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! 

Top down proteomics by EAD!

While I am skeptical of the currently accepted machanism for how those magnets induce electron activated dissociation (EAD), it's a super cool new technology. ETD but with basically no reaction time and no scary reagents?  Wins all around.

In this newest application of the technology these authors apply it to top down proteomics

They mix up some commercial histone standards and show they can resolve and identify their PTM sites, then they do some bigger proteins (heavy chain might be the biggest protein they try in the paper) and then also resolve some intact glycosylated proteoforms.

I thought this was a neat trick for overriding the default limiter for the ZenoPulsing (for the shotgun proteomics methods it was designed for you're more worried about saturating the detectors than what happens if your signal is a little lower, which is a very different problem than for intact proteins that really really don't want to fly). 

My very first question was "cool, but what can process these files?" and they used OpenMS and Flash Deconvolv (something like that? I might have the name partially mixed up with another similar thing and I'm not going back to the paper to look it up - definitely had Flash in the title). I'm pretty sure that if you can master the OpenMS framework that thing can process basically anything and this supports that assumption.

Holy crap - US HUPO starts this week (Saturday! 2/22/25)!

 

Ummmmm.....that's this week! 

Fortunately, I only have responsibilities on Sunday (Early Career Researcher "Difficult Conversations in Science" - woo!) Monday - co-chairing the most popular and absolute best session - Tuesday....ummm ... should probably make a slide or two....

And the astrologer they use to provide weather forecasts to my telephone says that we'll only have 15 inches of snow on Thursday. Which, given current accuracy means absolutely no precipitation whatsoever and I probably won't even need a jacket! 

US HUPO sold out of the conference block of rooms, but you can still totally go. I registered last week! 

https://www.ushupoconference.org/registration

Oh yeah, and these are the 2025 US HUPO award winners! If you are a podcast person, check out the new The Proteomics Show drops! We get to talk to all of them and Professors Cristae and Fenyo are up wherever you get podcasts! 

Single cell lipidomics by nanoLC TIMSTOF!

 

Wow, am I super pumped to see this one out! 

You know all those minimum handling techniques for single cells generally means that EVERYTHING is injected into the mass spectrometer, right? 

This is a pretty minor alteration from my favorite paper of 2024 - CellenOne pulls out single cells, but instead of digesting them you drop in lipid standards and methanol and then run the TIMSTOF optimized for larger small molecules (lipids). 

They have to do optimization in both positive and negative mode, the latter I've never even tried on a TIMSTOF unless I was doing MALDI, and then the entire screen changes. Fortunately, we don't have to do a darned thing, we'll just steal this method entirely. 

Immunopeptidomics on low cell numbers without crappy antibodies!

 

Big shoutout to Anthony for sending this preprint over! 

We spend a lot of time talking about how much specific antibodies can hurt science. We've been talking about it for as long as I've been able to have somewhat intelligent conversations about science. 

What hasn't gotten as much attention is how terrible "pan" antibodies are. I see papers all the time where they still do phospho-tyrosine enrichment with the 4G10 antibody- and I first used that thing 15+ years ago it wasn't very good then, and I ask at conferences all the time if it's improved and I don't think it has. 

You know what is way way worse than 4G10? The absolute pile of poo that is used to pull down the conserved regions of HLA (MHC) proteins. They are clearly biased toward specific epitopes, if you want them to work well you have to slowly rock the antibodies with 4 kilograms of cell lysate, and it helps if you do it a few times and the same procedure carried out by 2 people following identical protocols get very different results. (This last part I saw at a meeting in 2023 but if the presenting author has published those results -she was in pharma R&D - I don't see it on a quick search). 

CAN WE DO IMMUNOPEPTIDOMICS WITHOUT GARBAGE ANTIBODIES? 

No joke, this is worth taking a look at. 

Mild acid elution has been around forever. Heck, the best way to get a zillion "immunopeptides" identified is to use a not-so-mild elution that will break open the cells and release the endogenous internal peptides and then to turn off your peptide FDR --> boom! a zillion peptides. To be clear, I'm only recommending this strategy because there are at least a couple "CRO"s out there that will do something similar and charge you $10k/sample for completely and totally useless garbage results that hurt our field while making people I can't tell are unscrupulous, dumb, or both a whole lot of money. 

Actual mild acid elution, though, makes a lot of sense if you can fine tune it in. Now, there is some controversy out there about whether that has a bias. It makes sense that from the shape of these little presentation "binding" pockets that 3D structural stuff would weigh heavily in  on what pH conditions would cause an effective mishaping and release of the presented antigen. (Stole this image from here) 

It's actually helpful to this rant because MHC II (the ones that are way easier to analyze by LCMS because they're bigger) is generally thought to be much more resistant to acid antigen release stuff. Although you'll find people who will contest this one as well.

What was I typing about when I should have been working on a proposal on my desk....oh yeah! This paper! 

So these authors do a very standard crappy antibody bulk pulldown from your typical 800 billion cells or whatever and they look at it on a TIMSTOF Pro. Then they do some comparisons to their acid elution thing which is helpfully contained within a simple little tip that then goes right on the EvoTip (wooo!) to get down to the 50,000 cell and below they do go to a TIMSTOF SCP and run the nanoflow (Whisper) methods on the EvoSep. They stick to DDA with fine tuned polygon IMS/m/z windows for immunopeptides (rather than just no polygon, which some people use for endogenous peptides on the TIMSTOFs). They also compare this to the super small window DIA stuff that they recently developed. That was called something confusingly similar to diaPASEF methods by other groups, but I think they decided to call it Thunder, because if there is anything the proteomics field needs more than confusing new nomenclature is is absolutely and without a doubt the same method from the same group with different names. 

Seriously cool method and I'm absolutely going to be trying it myself this spring. Probably step by step, with the exception that I've got an Ultra2 arriving in 25 days! 

Was your LC column provider acquired? Try a new vendor out, save some $$ and get a free column!

A while back my nanoLC column provider was acquired. There are always growing pains with those things, particularly when production is shifted to a new location or continent. 

I switched to a nice small company with super friendly people - 

and some techbro bought that one too! I've been part of a couple of failed startups. I know a techbro when I run into one. I was so enamored that I immediately got a column bomb and dug out 15 year old notes on how to pack columns. As you might be aware, I have a blind spot in my right eye from accidentally ramming a rusty screwdriver into it during a bathroom remodel in 2020 that still isn't done. Maybe packing columns isn't something I should be doing, but desperate times...

Wait - Premium columns at affordable prices? For real. Check this out.  

I'm impatiently waiting on my first ever Ben owned mass spectrometer. A TIMSTOF Ultra2 with EvoSep. I ain't gonna blow it up with some dude's first column he ever packed. I contacted the owner and - dude's been making nanoLC columns for like 20+ years. He's US based (so...tarriffs shouldn't blow up my budget) and if you order a column they'll send you a second one for free. The columns are also lower priced than anyone else's to start with. 

The two columns I use the most are on that bottom row. These are close matches for the EvoSep recommended part EV1137 and EV1109 which I used to get the equivalent of from the newly acquired company. Stay tuned for a head to head when my stuff arrives in a few weeks! 

Again - Disclaimers over there --> PremierLCMS didn't give me anything for this, except they're sending me a free column (and Kerry is going to help me find an ideal matrix for a mixed mode application we're developing) and they'll send you one too if you move fast 

Multi-plasma plasma proteomics study will make you want to invest in O-link!

 

Look at these nerdy Europeans and their multi-institute studies! This one is on the achilles heel of mass spectrometry based proteomics - non-depleted plasma! 

It looks to me like they prepped plasma and sent it out and everyone could basically run it however they wanted on whatever they wanted - and 

....400 proteins is the high water mark.... now, this isn't enough for me to stop running plasma on mass specs, but it is enough for me to go across the hall later and ask how that O-link thingamabob we have here works! 

Formaldehyde fixed single cells maintain proteomic alterations!???!

 

Leaving this link here so I can find it. There is a point sometime when an ASAP JPR paper appears google searchable and may not make the ASAP page tab on the JPR website. Thanks @cportgrrl.bsky.social for helping me find it! 

This straight up surprised the fuck out of me, btw. I assume when you dump in formaldehyde for any length of time you just stuck all your lysines together and globbed up the nucleotides and left oxidative secondary changes all over the place.

This does not appear to be the case! The authors take some HeLa cells and hit them with a DNA chelator (5-FU does that, I think, don't quote me, haven't used in years) and some other drug and looked at fixed vs. unfixed cells. You'll see some huge numbers in the early figures. 8,000 protein groups. That is from 1,000 cells digested in the top of an EvoTip at a time. 

Protein groups drop a little with the brief formaldehyde treatment, but the differential proteins upon drug treatment look about the same - in some cases better - with formaldehyde rather than no treatment.

Does that mean you should quick formaldehyde your single cells? Maybe not - but maybe that would stabilize them if you can't analyze them immediately? Maybe that helps with shipping? 

MORE IMPORTANTLY! Of the tens of thousands of samples I can access through my super cool new blanket IRB and hospital repositories at my university - about 80% of them are formalin fixed! I'm getting very bad ideas thanks to this really cool paper. I need to reproduce it first before I go spend money and time on said bad ideas, but - hot dog, yo, this could be huge. 

An Agilent ESI source that might work?

 

Since this is a proteomics blog you probably aren't aware that Agilent makes mass spectrometers. While most of my commercial dealings with this company by, unit order, is the 5 qPCR/rtPCR instruments that we piled up on a loading dock during the pandemic that kept showing up functional for somewhere between 0 and 60 days - I have gotten hands on time with both Agilent QQQs and QTOFs in the last couple of years. 

The ESI sources these instruments arrive with are....unique... and interesting..... and quite honestly make the nanoESI sources of the early 2000s seem both mature and intelligently engineered. People I know using Agilent QTOFs leave the sources that come with the instruments in the boxes unopened and build their own.

NewOmics is one of those innovative little companies that finds fun little niches in which to operate. They decided "wait, these instruments don't have ESI sources? Let's sell one!"

There is a webinar coming up about it here, but it sounds like you can see them in person at multiple upcoming meetings. 

https://www.newomics.com/lc-ms-webinars/large-scale-targeted-proteomics-using-agilent-6495d-lctq-with-newomics-ion-source/

How do ApoE isoforms affect protein turnover (for 4,900 proteins) in the mouse brain??

 

I missed this one with the move and never ending sickness 3 (now 4? eeeek!) year olds create when they climb all over each other in "class" and big open gyms. 

Thankfully it came up during the new US HUPO sponsored podcast mini-series "The Undergrad Expo" which highlighted shockingly capable undergraduates (which you might have gotten from context clues) and their research. 

If you want an intro, Noah Earls is the guy who brought it up and his podcast is a cool 30 short listen. My understanding is that there are 3 main isoforms of ApoE and one is good, one is neutral and one is really really bad. This team took mice with each isoform and deuterium labeled them (!!) and did biochemistry at a massive scale. For people who do ApoE research, I suspect the well curated Excel tables in the supplemental are going to be a really useful resource.