Single bacterium proteomics - round 2 - label free!

 

Whew...what a month..... if only the highest numerical % of your grant was the one that got you funded, I'd be looking at catching my breath and starting a deep dive into some amazingly cool single cells for a couple of years. It is, however, the lowest number that gets funded, which is both seemingly weird (totally weird....nerds....) and it's funny to joke about it and not funny to be a little sad.

While I was doing ALL THE THINGS the world kept moving and I kept mostly meeting my daily reading goal, so I'll back print some things like -

SINGLE BACTERIUM PROTEOMICS - ROUND 2 - LABEL FREE??? Yikes. That's crazy.

I can't remember, but I think Akos's group got 12 good solid E.coli proteins. 

IMP-Vienna got 50 without TMT!   That's crazy. It's so so so little protein. I'm really impressed that it all didn't end up permanently trapped to the plastic of the 384 well plates they used. Super cool to see what we could do if we really really wanted to make a statement. 

DESI - ALL THE THINGS!

 

This is amazing. Not only did this group use a commercial DESI equipped instrument, they found an Orbi XL somewhere and put a DESI on it, AND they put one on an Exploris 240! 

Deeper is not always better in plasma proteomics!

 

So...this came up with some incredible scientists I met at the University of North Carolina this week...

And here is a really cool review/perspective on the same issues. 

UNC's core is getting WAY higher plasma proteome coverage than I ever have with their amazing robots and magic nanoparticle things. But when they do quantitative comparisons and have rigorous restrictions on their quantitative accuracy, the numbers drop.

Is it as bad as an aptamer? Of course not. Nothing is as bad at measuring the abundance of a protein as an aptamer. Might as well flip a coin ;) 

But this is a smart look at different proteomics technologies for plasma enrichment that...wait....did they only give 5 stars to the one they developed...? Hmmm.... I mean...I'm not going to make fun of the stuff I developed either.... hmmm.... okay, but they make some incredible points about a whole lot of this stuff.

AND - BTW - when you're drawing blood where does the stuff go that you stabbed a needle through? Does the needle just perfectly part it's way through skin and blood vessels? It must, right? There's not just a big chunk of human skin floating around in there, right? 

GlycoDiveR - Actually make sense of glycoproteomics data?

 

We were JUST talking about this in lab meeting last week! I swear.

I said something like "well...sure...we can generate loads of good glycoproteomics data (I've got a tattoo that is almost old enough to drive that shows I've successfully pulled it off at least once on some pretty crappy instrumentation)....but you can't actually interpret what that big pile of glycopeptide stuff means....

And....well...there went that argument! 

Deep Visual Proteomics of Pain!

 

Wow.... I do just have to leave this here and move on. I've already forwarded the paper to a bunch of people, though, and can't wait to spend more time on it. 

We need to figure out how much DDM you can use before it's a bad thing, though! This group used 6-8x more than what we use, and they get a lot more membrane proteins.....

Totally worth taking a look at! 

DIA-NN 2.5! Now with 70% more ....70%?? ...more peptides!?!?

 

Okay, we have to take a look at this for real. I do like the color scheme on these plots, though...

As an aside, I ran a commercial program for some people recently and it gave me 20% more protein groups than the ones I currently use. Those extra 20% really annoyed my collaborators. They were ...like... biologically very very unlikely...? Not DIA-NN, a commercial thing, but I did re-learn a lesson that more peptides isn't always a better. But DIA-NN has built enough credibility for me to be hesitantly optimistic that I will like this new version. 

Get it where you get DIA-NN! Probably here https://github.com/vdemichev/diann

Troubleshoot your EvoSep step by step with this cool online thing!

 

For the first time in a long time, I had to do some EvoSep troubleshooting. Turns out that ceramic needle thing can get clogged! 

Gabriel at EvoSep led me to this super useful online resource that walks you through step by step to get it all worked out. 

It's amazingly clear with pictures and "did it work? click here!" AND 4 MILLION PERCENT BETTER than letting Adobe's class trailing LLM help you dig through the user manual. If you see a button to turn that pile of poo off, please let me know where that is! 

MR-SP2 - Super affordable high recovery low input spatial proteomics!

 

Yeah! I love this new method at JPR! 

There used to be LCM(s) (laser capture microdissection thingies) everywhere! No joke, they almost died out to the point that one of the leading companies was briefly for sale at the price of a Baltimore/DC suburb house. At ABRF I looked at two very nice new ones and individual systems were in the very nice Pittsburgh house sort of price range. 

MR-SP2 takes one of the legacy systems that you can't buy new anymore and optimizes it up for spatial proteomics with all the details you'd need to set it up yourself.

At 1-2 cell cuts they gett over 600 protein groups on a TIMSTOF Flex system equipped with an EvoSep. I had this exact same setup for ore than 2 years and I think my record is less than 400 proteins on a cancer cell. That might have been Whisper (100nL/min) 40SPD or maybe even 20SPD. 

AND they get this out of FFPE tissue! Incredible approachable spatial proteomics without buying all brand new stuff. 

Proteomics Show 102 - Dr. Jan Mulder and BRAINS

 

Lazy post day! Reviewer comments (....some a little past the due date...sorry....) on ....I shouldn't type how many papers....it'll make me a little stressed out...... on my desktop.....

I am legitimately loving recording this new season. Thank you US HUPO and these amazing guests we have lined up. Did you know a brain...just....unfolds....? I did not know this. With the podcast actually now racking up more listens than this blog, I should probably advertise this on the other thing.... But this took 6 minutes and most of that was trying to decide on a gif. 

Why do immunopeptidomics anyway? And what comes after?

 

Do you wonder why we don't just do immunopeptidomics by genomics technologies? Besides the obvious fact that it's impossible? Or just wonder what happens after you've spent a really long time working on the crappiest peptides you've ever tried to fragment?  

Then this is the review for you (and you)!