MaxQuant updates for isobaric quan - TMTPro/MBR and no reference channels(?)!!

 

Oh yeah! Can't wait to try this out! 

Most interesting, maybe is that in the application of isobaric match between runs and new stats with/without reference normalization - it looks like pooled reference channels between plexes may not be necessary. The link above is my kluged together solution for matching between TMT plexes without reference/pools using high abundance "boring" proteins and I largely put that post there so I could find those same data and quickly compare the two. 

Something I hoped to get a good answer to from the paper is whether the TMT MBR was working well for non-Orbitrap data. I used it for a relatively small TIMSTOF TMT set from early 2021 and it did appear to decrease missing values, but n=1 and I could have just got one of my settings off between runs 1 and 2, but it could be a gamechanger for some of the stuff we're working on. You want to hear that it is working before you ask MaxQuant to load up 600 Bruker .d files, though, but sometimes you just have to YOLO and apologize to the other users of that PC later. 

US HUPO Video Contest Award Winners are live!

HAHAHAHAHHAHAHA! 

We didn't have the time to do an unveiling of the winners of this year's US HUPO video competition. 

But now they're live! 

The Tragedy of The Quadrupole - from Mobilion

 and Omics Family Therapy from UC Davis!  

Spend 3 days doing a pulldown and analyze it in 7 minutes of LCMS time!

 

Okay - I won't lie - 7 minutes to fully characterize an immuno-affinity enrichment experiment (IP-MS or AE-MS or whatever) is pretty cool.

I do have to wonder if that is the most effective use of resources. And - yes - I'd have the same question if this was a TIMSTOF Ultra or other super high end instruments, but only because the pull-downs I've seen seem to take students 2 days to do a pair. 

Presumably...you know....the instrument isn't just sitting there after it does it's 14 minutes of work....

It is super cool that the bottlenecks would fall back on sample prep and maybe through robotics and stuff you could find antibodies to do pull-downs on all the things. 

But you do have to wonder if maybe it is a tiny bit of overkill? Like...could you do this on an Exploris 240 for 1/4 the price and just run a 30 minute gradient instead of a 7min? It's tough to imagine a scenario where this is a superb use of resources. 

However - if you're in a busy core facility and you've got 40 people who do IPs and you the 3rd Thursday of every month is IP day (yay!) knocking out 40 pairs of IPs in triplicate in 

40 x 2 x 3 x 7 / 60 in 28 hours would be pretty great, particularly if you're paid by sample injection for 240 in about a day's time. 

The Harvard thing where they've done just an absurd number of pull-downs is one of my favorite resources ever (and completely under-utilized by the scientific community) - PlexBio or something? And the thought that you could do that label free between now and ABRF (if you had the samples) is super compelling. 

The Proteomics Show is officially top 3 proteomics podcasts on a service that lists...one!

 

This guy wrote me this morning from something I'd never heard of to tell me the good news! Out of a field of 1 proteomics podcasts - THE Proteomics Show had made THE TOP 3! 

On further investigation, the show is actually ranked #1 out of all 1 proteomics podcasts! 

Huge thank you to whatever this thing is called and whoever votes and for not picking up the much more professional proteomics podcasts by Nautilus and O-Link. 

In the latter case, we were just on that show! 

As always, thank you US HUPO for paying our editing fees and annual software subscription and for our new microphones. Well....I got an open box/refurbished microphone and saved US HUPO $15 and that is starting to seem like a mistake, as you might catch from the show from time to time. I'll be super bummed if I have to buy a new one for $90 later and use this glitchy one for parts. 

Most of all, thank you to everyone who replies to emails when we invite you on the show! Without 60 amazing and often famous guests over the last few years, it would just be Ben and I rambling at each other and we can't really get away with that more than once each season. The magic of the show is that we have smart guests saying smart things and then Ben and I dilute it down with our mouths and brains. 

Fingers crossed we just proposed something completely different for a Season 7! 

Also for not podcast people - your phone probably has a podcast app and you can just open it and type "proteomics" and ...that's probably us.... or Amazon music or Spotify or other things. 

Phosphoproteomic analysis of different CHO - Antibody producing cell lines!

 

I don't manufacture antibodies, and I honestly only have a loose understanding of the process. If I really like you, or your project I've got a 7 minute gradient, really expensive Thermo column and even more expensive high temperature heater necessary to make the column work properly to knock out standards that look like this 

...and data from your mAB that look more like this ...

Which is clearly your protein, cause my standard looks a great. It isn't that it was selected from a broad range of possible proteins to find the one that was absolutely the most amenable to mass spectrometry, or at a level of clean that is completely impossible in a production environment.

I know that CHO cells (Canadian Hippo Organoid - name suggested by a 4 year old, it actually might be something that sounds far more disturbing) cells are often used for production. 

Want to know more about the process? Check out this new paper! 

It turns out that there are multiple lines of CHO cells and some are good at making some antibodies and not others. This group investigated some cell lines that were good and less good at making particular proteins to see what the difference is. I'm not entirely clear on why they went straight to phosphoproteomics, but US HUPO 2025 was amazing but my work backlog wasn't and my brain is still toasty from digging myself out. 

Turns out, though, that they are really excited by the patterns of phosphoproteomic alterations. From a production standpoint I'm not sure if the idea is that they could leverage some cell types and selected conditions to force advantageous production conditions - or if the idea is better applied to selecting the right cell lines for product production based on these information. Either way - antibody based drugs aren't going anywhere and it's cool to see proteomics addressing riddles beyond host cell contamination! 

Proteomics reveals new cell phenotypes in a marfan model!

 

Geez. Cedar Sinai is starting to show off what they've been working on the last couple of years -and this is another complete gem. 

True story - I completely changed my plans for my first ever solo R01 submission (which I submitted whether or not anyone is there to read it) based on the paper they just dropped in MCP. 

I should really really get back to reviewing large instrument proposals cause they're like - due - soon, again - I'm already several hundred pages of meticulous writing (not mine, other people) and I'm going to turn them in when I said I would regardless of my suspicions I'll receive some email bounces. 

This preprint is stunning, though. And...intimidating....now I need to figure out how to make plots that look this fucking great? Yikes!

Wait. Clearly I'm sleepy and the foucs here is obviously that a team of top notch people put a shitload of work into a terrifying (and relatively rare) disease like Marfan? So so so so so so so cool. 

So cool that I didn't put the word "single" in the title so that it won't lower traffic to the post. For real, it is still a thing. 

FFPE vs Fresh frozen tissues - very few differences?!?!?

 

I'll probably eventually convert my pile of notes from #USHUPO2025 into ...something.... but I need to knock out some papers that you should read (and leave them on this site so I can find them later) 

Let's start with this "WAIT. THAT DOESN'T MAKE ANY SENSE, BUT IT'S AMAZING!" study

This little team answered a question that I'd assumed I wouldn't want to know the answer to - FFPE vs frozen tissues - obviously there are nasty differences that will make everything impossible to compare, right?

They did a crapload of samples for whole proteome and phosph-proteome at depth and found that - actually, the proteome seems shockingly well preserved in both cases. Fresh frozen tissues have some advantages in phosphosite IDs, but I'm shocked by how similar the results are. I have honestly always figured that FFPE would be something you'd do because it's just simply easier and way cheaper to store these samples. But if there is truly limited loss in proteome integrity at scale? Priceless. 

PSCS - A tool for sharing reproducible single cell analyses!

 

There are tools out there for building pipelines and sharing single cell -omics data analyses....but I sure ain't seen one from a proteomics lab yet! 

Check out SCMS. I would have missed it due to all the #USHUPO2025 fun, but it was a key feature of Aleksandra Binek's excellent poster and she set me up with links to the preprint and the online server! 

If you've got some SCP data sitting around you're trying to make sense of, there are written and video tutorials already up and you just need an academic email address to register and get in and mess around here. Just don't overwhelm it while I'm trying to get the hang of it. 

Finally! A comparison of different modalities for plasma proteomics!

 

THIS is the study that I've been waiting for. I don't know how it wasn't blowing up the interwebs when it dropped, but here it is. 

MS2Bac - Explore, identify and classify bacteria across hundreds of species!

 

Despite the fact that bacteria have little genomes (1,500 protein coding regions for the tiny ones and 6,000 for the big complex ones like Pseudomonas?) not even all of the model bacteria have been explored really in depth by proteomics. 

This study goes a whole lot of different directions with some great findings pretty much every way the authors point it. 

The data from bacteria were acquired on a nanoflow Orbitrap Eclipse and a microflow Exploris (50uL/min @90min). There is a helpful table that tells you which experiments were performed on what and when. The authors also do classical 16s rRNA sequencing stuff on dairy products infected with unknown bacteria and acquire a bunch of data, including proteomics of over 300 bacterial species with data dependent analysis.

MS2Bac is the software and, suprisingly, it leans on spectral counts as a proxy for quantification. For younger people, spectral counts relies on the fact that the number of peptide spectral matches for each protein is an indirect function of that protein's relative abundance. Imagine bacteria A has 5,000 copies of EtfA protein and bacteria B has 100,000 copies. When you do label free proteomics of both, the bacteria B will probably have both higher sequence coverage of EtfA and a higher number of repeated fragmentation of the EtfA peptides. We used to do this because we didn't know how to do direct quantification. The spectral counting approach is also generally handicapped on high resolution instruments like the ones used here because an Orbitrap is smart enough to recognize that the +2 and +3 verions of peptide X are the same thing and will only fragment the most abundant ones, reducing the proxy for linear dynamic range of spectral counting techniques. Given the fact that there is a 150 Hz ion trap on the market right now, I suspect that we'll see more spectral counting.

I don't think many microbiologists will be surprised to know that you can tell bacteria apart pretty darned well by LCMS. You can put colonies of bacteria on a plate and hit them with some MALDI pulses and the spectra coming off is a decent enough signature that you can choose the next clinical intervention. There is a big difference between that and a tryptic digestion, however, and this is a bunch of bacteria that look different even when they are closely related. If you are a microbiologist, I don't think you'd be sad to have access to 300+ bacterial proteomes, either!