Saturday, May 1, 2021
Friday, April 30, 2021
I will lead with the fact that this appears to be another study where Dr. Makarov was in his lab having some fun with his somewhat famous invention, rather than a product we'll see soon.
There is, however, some precedence here with something that looked fun later becoming a reality. The closest example obviously being when he pushed an LTQ Orbitrap to one million resolution using a hand picked Orbitrap and some painful manual tuning, and now you can up and purchase a Fusion set up in that way, so you never know!
In this study the team pops a hand picked Orbitrap into an Exploris 480 and does a lot of fine tuning through the internal development package. It doesn't look or sound like fun to me at all, but they get over 2 million freaking resolution and show what you can do with that much resolving power. This is a short and fun read that I highly recommend.
Thursday, April 29, 2021
Now for something completely different....
Non mass-spec based proteomics is coming, or is here already. It's not going to be perfect, but LCMS proteomics isn't either.
Magnus discusses two such technologies and presents some surprising results. A high number of canonical human proteins can be identified with knowing the correct sequence of just 3 amino acids. Of course, the canonical human proteins are only part of the story but a lot of LCMS proteomics only considers those sequences, making it a fair comparison.
Wednesday, April 28, 2021
The effects of my over-exposure to Arabidopsis from my years at an agriculture focused universities might finally be fading a little. Or this great study from last fall beautifully demonstrates something I've aspired to pull off myself and never have this well, and if you can pull it off in this awful plant, you could apply this to interesting things!
If you don't want to read the preprint from the figure at the top, here is the hook.
There are a lot of studies out there that have brought together proteomics and transcriptomics. Most of them have required a bioinformagician on the team to pull it together using a series of tools that I am probably not smart enough to install.
TMT-NEAT, however, is a perfectly packages Shiny web application that you point at your MaxQuant output files and it pulls your data together.
To this day, virtually no tools out there autonormalize your phosphopeptide abundance to your protein abundance. That's why this lazy tool I made gets downloaded from the blog so often. TMT-Neat does this directly from the MaxQuant output files and does it the right way, not the lazy way!
Honestly, I'm guessing we haven't seen this paper in print yet because it's going to be in a big journal soon and I don't even think that what I'm impressed with is why it will publish so well. The majority of the paper is focused on what they actually found, which seems like kind of a big deal. I hope the reviewers at least appreciate the informatics.
Tuesday, April 27, 2021
Well...this post wrote itself!
Shoutout to Dr. Robitaille for the heads up, and for the better joke.
Monday, April 26, 2021
When I met with some applications scientists from this interesting little company, I told them that every time they referred to their instruments by the term "Iso" it made me involuntarily shudder.
Last year I was fortunate enough to lead a very small team of very smart people through a full multi-stage ISO17025 and then government audit process. There are exactly two ways to get multiple government bodies to bless Q Exactive assays.
#1: Put this flowchart on your wall and live by it:
#2) Put this exact same flowchart on your wall and use dry erase markers rather than more ...permanent... markers. (Why do I have so many pastel markers?) And live by it.
Name aside -- what's an ISOPlexis or ISOSpark? That link will take you to their very pretty website. They use thicker border lines on shapes than even I do.
It's possible proof that I should shut up and listen more, because it is a surprisingly powerful platform for looking at proteins in single cells without needing to master a 2,000 pound instrument.
What's it do?
It uses little flow microchips to sort a small number of cells (you typically start with 30,000) by basically their normal propensity to migrate and flow around into little microchannels. An imager looks through and discards empty wells or wells where more than 1 cell ends up. It's pretty efficient with something like 800-1200 wells ending up with one cell in them. Blank? There is your background subtraction. More than 1 cell? Ignore.
When the cells are stuck they're lysed (if the workflow is for what is inside the cell) and fluorescent antibody things are used to quantify the protein signal.
What's the catch? Well, it's antibodies, so you've got their variability to consider. And you're obviously limited by the number of antibodies, dyes, fluorescent thingies, that you can use at once.
However, they have panels that can quantify up to 32 proteins per cell! If your cells distributed well, 32 proteins per cell across maybe 1,000 cells? That could be very useful validation data. You can also work with them to build your own targets if they don't have a good panel for you.
The instrument is less expensive than a mass spec (unless you're talking Advion Expression) but the chips aren't free, but they're WAY less expensive than doing 1,000 western blots, particularly if I have to do them (...which probably means 75,000 attempts....)
ISOPlexis also realizes that we're out there (the ones with the half-ton machines) and they seem interested in bringing us all together. Last week they sponsored a really cool proteomics webinar with GenomeWeb where some great people from our side talked about where we are with our single cell -omics plans. I didn't find out about it until it was going on, but it's on demand here! This link might be glitchy because it's linked to my name, but I think it will get you there.
Sunday, April 25, 2021
We occasionally still hear (here? come one, brain) about Stable Isotopic Labeling (with) Amino (acids in cell) Culture (SILAC). Which is probably still considered by many to be the gold standard in proteomics quantification.
There is still a ton you can do with SILAC, but when you first start planning an experiment one of your first impressions will probably be
Saturday, April 24, 2021
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.
Friday, April 23, 2021
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.
Thursday, April 22, 2021
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?
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.
Wednesday, April 21, 2021
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?
Tuesday, April 20, 2021
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.
Monday, April 19, 2021
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.)
Or you can just mess around with their great Shiny app here!
Tuesday, April 13, 2021
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.