Friday, December 2, 2022

The incongruity (not incredulity) of "validating" mass spectrometry data with western blots!

 

I know some authors who just earned a citation in every paper that I clumsily write for whatever is left of my "career"! 

This is short. It is well-written. And they didn't mess around with references. 

They put one of the most important studies in our field's history as #1.



Finally -- the ZenoSWATH paper!

 


ZenoSWATH is an upgrade for the SCIEX 7600 that rolled out at ASMS 2022, and it looks like somebody got it enabled and took it for a spin!


The ZenoTrapping/ZenoPulsing thing is a neat trick inline after the quad and prior to the TOF. Rather than just running the ions into the accelerator thingy, they can accumulate (pulse?) prior to firing them into the TOF. My understanding is that it doesn't do it with full scan, just MS2, so you get the data looks sort of weird where your MS1 signal doesn't really go up, but your MS2s do. If you are doing data dependent you can probably imagine some drawbacks here, right? Maybe you don't have enough sample intensity to trigger what you want to, etc., but we're still finding it really useful for DDA. Where is absolutely shines is the PRM (mrmHR) functionality. 

However -- if you're DIAing/SWATH....buckling...?

Who cares about your MS1s anyway? (I mean, I do like to have MS1s, but they are clearly less important here).  

You get to a point where SWATH can't see anything, but ZenoSWATH keeps on going. I'm less impressed with the high end numbers (yo, just load more peptide) but the low levels are impressive. The copy number distributions shift decidedly in the correct direction. 

When you look at the numbers in this paper, keep in mind this isn't nanoflow! This is microflow (5uL/min) AND analytical flow (2.1mm column!!!!!!) 

Seriously cool trick and another big step in the right direction for a competitive landscape in LCMS proteomics technology. 

Thursday, December 1, 2022

Open up the plasma proteome with ultracentrifugation of EVs!

 

Every single time, plasma proteomics is disappointing. Haven't done it in a while? Have an instrument that is fast, more sensitive, and just better in every single way than the last one you tried it with? Fire it up .....

(drumroll)

....the same 400 proteins you were able to quantify in 2012 on that instrument you can get on Ebay for a pack of Big Red and paying for freight....

You can deplete your top 3, 10, 14, 31.23 most abundant proteins and see more, but now you're introducing a whole new can of worms (which is a weird phrase. who has a can of worms? gross). 

What if you could just centrifuge the heck out of the plasma and concentrate the EVs? Extracellular vesicles are something that has been leveraged before (there is some really cool work from Geiger lab rings a bell Promise-Quan?) but how does it compare on today's fast DIA methods?  

That's what this group went after in this fun new study! 


And it seems to work really really well. 1,400 proteins using DIA in relatively short gradients and these aren't just random proteins they directly correspond to what we currently know of the human plasma proteome. 

Wednesday, November 30, 2022

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!  

Tuesday, November 29, 2022

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! 



Monday, November 28, 2022

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! 

Sunday, November 27, 2022

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! 

Saturday, November 26, 2022

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. 

Friday, November 25, 2022

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! 

Thursday, November 24, 2022

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.

Wednesday, November 23, 2022

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! 

Monday, November 21, 2022

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

Sunday, November 20, 2022

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. 

Saturday, November 19, 2022

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?