Next week is the huge Brazilian Proteomics Meeting! Virtual appears to still be open!

 

It's OCTOBER?!? WTaF? 

So....Imma write a talk at some point today....cause this is the 12th Brazilian National Proteomics Meeting and it's huge! 

Day 1 opens with Jenny van Eyk! 

Day 2 opens with that Kuster guy! Not the Bernard I named an amazingly inspirational little dog with extremely sharp teeth after, but always worth listening to. (Unreleated) 

Ben Garcia is also speaking! Coincidentally, I think he's on THE Proteomics Show podcast that released today. US names I can remember off the top of my head are Gundry and THE Sue Weintraub and Alexey N. It's a big meeting.

If you're tired of hearing all the US and European people ramble about their stuff, why this meeting is cool is that it also prominently features the scientists doing cutting edge research in Brazil. Super cool stuff is on the table like Flavia Winck and microalgae work and Wagner Fontes purifying neotrophils for single cell type proteomics. The coolest part of any conference for me is the student presentations anyway and this one has sets of cool lightning talks to highlight the people in the labs actually doing the work. I'm super pumped for this one....and....should find some slides! 

Can you explain a proteomics concept in a VIDEO? Wanna win some money??

 

As I know I've bragged before, I'm somehow the chair of the super cool US HUPO VMO committee. Our quest. Nay, our destiny, is to make Proteomics less scary for the rest of the world.

We're going to cast off this persona of the mean scary mass spec wizard in the cave who just shouts things about ion suppression and "I said NANOgram NOT MILLigram" at unsuspecting biologists and doctors.

NEXT STAGE? 

Let's TOKTIK this ship, fellow kids! 

GOAL - explain something in proteomics in an approachable and entertaining format on video! 

Some weirdo already sent in a video of him trying to cover an old Mudvayne song by substituting in something about how transcript abundance and protein activity have no linear link. Fortunately, said weirdo, encrypted his IP so we have no idea who it was....because it was terrible. 

Since it's our only entry so far, it might win. I challenge you to diem that carp and put in your entry. 

You can register and upload your videos here! 

Is subcellular spatial imaging possible by shaking up the laser and prep?!?

Laser capture microdissection (LCM) is having a bigtime revival right now! But before you go checking surplus warehouses and Ebay for these things you should keep in mind that they are typically

(I have this perspective thanks to Ryan Kelly providing a crash course in LCM while I was on his beautiful campus in Provo) 

It would be really cool if one of the big innovators in sample hardware would take a whack at this, right?  I mean, it's sort of dumb that we have these absurdly fast mass specs but we can only cut like 2 samples per hour or something, right? 

Time to Ying Zhu LCM! 

It would make a catchy verb.... My robot is slow? Ying Zhu it! I have too many peptides binding to things! Ying Zhu it! 

What was I talking about while I was Ying Zhuing this bread machine? 

What about LCM at 30 (THIRTY!) CUTS PER HOUR! At 2 micron cuts??? Honestly, I can't follow this paper, but that's a whole lot faster and at higher resolution than anything I've ever heard of. The laser is swapped up and the transfer to NanoPots is faster. And get this - from these tiny cuts they're scoring 2k proteins.... sort of makes me want to stop this MALDI grant I'm working on, but I've got way too much inertia for that.

There are alternatives to PEEK Scientific for N2 generators, and they have to be better!

A common conversation at every conference I've been to the last year or two has been something like 

"what happened to PEEK Scientific?" They used to be so good! 

And I would have agreed. At this point the only conclusion is that Space Karen himself, Elmo Musc must have purchased the company. 

However - generating N2 is actually sort of easy, so you don't have to drop huge amounts of money on service plans for people who will literally never come on site no matter what. There are other alternatives! 

Check out LabTek! 

Pros? They aren't PEKE Scientific! 

Did you know VICI makes N2 generators? They sure do! 

Pros? They aren't PEECK Scientific! 

NIGEN will make you an N2 generator that you can hang out in if you want! Why would you hang out in your N2 generator? I don't know! 

I DO KNOW I'd rather hang out inside of my N2 generator than wait for PEACK Scientific to ignore every email for 19 months about my once new N2 generator that has - since install - had a warning light on and made a weird internal hissing sound. 

And PARKER is the big competitor, I think. I had a single PARKER once that could provide N2 for like 4 mass specs. 

Parker's website is klunky, but you can buy their generators from Restek and they're legit. 

And I'd rather hang out on a klunky website for Parker than try and convince PEACKE SCIENTIFIC that the serial number IN MY F'ING SERVICE CONTRACT THAT THEY WROTE is the correct S/N. And that after 11 months they should put a m'fer on site.

Disclaimers: This post has nothing to do with an actual N2 generator company that used to be good but now fucking sucks. The names used in this post are clearly different. 

ONE-TIP Preprint! Do your entire sample digestion protocol on your EvoTip!

You've probably seen talks that this was coming. Here it is! Wait. What month is it? I've been busy. Not the newest news, but still worth talking about! 

Don't get distracted by the Astral - that's not what I want to talk about here. What I want to talk about is how if you prime an EvoTip and then leave it sitting in warm water you can just put your lysis buffer and trypsin in the tip and load your cells. A mere hour later -- you can complete your EvoTip peptide loading and wash steps and run your tip! 

The numbers are awesome. Part of that is the Astral we aren't talking about, obviously. But if you're getting 8,000 proteins with a direct injection on anything your digestion worked pretty darned well.

Hell yes we're trying it right now. 

Now, if you want to do proteomics with me I pretty much require that you use S-Traps. The darned things work every time and people who have never done a prep in their life can buy the complete kit from ProtiFi and it almost always works. I LOVE that consistency, but a commercial S-Trap in bulk will set you back about $3/sample and one of those complete kits are closer to $20/sample. You throw in $7/sample for trypsin and then your $2 for each EvoTip and your sample prep isn't cheap. 

If you can load the EvoTip alone, you'll obviously only get one shot per tip, so replicates will pile up to make the S-Trap a much better bargain, but there are places where doing everything on one tip will be a huge cost and time savings! 

An (up to date) guide to MALDI imaging for tissues!

If you haven't heard me whine about it, I had a messed up bacterial infection in MY EYES because toddlers are little disease vectors and while my 2 year old recoved in 2 days, I required multiple different antibiotics to get to finally go back to work. F'ING FUN. I'm catching up on almost everything. Only one late peer review and four of our papers revisions to get out before Halloween. 

We get a lot of collaboration offers from people who want to use our awesome TIMSTOF Flex for the imaging things it does and I'm always trying to find ways to deter people from it. There are reasons for MALDI imaging. There really are, but most things are way easier if we cut the tissue, extract the metabolites an then measure them with HPLC. But the pictures....

(I do not know who this is, I just thought this was a funny gif. If this is a terrible person I should know and not post, please let me know) 

The MALDI pictures are super cool, but they can take like 24 hours to analyze like a cross section of a single mouse brain with decent resolution. When extracts from 6 mouse brain regions from 4 mice ran by LCMS can take way less time. When you really truly need that spatial profiling it's great, but most of the time you can do more with something else. 

What I need is a great modern tutorial to hand out to people! I'm sure a decent fraction of people will say "...this m'fer expects ME TO READ...?" and they'll go away, but that's secondary. You get better collaborators when they know what you're doing! 

And that is what this blog post is sort of about? This cool guide! 

Mango proteomics/glycoproteomics study shows we don't need genomics people anymore!

I liked this study so much that I wrote a commentary for this weird Proteomics journal, but I think they must have hated it because it's been sitting as "Accepted" for like 2 months. I might have been super jetlagged in Europe and forcing myself to stay up all day and mildly hallucinating while reading Tolkien and it might show in the commentary. 

So maybe what I'll type here about this great study will be more coherent. 

We've been looking at some less well annotated organisms recently and I feel like the genomics people are really sort of mailing it in right now. They're sequencing all the things, and as long as they deposit their half aligned crap on GigaDB and they get a BUSCO score of greater than 80% (this means they are able to find the majority of genes that we know need to be there for something to be alive, you know like the ribosomes and TCA cycle enzymes, that stuff) they can deposit crap.

Why would I think it's crap? Well...if you predict open reading frames and more than half of them for a eukaryote don't start with a methionine, chances are you found the wrong calling in your career. 

The reason I really liked this new study is that the mango genome is crap. And this team was like - we don't even care. Our proteomics is so good that we're not only going to identify proteins that change as mangos ripen, we're also going to do the glycoproteomics -- ON A CRAPPY FASTA? 

And it works. And it's really good. For mass spec nerds you might be more interested in the comparison of TMT-SPS-MS3 and LFQ based quan for underannotated organisms. Both datasets are really good and overlap well. The glyco is awesome, though. And they really tweak things to dig deep into the N-glycans. 

vDIA on the Exploris! Repost?

 I thought this was here, but I couldn't find it with a quick search! 

https://www.protocols.io/view/protocol-for-data-independent-acquisition-mass-spe-kxygxzrokv8j/v1

VENNY VENN HAS Moved!

NEW LINK FOR VENNY VENN IS HERE! 

EDIT - 11/4/2023. Thank you @cinderbio.bsky.social!!!!!

If you aren't familiar, the VENNY website by Juan Carlos Oliveros (2007) was the first time I realized that the internet was for more than just pirating music and seeing who could have the largest poo. That was before you could even really access papers online. Most journals were still - go to the library and photocopy that, you slacker. 

Venny allowed you to  - instantly - filter and compare lists of ANYTHING in a web application and it has stood up to this day as something just super important for the internet to uphold.

AND. IT. IS. DOWN.  AND IT HAS MOVED TO HERE! 

Yes, you can generate a Venn diagram in R or with the super cool PNNL tools but Venny is a scientific cultural icon.

If anyone knows Juan Carlos, can you put me in contact with him? It's a common name. I'll pay the hosting fees or whatever to get it back up.

Systematic discovery of neoepitope–HLA pairs!

 

Holy. shit. I think Genentech just drew a line in the sand and said that amateur hour is coming to a close for HLA neo-antigen discovery....

The amount of work in this is crazy. And the level of validation is extraordinary. Switching the top figure for the post to a protein I know pretty well. Not Megan Rigby well, but I acknowledge her in a lot of my papers for very good reasons. 

We know without any doubt at all that KRAS peptides makes it to the HLA expression system. Bert Vogelstein has 5 very good papers on this and has a brand new aggressive spinout company full of badasses who are working on exploiting this.

Rose's team exploited this in their validation by constructing cell lines where they can induce mutant KRAS expression! Those bar charts above show the specificity of their their toolkit! They can induce the system and measure the correct antigen production with absolute(?) quantification. 

This is important because...? If you've got one copy of a neo-antigen on the surface of a cell, that isn't enough to use to target it for destruction through CAR-T or whatever system. There is a threshold - I think of as almost the immune system false discovery rate. 1 copy doesn't trigger DESTROY THIS CELL. But 50 copies almost certainly does. 

I started with the validation that this method works. What you want to read it for is - that they used the validated system to identify a crapload of novel neoantigens. 

Seriously seriously cool work. 

If you have a comment on Thermo buying O-link - you can't share it on LinkedIn!

 

THERMO BOUGHT O-Link - WTaF (actual fourier)??

 

So......in today's news.....antibody based quan is officially "next generation" proteomics technology. What is discarded is new again. 

On the up-side I bet we finally get to see some data pitting something we know is quantitatively accurate against O-link soon, if it works! If it doesn't, I bet we'll be able to tell from...well...if we don't see the data....

I'm not allowed to own stock in privately held biotechnology companies, but I sure wouldn't have thought O-link was gonna DogeCoin today.

If you are new to this blog, sorry, this is really a venting ground for a sarcastic first gen scientist with filter issues. 

Yes, O-link is an antibody based technology, and those are very very very well known to be error prone, poorly quantitative and to have immensely frustrating off-target effects and batch to batch variation.

O-link has a smart double hit QC thing that reduces false discovery rates. And the antibodies have a nucleotide sticking off of them. You amplify the antibody by PCR for the targeting stuff or use next gen sequencing (cause it's faster) for the other stuff. 

Still an antibody based assay that relies on the successful reproducible creation of something billions of years of evolution has imbued with the ability to be ...not...reproducible....but in a smarter/better way that the exact same sort of technology (this stuff has been around for 20 years) would have been done.

I'm DYING TO SEE SOME REAL HEAD TO HEAD DATA but that doesn't mean this stuff doesn't work. And I sure as hell would take their double hit antibody technology over any single hit protein microarray of the past.  

Data please. Data please. Data please. Data please. 

We have a champion for the Proteomics Battle Royale! Webinar was recorded and will be posted.

Huge thank you Dr. Ling Hao, Dr. Devin Shweppe and BATTLE ROYALE ROUND 2 WINNER - 

DIA/ Which was well-championed by Dr. Lindsay Pino.

We didn't decide the winner, the almost 200 people logged in did! 

The Battle Royale was organized by the US HUPO VMO committee, in particular Dr. Amanda Smythers. Also thank you to Ben Neely, Jennifer Jones and Lydia Bradshaw of bigger US HUPO.  I helped by making the slides and biasing the questions to once I really wanted to know the answer for. 

Reminder, this video will be posted where you get US HUPO educational content. And...this totally counts. https://www.ushupo.org/Webinars

It is BATTLE ROYALE WEEK!!! Wednesday 1pm EDT!

BATTTTLLLLE ROOOOOOOOOOOYYYYYYYYYYYAAAAAAAAALLLLLLLLLE TIME! 

Register here!

THIS WEDNESDAY! 

Register here!

What is it? Oh - it's where we 3 competitors (ABOVE) get faced with different real life scenarios in proteomics and we see which one comes out on top. Can TMT do it? Can DIA do it better? Would label free DDA be the bestest choice? 

Interactive judging by YOU? You betcha! (I think...we haven't tested it)

Plus lasers? No.  Disco ball? Sure, I guess!  Hosted by a 70s gameshow host? No. Wait? Why would you think that? That doesn't even make sense. 

WHO WILL COME OUT ON TOP - LAST TIME WE DIDN'T LET TMT EVEN PLAY THE GAME. We were probably scared it would win every time? Or lose? I don't know. 

Update your reference library! The ProteoChip paper is out!

 

I keep getting flagged for having preprints in my reference libraries when the papers have come out.  You'd think the citation managers would have enough money to update those on their own but noooooooooooo. I have to manually delete them and then update them myself. BOOOOOOOOOO.

And this is a preprint I cite a lot. This is your reminder (and mine) to fix that reference! 

SAGE - Search your data 10x faster!

 

What if you could get data as good as MSFragger but AN ORDER OF MAGNITUDE FASTER? That's what SAGE promises. Funny story, I think there used to be something called a SAGE-N or something but since that company sort of went away before I think the author of SAGE was born, he clearly isn't responsible for keeping track of such things. 

You can read about SAGE here!

Okay, I know, there are all sorts of cool new tools informaticians come up with all the time, so why should YOU personally care about another one? You probably need to know how to write in PERL to use it or something, right? 

Bulk make a custom .FASTA file from anything (tutorial for me!) then deep learn it!

I swear I thought this was on here somewhere and then I had to go Googling all over the place.

Let's quickly get a FASTA (and then deep learn it for any program at all)

First get a protein list of any kind. UniProt, RefSeq, Gene Identifier, whatever and go here: 

https://www.uniprot.org/id-mapping

Easy! This is 3 KRAS isoforms (though the 3rd one I think has an A or B in it after the 3, so it might be wrong. Going off memory. 

Make sure that "From database" is correct, particularly if you're using a Gene because UniGene and Official Gene are different (I forget what they're really called). 

Keep the "to database" to UniProtKB - unless you need something weird at UniProt - this will get you to what you need

There is a "MAP IDs" button in the bottom. Give it a minute or two. Then click the "Completed" link that pops up. 

Go to Download and toggle off the "compressed" button. It's an annoying Linux compressed format. .gz or something indecipherable. 

BOOM -- FASTA! 

Wanna deep learn it? HAHA! The buttons in Prosit are more complex these days, but this 3 year old tutorial has the rest of it. 

After today's generation of spectral libraries exercise I was reminded that about 9/10 proteomic studies that are funded are for generating new and exciting spectral library formats! 

Thank you to all the study sections who immediately move a proteomics study to the front of the queue when someone proposes yet another way to display and store the same thing. There is nothing we need more today than more spectral library formats. 

Clean up SpectroNaut false discoveries from sample transfer with these simple tricks!

SpectroNaut is a bigtime player in the present/future of proteomics for very good reasons. It's really easy to hit some buttons, wait a really really long time and then get data. You can then hit some buttons and then get to some proof that what it told you was there is actually there. You don't actually have to read anything.

But -- is the data coming out of it always correct? Could you really fine tune it if you knew what you were doing? 

Of course you could! So this group took some yeast(?) and some mouse digests and mixed them together and didn't mix them together and cleverly adjusted parameters until they cleaned up false discoveries caused by sample to sample transfer. (This isn't "match between runs", I asked, they don't use that, but it does take hits identified in one run and look more cleverly at other runs. Is that what match between runs does? Yes, but that's not how they do it.) 

End result? You have to read stuff about how to use SpectroNaut - but you can dramatically improve the number of false positives you have in your data, preserve your reputation, and feel smarter for having read something so practical today. 

SpectroScape -- Real time spectral query and visualization in proteomics!

The outside world (outside of proteomics) is generally a lot better at computer things than we are. We're a tiny little industry compared to things like social media where inserting an ad for a heated toilet seat in-between someone's wedding photos can simultaneously trigger revulsion and $100 million in bathroom product sales. 

A great strategy for a tiny innovative industry okay with borrowing ideas is to - borrow everything we possibly can! And SpectoScape is a superb example! 

SpectroScape borrows a central FaceBook algorithm called FAISS which is: 

"This is made possible by an indexing scheme based on the inverted file and product quantization encoding (IVF-PQ) algorithm in the Facebook AI Similarity Search (FAISS)22 library that groups spectra in neighborhoods in high-dimensional space, defined by approximate spectral similarity. Given any query spectrum that the user supplies, the method efficiently retrieves all its approximate nearest neighbors in the entire repository and performs real-time spectrum clustering by computing accurate pairwise distances among the query and the neighbors to reveal any cluster(s) in the neighborhood. The user can then visualize the result in an interactive web-based user interface. " 

Right?!?!? (This is a link to reference 22) 

The end result being that you can real time hunt down a spectra matching one that you're interested in  -- OR - one that is SIMILAR TO the one you've got across something as big as MASSIVE.

The authors have a lot of big plans for something that allows data analysis/clustering/visualization at this kind of scale, but the first thing that pops out to me is the whole dark proteome thing and how valuable this can be.

Imagine if we flipped proteomics sideways and did it the way we do Metabolomics most of the time. What we do there is basically say "this ion comes off my column at 2.3 minutes and has this mass and fragment pattern and it is important under these conditions. No fucking idea what it is, but it is clearly involved" Then you go and try to figure out what that thing is. I accidentally agreed to do another natural product discovery study because one I was sure there was no chance in hell would work totally ended up being one of the biggest papers I've ever been on. That's how you do that as well. Here is this peak, wtf is it? I commonly wonder if we're just being dense doing proteomics the opposite way. 

This is something I think could truly be enabled by this truly awesome new resource. They envision the almost instantaneous generation of new spectral libraries and the integration of new data into our comprehensive knowledge of the proteomes of life on this planet and that's also pretty cool.

FDA Omics Days Start today (October 10 and 11, 2023!)

 

Are you interested in whatever it is FDA does in -omics? 

Are you interested in showing off what you do to the FDA -omics people? 

Are you interested in seeing what people want to show off to the FDA -omics people that they do? 

You're probably too late for the second one, but the other 2 are still a possibility, because FDA -Omics Days start today! 

You can register/sign in here. 

HINT HINT - FDA might have sort of figured out that transcriptomics is not where it's at. They have to protect people from bad molecules and bad proteins, and they are spending a lot less time and money worrying about theoretical things that might be there (because that's basically what the transcripts are -- things that might theoretically lead to the creation of something that does stuff). 

Cut from Day 2 schedule here. 

The Effects of Cell Culture Conditions on the Proteome and Transcriptome!

 If you've seen me speak anywhere recently, you've probably heard more f-bombs when talking about single cell proteomics than maybe if you heard me last year. Our field is going great, but there are a lot of things on the basic level that complicate even the most basic type of research. 

Here is an example - 

This is a really cool resource where a vendor has compiled 7 cell lines derived from patients with the same disease and these cells were selected because the mutations driving the original cancer are different. 

Super cool, right? Particularly if you are using a TOF for multiplexing and your maximum number of samples you can plex (after carrier and 2 blanks) is 7. 

Imagine that you are sort of dumb and you think that "wow! what a great thing, I can do a proteomics (and/or single cell proteomics) study to investigate what effects the mutational status of each of the cell lines has on the proteome! 

Maybe you'll spend $4k on this kit and go to start culturing these cells to find that EVERY ONE OF THEM REQUIRES A DIFFERENT GROWTH MEDIA????

3 shown as an example. One needs something called "McCoy" + 10% baby cow blood extract and the next one needs RPMI-1640 and the same amount of baby cow blood and the third one needs RPMI + 15% cow serum, but also insulin. 

For someone who basically learned cell culture from YouTube and asking some friends and students really dumb questions, what do you do next? 

More importantly, how do you tell that the changes you're seeing are due to the genetics of your cell lines versus what are just the fact you put insulin in one cell line? 

What things would you worry that would change? 

Guess what - this guy did an entire PhD thesis on exactly this question and it's a gold mine! 

iSCMS Workshop - Learn to do single cell proteomics (and other things!)

 

I had the best idea ever - I did not stay out for a really fun punk rock band in Provo and, instead, went straight to bed, got up hella early, packed and crashed the iSCMS (hands on single cell proteomics workshops for a couple hours before driving the very highest legal speeds to the airport to get on my plane just in time. 

Imagine that you have read a bunch of papers on single cell proteomics but you still didn't get the magic behind it. iSCMS arranged rotating groups of demos where you could see these processes or even get hands on with things....like LEVCELL! 

Nikolai Slavov got there when I did so he got stuck in my group, and here he is loading droplets into the LevCell. 

You might have to zoom to see them but this is after our group got done loading ours. How sick is that. The sonic fields just sort of ...feel...right...?...as you're putting the droplets into the correct space. Gao lab is very close to having all the stuff together to release these plans to the community! 

There were also Tecan UNO demos that were very thorough (top panel). More on this soon, I figure since we'll be running cells on ours this week, and CellenOne sent THE Joshua Cantlon himself for live demonstrations on the mighty CellenOne system. 

You know the double trap elute system the Kelly lab developed for ultralow flow on the regular Dionex nano systems? Woooooweeeeee! 

It's even more intimidating than in the diagrams in the paper! But it makes sense when you see it and the diagrams side by side. They're working on making it more easy for others to adopt. Stay tuned, I think. 

On the LC topic -- and this was a big point of Vici's talk -- if we're going ultra low flow, we might need to start thinking hard about whether the relatively huge 1/16th fittings make sense. All of the HPLCs used for single cell that I saw had this much much smaller plumbing systems. There were hands on demos on how to swap those out and how to work with these smaller fittings! 

On the instrument side, we know Kelly lab/BYU is vendor agnostic. I saw a 480, Lumos, the big high end Agilent and I finally got hands on with an Ultra and have a better idea of what makes it special. 

Bonus tour point - I've never actually seend a laser capture microdissection thing before and Ryan gave us a personal tour of his equipment! 

There were other stops on this tour with great Q/A, cool people to interact with, lunch and then afternoon sessions, but - again - I had to get to SLC at very legal speeds to get home. 

Again, just stellar stuff. I took a ton of notes and I swear sometimes you just have to see the things up close or ask 10 stupid questions (that's probably just me) to finally understand just get it. Super cool stuff and I sincerely hope that there is something like this at iSCMS 2024!