Decompress ratio compression with this one simple trick!

 

I'm trying to wrap up a tutorial to support the development of SCoPE-MS type devices on "non-traditional" mass analyzers, but --wow-- is that deadline coming up fast. I'm getting this worked out, I guess because the TIMSTOF SCoPE-MS study finally published (...whew...) and I'll be talking about SCoPE-MS on another QTOF in quiet rural Quintana Roo next week.

One thing that we've got to think about is the dreaded zero value, or the Interference Free Index! I'm using the methods described by Paulo et al., and those are awesome. Though...worth noting, you used to be able to upload any LCMS data into their toolkit, but now it has to be from one specific vendor. I had to build my own. That will be in the tutorial if I ever finish it. 

The goal of the IFI is to understand how much background you're acquiring and when you've got a ground truth like "this yeast DOES NOT EXPRESS" this protein, that's a darned nice zero point.

As you might expect, MS3 based approaches for reporter ion quan outperform anything MS2 I've seen, though really tuning in your chromatography and high resolution ion mobility can have a dramatic effect. 

But -- if you understand your zero point ground truth fully (aka, zero looks like this number) and you have some values that you absolutely know the quantification on, can you do something with that? 

Could you, for example, build a causal model? And use that to readjust your quantification? 

Wooooo.....that's some grown up math......and probably not for me. I'd rather just draw an arbitrary line, let's go with...I dunno....2-fold and then just send that out to people!

These nerds, however, decided to see what they could do with the grown up math.

They took knowns standards and modeled it out (using FAIMS + MS2 based quan), then built out that statistical model to differentiate their known values.

Guess what -- it looks AWESOME, and it almost completely decompresses the dreaded reporter ion quan ratio compression.

This isn't the first attempt at something like this, but it may very well be the simplest. Could I reproduce this? Not today, I got up at 3am and I'm still super behind. I skipped breakfast and lunch so I could type this. But this group has a bad habit of making their tools freely and publicly available, so maybe I don't even have to try. I guess we'll see!  

Did someone do the obvious SOMASCAN vs OLink vs LCMS experiment!??!

 

I can't read this fully yet, my gosh my calendar is at "time to move to Costa Rica" crisis levels. I will find time because it is: 

FINALLY TIME! 

O-link vs SOMASCAN vs LCMS proteomics. In Cerebral Spinal Fluid! 

Meta-fold absolutely any protein in this super easy web interface!

Shoutout to @MatthewCollins@mastodon.social for his post on this crazy awseome thing! 

Do you want to just 3D model any protein you feel like? It has never been easier than this thing that is powered by Meta! 

Not only can you look up your specific protein, you can essentially blast a FASTA sequence against their database and then 3D model that.

Check out what happens when I model an essential DNA damage protein I can't seem to get very good coverage of and I don't know why (possibly PTMs everywhere, and maybe hyperphosphorylated, but I don't know yet). 

Now, we can go to SWISS-Model and pull a couple of Cryo-EM based ones! 

It looks bad at first, but if you ignore the first 51 amino acids you can see it actually did a really good job. The three main Rotinis line up reasonably well, though there is a difference in whether they are linked by Macaroni or Mafaldine folds. The cryoEM that I pulled had a short ligand in it that appears to interact with the wobbly Trofie terminus predicted by Meta which may be what stabilized it into the Tagliatelle structure.

YOU KNOW YOU WANT TO MODEL THINGS IN IT! GET GOING! 

Mapping viral infection with imaging proteomics!

 

I'm no expert in imaging mass spectrometry (yet. I hope to get there since I've got two!) but I've been suprised/shocked by some of the limitations in the technology in 2022. This group of experts talk about these limitations in a refreshingly frank manner... 

...and how they try to work around them. 

This study is largely evidence that their current procedure of working exclusively with MS1 medium resolution TOF can differentiate viral peptides in a known model. I won't lie, it sounds like a pain in the butt with overlaying slices and multiple data types, but those images look awesome! 

How to move to Mastodon for #TeamMassSpec and other science social media!

(Original image, believe it or not). 

So...Twitter is not getting better. It is doing the opposite of doing better....

I GET SO MUCH OF MY CONTENT FROM TWITTER! Where to next??

Introducing Mastodon Social. Nope, I didn't know about it either! 

What if something was sort of like Twitter but was decentralized so that no one could buy it yet and there weren't ads? That's what it's like.

It isn't, however, as easy as Twitter, but if you're here you are a scientist or really really strange. In either case you are probably smart enough to sort it out! 

I, however, needed a tutorial. Or three. 

#TeamMassSpec to the rescue! 

Where I started was this Github which has some great tips from some dude as he was getting set up, and it largely draws from a crazy awesome resource that Cris Lapthorn set up.

If you're on the Twitter, here is a link to a Tweet from Cris. 

Check out how cool this is: if you go to this survey that Cris set up and enter your details after you're set up on Mastodon it will add you to this big #TeamMassSpec google sheet, which you can download as a CSV file. 

Then when you get into your Mastodon account you can go to your personal profile and upload that CSV (you do have to delete the first line of that CSV file).

VIOLIN! You now have a whole bunch of cool people that you're linked to through the social media stuff so now you can see what they think is important. As I'm typing this now it looks like you can download the info from like 75 people there. 

You do have to pick a server. I went with genomic.social because that sounded fancy, but it isn't a big deal which one you set up under. You can swap it later. 

Need more ionization signal? DROP THE BASS!

 

I owe Faith Robison and Valerie Gabelica for bringing this one to my attention. 

Wooohooo! And reuse of these images are allowed with proper credits! Check out how cool this is. 

The analyzer in question is a Shimadzu 8030 and all you have to do is wire in a subwoofer to the source and drop the sickest track you can think of -- and magically improve ionization! Absolutely makes sense, right? 

Okay -- that isn't actually true...what they do is directly control the woofer to maintain constant frequencies. They tuned these in first off the instrument and performed image analysis to determine optimum settings, before actually setting it up on the instrument. The number of iterations they go through are pretty impressive as they tune this in. 

Y'all know that something like 1% of the ion current ends up doing anything helpful, right? It's a serious problem that is more fun to ignore.

Okay, so is this just a weird trick that makes sense? We can aim, alter and focus ion plumes with sound? Who cares? 

They tune it in for specific molecules to boost their signal! Including a couple of peptides I'd figure you'd never get to ionize efficiently in a half million years. One particularly crazy looking one that is all HPFpeptide repeats gets over 1,000x signal increase, suggesting that maybe they have a specific application in mind for all this work?

ABRF Video series on Cloud Computing is now live on Youtube!

 

Gotta move fast I'm hoping to get in and out of my lab today without being killed by someone on an adrenaline rush from the $14 they saved on a television by camping overnight in a Target parking lot. Sleep deprived Black Friday Shoppers driving in the rain, what a great morning to see that instrument signal to dropped precipitously! 

Wanna learn something about Cloud Computing? Betcha wouldn't have guessed ABRF was the place to turn for it! Click on the image above or here to check it out! 

Don't bother entering the US HUPO 2023 T-shirt competition by December 2nd!

If you are unfortunate enough to have been at this blog over the years, you might know that I've mysteriously somehow struck out on winning the US HUPO T-shirt design competition. How is this possible for someone who has personally created every one of the absolutely permanent tattoos on his body, completely by himself? Every single time, the person who takes what I've drawn and permanently embeds it into my skin has said something like "....are you sure about this...?"  Obviously because they've wondered if they have a level of mastery of their craft necessary to follow the lines of what I've created with sharp rapidly moving needles to place them in my skin forever. 

While there is still officially a chance that you could enter a design by December 2nd by clicking on this link and uploading it, you might want to save it for next year when you see my entry for this year. 

Intimidated? 

You should be. 

It has it all. Did the greatest rebounder of all time work out that protein structure while 34 hours into a quick 4 hour trip to Vegas during the playoffs? Or did deepmind AlphaFold 2? (Shoutout to Robbin Bouwmeester because his protein looks even wackier than my favorite protein which I can absolutely guarantee with no question whatsover has not been predicted anywhere near accurately. Robbin's seems....maybe a little unlikely as well...

Again, who knows. Maybe the secret judges of this competition are visually impaired or have no taste at all, and you'd have a shot.

Revealing the Mucinome!

 

This one has been on my desktop since the summer and keeps slipping through the cracks to talk about.

The study is open access so I'm going to zoom in and highlight something that is strongly implied by the figure abstract image at the top.

LOOK AT HOW GLYCOSYLATED THESE F'ING THINGS ARE! Why would anyone even consider working on the mucinome? 

Unfortunately, these things are linked to all sorts of diseases -- like --

and what this team has pulled together is an enrichment method using a slightly modified protein (single aa substitution, I think) that allows them to selectively enrich these awful things. 

Through some serious work they selectively enrich and analyze these proteins through both the identification of the unmodified regions and an O-PAIR (MetaMorpheus) analysis of the glycopeptides. They exclusively use HCD and demonstrate some new tools that can work biological data out of these awful modified proteins! 

How to set up FAIMS PRMs (and PREDICT the right CVs)!

 

The figures in this new ACS study are really pretty and demonstrate how to and why FAIMS can improve PRMs! 

Now -- all you need to do is run a bunch of multi-FAIMS CV DDA methods and pull that data out of your results!  

It sure would be cooler if there was a way to predict the right CV for your peptide, right? 

You can with this R package! 

Reddit.com/r/proteomics forum is up to 944 members!

 

With the whole Twitter implosion thing in high gear and me stuck on a bunch of paper proofs rather than getting my Mastodon socials set up, it is cool to see that the subreddits for our community are really starting to grow.  r/massspectrometry forum is at almost 5,000 members as of this morning. 

More importantly, I'm super pumped to see that r/proteomics is continuing to grow. 944 this morning!! 

Blog rules state that this video is a mandatory insertion. 

For real, though, even with a relatively small community we've seen some increasingly sophisticated questions posted here and get answers. However -- no joke -- don't be afraid to post questions that maybe aren't as sophisticated. Proteomics has been moving really fast, too fast for books and tutorials to really keep up. It is fun to see a question from someone just getting started in the field get immediately answered with links to relevant and current resources. 

TMT-labeled peptidomics of plant stress suggests we might be overcomplicating some of our workflows!

 

It took a little while to wrap my head around this method, but in the end what you have is a really simple way to quantitatively study endogenous peptides. While this was used to study salt stress in plants, I don't see why it couldn't be applied to just about anything. 

Salt stress in plants is a critical area of study right now with the climate changing and water availability becoming increasingly questionable just about everywhere. So definitely pay attention to that.

What I'm selfishly interested in is how this team extracted proteins (froze and ground the plant tissues with a mortar/pestle, then TCA precipitated to clean up to amino acid containing things. Once they resolubilized the proteins they used that to normalize their abundances (x mg of protein per sample). To get the endogenous peptides they simply used a 10kDa filter.

That's it. Then they cleaned them up, TMT labeled and ran everything the normal way. The TMT on the terminus helps add some mass and a partial charge to the peptides so they both fragment better and fit within our normal proteomics type methods if they're small.

My first thoughts were things like "what about small proteins or proteins that are degraded by the sampler harvest, lysis, extraction". And I'm sure you absolutely see those things as well, but if you control the conditions right, those are probably all 1:1s. What they find under salt stress is a population of peptides that are differentially regulated and they backtrack them to make a nice story.

If you often think "man, this peptidomics workflow seems way way too complicated" maybe it is and maybe it is worth thinking about this one? 

THE PROTEOMICS SHOW is now live, wherever you get podcasts!

 

Have you ever thought "wow, I sure wish I could listen to Dr. Neely and a dude who sounds like a Muppet interview some super interesting proteomics people on my commute?"

I sure as hell have and Dr. Neely and I made a pact at US HUPO 2021 that if no one else was gonna do it, it was obviously our responsibility to make this happen. Fast forward like 8 months ---

The Proteomics Show Series "The Road to Chicago" series episode #1 is now live. 

The goal of this podcast series is to let you in on the awesome talks that are happening in sunny Chicago in March and why you want to join us at US HUPO 2023! 

Don't know how to do podcasts? Me neither. But, honest to dog, if you go to your Macintosh phone, there is a Podcast App on it!. And you type in "the proteomics show" or even just "proteomics show" or maybe "just proteomics" and -- BOOOOM.

Don't like the phones with the weird bitten apple on the back? Too bad. Psyche. A few minutes ago episode #1 went live on Amazon and Spotify (Google is being slow).

Spotify link!

Amazon link! 

This couldn't have happened without the people we try hard to credit in every upcoming episode:

Johannes W. Bagnoli who made the totally sick death metal intro. If you think you've heard something cooler than this dude screaming "PROOOOOOOOOOOOTEOOOOOOOOMICS" over a killer guitar riff, something is wrong with one of us. 

Huge shoutout to Kaylie Kirkwood (@KaylieKirkwood) for making this super cool and professional artwork. 

AND to US HUPO, especially the VMO group for helping us find guests and letting us do this and for sponsoring the costs, which are primarily a professional to edit whatever Dr. Neely is rambling about into some semblance of reason. 

AND ESPECIALLY OF COURSE our guests who have taken time out of their lives to tell us cool stuff while we clearly have no idea what we're doing. 

A bunch are recorded. Several are edited. And we'll keep posting these. 

The Chalf (C-half) paper is out! Protein structural stability at proteome scale by LCMS!

 

This is on the list of papers I've been looking forward to reading since USHUPO 2021! 

For some reason, people actually sometimes care about more than the linear protein sequence. They care about what it looks like before you break all the bonds and cover it in SDS and cut it into little pieces. I did this study section thing I can't tell you about, mostly because the sleep deprivation it required left me unable to form long term memories. Based on what I can read of my notes I'm sorry to say NMR and baseball still appear to be a things that people do or go watch or however that works and the flashiness of CryoEM is definitely able to distract people from really nice LCMS proteomics work.

Chalf (See-Half) is part of a pipeline that looks really complicated at first, but is really elegant and suprisingly simple to set up. 

The best part is that you don't have to do 1 purified protein at a time (I bet those 6-10 histidines at the end don't actually alter those folding or melting dynamics anyway..... ) With this, you can do ALL the proteins. Want proof?  This team did HUMAN PLASMA. 

Sounds impossible, right? 

You remember guanidine? For some reason it was a component of some soft drinks in the 1990s, and the rumor was that it was derived from bat poo. One of the reasons that the 90s was the greatest decade in the history of mankind was that our population density had just gotten to a point where urban legends were passed around at their peak level and access to ways to check whether there actually was bat poo in that can of soda were very limited. Maybe that information was on the message boards somewhere? Who knows?  

Guanidine resurfaced in mass spectrometry for a while as a way to linearize proteins! Focus on that because I think the soft drink might have been something different. GuanOdine? I don't know and I'm not going to look it up. 

Cool part. Iodine apparently can't get in to react with amino acids if they're inside a 3D protein structure. It only works right if that amino acid is exposed to the liquid surface interface thingamabob. 

As you add increasing amounts of guanidine proteins unfold to expose amino acids to iodine reaction as a direct function of their structural stability. RIGHT?!?!?!

Okay, but how would you process that data? Now you've got iodines on everything? It is actually more complicated than that, unfortunately. The free Iodine doesn't just bind to amino acids. Well, it does for some, adding two I to tyrosine and histidine, but it does other weird stuff to other amino acids. 

The modified amino acid masses are all here in the paper, and they processed the data with PEAKs for both label free and TMT labeled samples (quan!). You don't have to do the whole structural stability caclulations yourself (thank you and whoever did the experiment that discovered you get extra energy from bat poo!) because you can get the software that does that here. 

And that is actually what C-half or Chalf is. The workflow is called -I-P-S-A-, but that is the exact acronym for something else I use just about every day that I can only find by searching my blog for the link to it and I didn't want to make it difficult for me to find. 

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: An Amazing Tutorial

 

Since you've got some HPLCs in your lab connected to your mass spectrometers, you run the risk of people thinking that you have some idea how to do chromatography. Depending on how good your job security is you can either be honest "I actually don't know anything about chromatography, but MY DOG, I hate every one of those things" or you can pretend you actuall know what you're doing. 

What if there was a 3rd way? Could you learn about chromatography? Does anyone out there know enough about chromatography to make a tutorial dumded down to the level mass spectrometrists might understand it? 

Hell yeah there is! 

Well....I got to the point where there were prefixes and suffixes in the same formula and figured that was far enough to write a blogpost on it.

However, someone in this group clearly understands chromatography and they tried really hard to get this information into something that we can reference. As they note repeatedly, to get the maximum level of performance out of these instruments you absolutely need to get great chromatography and this is THE tutorial that can help you get from meh to amazing. 

Contactless (lossless??) proteomics sample prep by levitation!

If me embedding it above didn't work, this is the link for the video. 

And this is the link to the details! 

I've said this before and I'll keep saying it -- Single cell proteomics might be most important right now because it is our field's moonshot. 

We're getting 1) attention 2) a desperately needed influx of new informatics for dealing with high n samples and 3) innovation from all fronts that are questioning how we do everything.

It doesn't matter how you are prepping ultralow level samples right now. You are getting losses. Everyone is trying hard to minimize them, but peptides stick to things. Picoliter robotics like the CellenOne preps and NanoPots and nPoP etc., are all reducing them the best they can, but you are still touching things and peptides stick to those things. 

What if you could prep your samples without touching anything at all?!?!? 

This group isn't touching things. They levitate their samples -- including single cells -- in space with acoustic fields and lyse, digest, and transfer them without physical contact. 

What are the improvements versus basically exactly how we prep our single cells here? It looks like a 30% reduction in loss at the peptide and protein level. I guess the inverse is more clear. 30% more peptides!!

You can read a short preprint about this ridiculously cool technique here. Did I already extend a collaboration offer? Hell yes I did. I know exacty what I would do with 30% more peptide signal!!

An infinite resource of human phospho(serine) peptides!

 

Big shoutout to the recently official Dr. Eric Mosher for sending this over to me this morning!

Phosphopeptide standards are:

1) Expensive

2) Hard to freaking find

3) Something with a finite expiration date.

What if you could have an infinite (regenerable?) source of human phosphoserine peptides? 

Might get this part wrong. 

What they did was design some big ol' E coli plasmids (?) that, once inserted into this unfortunate little organism, forces it to produce human phosphopeptides on 6xHisTags! 

Check this out, though, this is where it is weird. This system, called pSERots or something, actually forces the insertion of phosphorylated serine as an amino acid. You don't have to put in a serine then phosphorylate it. It comes inserted in the protein. Now you know EXACTLY where that phosphorylation site is. 

Need more phospopeptide standards? Grow more of that Ecoli. Extract the proteins and digest them -- phosphopeptides. 

Rough cost estimates are that the process will generate around 600 micrograms of human phosphoserine standards for about $175 when they submitted the paper in February. Given inflation in the US caused entirely by the fact we don't tax the rich or control, in any way, the profits corporations are allowed to make, that is now $430. But either $0.02 per phosphopeptide or $0.05 per phosphopeptide is sure a lot less expensive than absolutely anything else!

There are other gems in this paper, like a new AScorePro algorithm for HCD and a cool website for helping to localize phosphosites!

How to use Science Social Media without ruining your career workshop!

 

I FREQUENTLY consider how lucky I am that people just had film based cameras and NO social media when I was a young adult. Whew. 

Social media enabled science can be VERY POWERFUL and fraught with perils! Learn from Dr. David Tabb and Dr. Ann van der Jeugd, two intelligent people who have successfully used social medai to benefit the world.

I think I'll do the scared straight thing. 

YOU WANNA GET TURNED DOWN FOR TENURE TRACK BY EVERYONE? 

YOU WANNA WORK 80 HOURS A WEEK FOR LESS THAN YOU MADE AS A TECH IN THE SAME PLACE I5 YEARS AGO?!? 

Sign up for the HUPO ECR SCICOMM -- Who, how, where, and why (DON'T MEET THE BILLIONAIRES AND THEN TELL EVERYONE HOW BAD THEY SMELL ON THEIR OWN PLATFORMS) workshop! 

Phil Wilmarth and my favorite meeting in the world is only 1 month away!

 

One of my favorite meetings ever is in the beautiful Christmas city of Bremen!  Since we're taking a 3 foot tall one-year old to a pleasant quiet rural place in Mexico for something super boring that month already, it is not a possibility for me.

However, since Phil loves the funnest to say proteomics data processing software packages, you can join him there! 

Why go? Well, that's where all the cool people making their own nodes hang out! 

My advice -- you should register and you should NOT eat a blood sausage. Yes. Super metal sounding. Acquired Taste. 

Optimized diaPASEF for phosphoproteomics!

 

This is great! Does anyone have one of these for ADP-ribosylations? I have yet to see one in our data, and we can't tell if the TIMS is too rough on the mod (in-source decay?) or if they move funny in ion mobility space. 

This group claims 35,000 phosphorylation sites in 44 minutes! Out of DIA data? 

They did this by creating project specific phosphoproteomics data with DDA processed through MSFragger (!!!) and then they used this program (pyDIAid) to create their method (!!!)

The Molecular Human!

 

I suspect that we're about to see the floodgates open wide now that SomaScan and O-link have gotten some level of acceptance for their ability to do huge throughput studies and here is a taste of what we can expect! 

A moderately sized study that was obviously really expensive and designed by a committee with some really great intentions and some solid bioinformatics visualization people! 

What did they do? Well, they somehow got 300 or so participant samples of 3 types (blood, plasma, and urine) and they did all the -omics! Then they built this Shiny app!

Before you do anything else, I STRONGLY suggest you make a network and then click on the center thing in that network and drag it around. 

I wish I had time to make a .gif of it! Here I've clicked on the star and then just hold your mouse clicker thing and drag it around the screen really fast. I don't know how they did, that and I know someone to ask.  It is really really fun and, unless I'm missing something big in this preprint, supplemental data or clicking around in the app, it might be all that they did with this patient cohort.

I swear, every one of these next gen proteomics papers that I've really put any time in makes me feel like almost 30 years of headbanging finally did catch up to me the way my Mom said it would. I get to the end and I can't figure out why they did it, what they were trying to accomplish and how it could possibly benefit anyone or anything in any possible way. 

Again. Probably just mushed my brain, but I did enjoy this cool Shiny web effect and I'm using it later for sure! 

Localize all the PTMs with a little python script you can drop anywhere!

 

Not every software goes above and beyond in localizing a PTM. And, I tell you what, some of these pesky biologists seem to think knowing where that PTM is critically important information.

Of course, we have things like AScore (well...some people...do...) and ptmRS for some pipelines.

Why would we need another? 

I can easily email you pyAscore. Hell, I can mail you a floppy disk if you really need it and that's how you roll. Even after you unzip the program it is kB size! 

Drop this little bit of code in any pipeline you want. It is compatible with everything and now you can get that pesky biologist off your back without manually annotating the spectra they care about (maybe at least look at it, particularly if they are really pesky and S-174 turns the thing they care about on and T-177 turns the stupid thing off. 

Brainprot portal! Where are the phosphorylations (and happy ducks) in the human brain?

Imma fall asleep at this keyboard for real, so I'm going to drop this link to the paper above. 

Check it out! 

 

El Fragmentador -- Neural network fragment prediction in the easiest interface ever!

 

We're all using deep learned machines for artificially inflating our intelligence in some way or another right now, but a lot of those thingamabobs are kind of convoluted.

Have you ever wished that you could just type in just one peptide sequence into a handy little box for a quick additional confirmation that what you are looking at is what you think it is? I sure have! 

Check out the ultra nifty El Fragmentador here! 

I made a brief visual tutorial here. 

Did it just predict the fragmentation of a phosphopeptide for me? 

Heck yeah it did! 

Did it do a good job? 

There is plenty of documentation on this awesome Github! 

What I've found by trial and error is that you have to use all capital letters for your modification, and you want to get the square brackets and not whatever not square brackets are called. And it will do at least phosphorylations and lysine acetylations. I chatted briefly with the author [Sebastian Paez (@jspaezp1)] and he's looking for community feedback to improve it, so check it out! 

JINX! Another way to run a TIMSTOF! Introducing Synchro-PASEF!

 

(Blazenly stolen without permission, thank you in advance @NotValdemaras)

Are you ready to RuuuUuuuUUuuuuuumble.......?????

Synchro-PASEF! 

OMG! What a great day for one of my TIMSTOFs to be working again!

Okay, so maybe we've got two smarter ways to run these things. Now all we have to do is get rid of a nanospray source that requires that you carefully slide a $400 gold ring down a long fragile glass tube and then secure it with a tiny hex screw! 

IS THIS IT? IS THIS THE SOLUTION?

I think they're going to send me one to try out!