Reversing stem cell state differentiation!

Warning: This is a stem cell paper. In order to read it you must get a spare sheet of paper on which to write all the acronyms and tape it to the side of your monitor. It's like reading an LCMS proteomics paper if you haven't been running your own system for 3 or more years. 

I don't get all the biology, of course, but I kind of get the gist of it. Primary cells in culture get old and you can't use them anymore. This group has a way to push the cells that they're interested in back into doing what they were originally doing before they got old. Super cool, right?!? I can draw all sorts of ignorant implications from this and I'll keep them to myself.

Okay, while it's cool to be able to do this, the next obvious question is: what is the responsible element(s)? Can you hunt it down and put it in a vial? 

Using a combination of RNASeq and proteomics (Q Exactive HF) they come up with a list of potential candidates. The RAW and processed files are up on ProteomeXchange at this identifier: PXD021069

WHAT? Single LIVE cell NATIVE intact protein mass spec??

I'm super impressed. All the pieces to do work like this already exist but you have to pull techniques from all over today's best analytical techniques to put it together. This team did and...wow...

The single cell metabolomics people have live cell probes where they use a microscope and run a tiny object into the cell, pull out the cytoplasm and direct infuse it. It looks amazingly difficult to do right, but I know a lot of work is going into making it easier. The last couple talks I sat in on made it seem like something I probably couldn't do successfully rather than something there is no way in the world I could do successfully. I mean, you've got to get a cell and then stab it. 

How do you even find it? 

Super fancy microscopy or something. 

Not to put down the metabolomics people but small molecules are pretty easy to electrospray. You go to nanospray and you've got plenty of signal to work off of.

You know what isn't easy to ionize with electrospray? NATIVE INTACT PROTEINS. For native I like about 10 more protein than with reduced proteins. 

The fact that this group can resolve native hemoglobin from a single red blood cell is just nuts. Yes, there isn't much in an RBC but hemoglobin, but those cells are tiny. 

For the LCMS work you need to go to the supplemental where you really see how much optimization went into making this work. The instrument used was a Waters Synapt G2 HDMS. 

FAIMS Pro for Top Down Proteomics!

Okay, so it obviously isn't revolutionary or anything to think that FAIMS is good for cleaning up your top down background and boosting your signal. Where do you start, though?

Right here! 

Okay, so for those of us who might not be able to upgrade to the newest instruments, I would like to direct you toward an extremely interesting submission at PRIDE from some Thibault guy that I've heard knows something about FAIMS.

The submission is called "Integration of FAIMS Pro on a Q Exactive mass spectrometer" and you should search the files, they're impressive. 

An amazing new automated platform for single cell proteomics!

Hmmm....I wonder what Mechtler Lab is up to these days with 3 single cell papers in as many months, and a single cell proteomics conference coming up right around the corner in Vienna? 

This new preprint is ridiculous in a number of ways. First of all, what is even happening with the sample prep above? I pulled the links to the hardware they're using and it looks to me like this thing is doing the sorting, and they go right into a stream for digestion labeling AND injection to LC? 

The LCMS is an Exploris 480 with FAIMS and surprisingly they appear to use a trap column, which -- in my hands -- has been disastrous for my signal, but I pulled some files from ProteomeXchange and these files are intimidatingly good. 

E4 fragment ions?!? Geez....

Okay, so this is hands down the best SCoPE-MS data that I've seen on a D20 Orbitrap. 

There are no tricks here, they compare NORMAL SIZED HUMAN CELLS, (shoutout to anyone out there using 7 foot long giraffe neurons for single cell.) 

This sample prep method should be investigated! 

INTERNAL fragment assessment in top-down proteomics!

 I'll be honest. I don't fully understand this cool new study. What I do understand is that when I fragment an intact protein I get fragment ions from the N and C terminus(es) and only in the very best cases do those fragments extend to the middle. 

What they've worked on here is alleviating this by optimizing parameters and considering that the fragment ions might not be working all the way from each terminus. They use a special Mascot version (TD?) but explain how you can process this data in a similar way using other tools with a link to an older study (direct link to that study here). 

It makes a lot of sense to me, we're hitting these intact proteins with a lot of energy and why wouldn't we get internal fragments? Thinking we're exclusively seeing b and y ions is convenient, but we see secondary fragmentation in some large peptides and definitely in peptides with PTMs, so why not proteins? All the work is performed with a Bruker FTICR so they use some pretty amazing mass tolerances to demonstrate that they can actually observe fragment matches. 

Assess "missingness" in DIA data with OptiMissP!

Missing values?!? Isn't avoiding those the whole reason we even do DIA? I thought there weren't any?!?

Yeah, there still are, particularly in really complex matrices. If you want to assess the levels of it, you can manually search or you can use -- OptiMissP! 

Don't feel like reading? Me either! (Just kidding, it's a pretty cool paper.) 

You can get all the code at github here. 

Or you can just mess around with their great Shiny app here! 

A nice preprint that breaks down the Exploris'es

This was not what I was looking for at all, but it is interesting! 

What's an Exploris 420, 680 and/or 102? What makes them different? 

This preprint from June has all the schematics and some data showing what the different models can do. 

Donkey milk proteomics!

Did you think about donkey milk today?  Me neither! And I wouldn't have guessed it was useful. Turns out that it is: 

1) Good for children with allergies to cow milk

2) Has a long history of being utilized for various medicinal purposes.

The first seems less fantastical than the other, right? 

This group went out of their way to study the donkey milk proteome and reveals an interesting story. The start off by doing intact proteomics, and double back to fill in the blanks with LysC and Tryptic digestions. 

1) They explore both the proteins that could explain why people who are allergic to cow's milk may not have the allergy to donkey's milk

2) The antimicrobial properties? They totally confirm it. You can even leave donkey milk out for days longer than cow's milk. Antimicrobial super powers? Sure looks like it and this group points at what might be causing it. 

Altogether it's a really nice study with some cool biology explained by some really good mass spectrometry. 

Were you dying to know about the VAMPIRE BAT serum proteome?!?

You know, prior to this recent little thing that happened with a bat virus  I might have cast this paper in a different and less serious light. However, it seems like we should probably know everything we can possible know about anything. 

Therefore, I present a no-nonsense blog post about this great new study. 

The study focuses on vampire bats in Belize and samples come in as part of a longitudinal study. Rabies is involved and so is livestock due to agricultural alterations of bat habitats. Yikes. See. Totally serious stuff. 

All LCMS is performed on an Orbitrap Fusion 2 "Lumos" using DIA. As you might imagine the vampire bat FASTAs may not be as well developed and annotated as some other organisms, so the informatics section...

...better just skip that part. 

Do you want to know if they found high quality MS/MS matches to peptides that match the middle eastern coronavirus? Or should I ...lay this one to rest... already? 

Predict peptides with PROSIT and MSPiP for proteogenomics!

Wow. Okay, of course this is one way to do it! Actually, it seems blindingly obvious to me right this second, but I certainly hadn't thought to do it the way they did in this new study!  

There are several smart ways to get around proteogenomic challenges that we're increasingly being encouraged to think about. It's a lot easier if we can just use the UniProt SwissProt little tiny library of protein sequences, but they ignore:

mutations,

individual biological variation,

the fact that some of those sequences might have been Craig Venter's and if you were picking a normal human being to pull sequences from, would he really be the best pick? 

What this group does? They pull out their little nanopore gizmos and they do some sequencing. Instead of feeding their Variant Call File (vcf) or whatever is the same thing in Nanopore world into a search engine, they feed those sequences to deep learning peptide fragment and creation tools. 

BOOM! Modeled MS/MS spectra that they can match against theoretical! Smart, right?