Zoom. Higher sensitivity, 270 Hz max speed (up from 200), capable of doing TMT 32-plex -with some important caveats. Like the TIMSTOFs you have to do 2 scans for TMT. One for the peptide sequencing and one for the low mass fragment ions. In the second scan the fragment ions spend more time in the TOF region so they make more passes and then they get the minimum resolution to separate out the ions. The bonus is you can optimize the high mass and low mass fragment acquisitions - and - of course, Proteome Discoverer first launched when the first Orbitrap XL was out there and it had to do 2 MS/MS for every TMT/iTRAQ, so it can take these data. The Astral is super fast, so even 2 scans/peptide and even when one scan is a little slower, is probably really fast.

Thermo also had the Excedrin? Excedion? Benchtop high resolution with higher scan speeds. You can run your Orbitrap at 3,000 resolution and get 70 scans/second. It also has ETD built in. Who would want 3,000 resolution and ETD? Anyone worried about the collapse of their transient in the Orbitrap! Big intact protein, people, for sure! The signal optimization seemed to allow bottom up where the MS/MS actually looks pretty good at 3,000 resolution - from a signal perspective. Some people have noted the mass accuracy seems a bit wonky. Not something I'd worry about, Makarov probably has that fixed already, but it's not like you can't run a higher resolution on it. 

EvoSep had the EvoSep 2 (Eno). Higher pressure loading pumps, better chromatography and higher signal. Looked very plug in play for anyone with and EvoSep One.

Bruker had a pile of launches as detailed in earlier blog posts. The TIMSOmni seemed to be stealing the show on the proteomic side, but we also saw an improvement on the TIMSTOF Ultra2 in the addition of the Athena Ion Processor. (Box now says TT Ultra2 AIP). From van Eyk lab stuff, it looks like a solid signal boost but it also adds some interesting level of control to the Ultra2 that seems under-explored. Imma send some samples up. The fun thing about the TIMSOmni is that you just turn off the OmniTrap and you've got a TT Ultra2 AIP. 

Man - was there some cool automation stuff - everywhere! Robots for proteomics!! And downstream data analysis. I'm currently demo'ing Mass Dynamics stuff and I'll post how it goes here. 

OH. And this was a big one for me. New vendors for N2 generators!! Parker was there and excited to talk to anyone who was tired of the generator that came with their instruments but Swissgas was a completely new vendor to me and we talked a lot about longterm cost breakdowns. It's one thing for me to whine about my N2 generator. It's nice to have useful information to share with people when they say "okay, what are my other options?" 

Alternative suppliers for some things you use a lot? That probably shouldn't go here, but if you were there, you could see where maybe someone else was like "we're also very good at making stage tips..." or "would you like to try one of these things?" I'm rarely going to turn down free stuff even if it is a stranger offering me something that looks suspiciously like a captivespray emitter. I'm probably not going to try it out, though. 

I was part of the Mobilion BILLIE launch (and, again, I'm a compensated member of advisory board) and that was really fun. Hopefully data will be up on PRIDE super soon so you can look at it. All of the data showed was on Agilent QTOFs that were not designed for proteomics, so it's super impressive to see the instrument doing competitive proteomics. It was super cool to work with that team and to field questions like "what could that front end do to a system that was already a good proteomics system??" And I'd also like to know that answer! It's currently a completely vendor neutral system, so hopefully we'll all know sometime soon.

Now, more than ever, proteomics needs better chromatography! ASMS 2025 edition!

 

As my very first takeaway from ASMS2025, I'd like to start with this paper that is just a little bit older than I thought it was when it came up in my talk on Monday. 

Oh man, did we ever see some awful chromatography this week. Whole proteomics on 5cm columns was often higher resolution LC separations. There are 4cm columns and probably shorter. And often at low pressure. Now, maybe all the hardware advances that have happened on the gas phase should negate the statements in this commentary? But I'm skeptical that is truly the case. And maybe I'm just old and old  fashioned, but proteomics did seem to be the one place where the LC seemed to be left the most behind. I popped in on some food chemistry talks and a couple environmental and - they also have big complex matrices - and they were still leaning on UHPLC - often with very fast gradients and the discrepancy was a little jarring. 

Time will tell, I guess! I just wanted to drop this overall impression while I had a second - not long enough to write anything more involved. 

How much ion mobility would be necessary to replace the quadrupole?

 

Conflict of interest statement on this one - I'm both a compensated member of the Proteomics Advisory Board for Mobilion Systems and the first author of this preprint met weekly with me for 4 or 5 years while getting her PhD. I'm even more impressed by her work than by the quality of her education. ;)

Okay, but here is the real question and I know I've certainly wondered about this..... at what point would ion mobility and quadrupole isolation overlap? Or maybe this? Could you crank up your ion mobility resolution to a point you could widen your quadrupole and get equivalent data? Or if you had 1,000 resolving power ion mobility, would the quad just slow things down? 

Those are the questions tackled here. 

The short answers appear to be - absolutely, yes, and maybe. There is certainly a point where you can get enough ion mobility to get the same isolation you would get with a quadrupole alone. On the SLIM system the coisolation can be limited to about the coisolation of a quad running 5 Th windows. If you want the cleanest data you've ever seen, SLIM + 25 Th windows would get you there. And....yeah... in the world of wide window DIA or wide window DDA and now wide window PRM...these numbers are relevant. 

New Exploris confirmed! And...print cartridge LC columns....?

 

Just stealing Chris's post! 

Bonus, if you see Ian watch what he's eating and model your diet after it. He's at least 114 and he's out there on stage giving talks at 8 in the morning? 

Oh! Website went up on the Astral 2 "Zoomie"

270 Hz and the resolution to do TMT 32-plex (I thought it was 35? Does anyone know?) Big question should be - can it do the 32-plex at the highest acquisition rate? Or do you need to slow it down? 

We're going to see a lot of 300SPD proteomics on it, I guess. Probably with a 4cm column so everyone get excited to do label free quantification at 2 scans/peak! Still better than an aptamer!