Moving fast and maybe just leaving this here for myself to actually read later, but this looks kind of crazy/amazing.
Okay, this might take some time, because!! WOW!! what an ASMS hardware rollout!
If I'm not covering your product, I apologize, its been a long year (and ASMS starts on Halloween!)
There were some seriously big vendor rollouts on the hardware front which got me to thinking ---
1) That there are people out there who didn't realize they were being eccentric with their low signal mediocre resolution data. 😇
2) It was kind of a tie between software and your relationship with your sales and support team. Which...probably doesn't bode well for Thermo since they recently got rid of their subject matter expert sales teams for their products in the US. If you weren't aware, they now sell by geography. So...your local rep who got her/his PhD from the famous proteomics lab? He/she might be selling ICP-OES systems now. Strategery from the company that can't seem to find a way to give up market share in proteomics fast enough to keep their executives happy!
VENDOR LAUNCH 1: BRUKER!
Bruker's launch was tied for the longest (3 days, thank goodness for how much time off we get in the USA) and was centered on the "TIMSTOF
TRUE SINGLE CELL"
The response was extremely warm from the single cell proteomics community.
After "Fake single cell proteomics" was legit trending, Bruker altered the name a little. It turned out to be a translation issue, but the ions are still entering the instrument at the wrong place on the instrument.
In the sadness of this release was some cells spread out on a plate and someone hitting them with a MALDI laser in some sort of a "single cell" approach which I think it's best we destroy the video evidence of and we never consider it again. I wasn't the only person that worried that Bruker was holding his dog hostage. I'd hit cells with a 20 um laser and sign things saying that it was 5 um laser if someone had my dog.
Legit, I think both of these things are totally cool, my job is to be a critic, I think
ASMS Launch 2 and 2.5
The next week was coinciding launches by Waters and the first of 3 days of Thermo!
I've been asked by multiple people "wow, you seem really pumped about this one" and I am from a theoretical standpoint. I'm extremely clear in my "mass spec physics taught by a dumb person" slide deck that you can't get above 70,000 resolution on a Time of Flight instrument. The vacuum on the flight tube just becomes far too long to make any sense at all.
Waters has a MALDI/DESI-TOF that can hit 200,000 resolution against the whole goshdarned mass range. Speed? About 10 scans/second.
1) How much time does that take? I don't feel like doing math, but it's probably about a second. (You can download my calculator here)
2) What about resolution degradation across a mass range? What?
Okay, so there are a bunch of secret rules in mass spectrometry vendors that they must stick to. I can't tell you all of them, but:
1) The compounds that you are provided for calibration must be the stupidest thing they can come up with. A peptide that will only singly charge and will rapidly oxidize? PERFECT! A polymer that will stick in your system until the fucking end of goddamned time and will always affect your sensitivity? BINGO!
2) Vendors can pick any one of these stupid inconvenient molecules and use that for the point where they provide their mass accuracy cutoff. As long as that molecule hurts the end user, it's fair game.
On Orbitrap instruments, the resolution DECREASES as m/z INCREASES. Thermo, therefore specs their instruments on the lowest molecule they can get away with (which, to be fair, they use 200, if they really wanted to cheat, they'd go to 40 m/z where the resolutions is insane!)
On TOF instruments they generally spec on that polymer you'll never ever get out of your system (acetone helps) and they aim for 1222 or maybe 922) and resolution slips (but not to the same level) as m/z decreases.
For real, if you're doing stable isotope labeling (heavy glucose/glutamine incorporation, for example) on an Orbitrap) around 800 m/z you're out of luck. At max resolution (if you aren't running the 1M option) your heavy ATPs are all mixed up because your resolution has fallen off a cliff. You can't tell that heavy N from heavy C. That's a huge deal for metabolism researchers.
So...200,000 resolution is a big deal to anyone in the medium mass molecule range who needs to resolve nitrogen from carbon isotopes. And 10Hz? That's fast. Yes, right now it's just MALDI and DESI, but how does DESI work? The last 3 letters are ESI! I'd be shocked if Waters doesn't have an LCMS super high resolution TOF out soon. I'd volunteer to test it!
Ummm....okay...so due to the preprinting time of a really cool study from some ultra marathoning dude with who has great taste in high pressure turbochargers in the Seattle area, I think a lot of people were expecting somethig really super ultra cool for ASMS.
What did we get? An ultra expensive metabolomics unit and a new FAIMS unit!
(Again, my job is to be a critic. Who hired me, again? That's right! Nobody! Whassssuppp?)
Jokes aside, the FAIMS is super cool.
It can do high flow without you having to hack it to do high flow yourself! And it's higher resolution or something. I don't have an instrument that it will go on right now, so I didn't pay attention. Again, anything that will add more capabilities to a currently existing instrument is awesome and there are tons of Thermo boxes out there with the next generation ion source that can get much cleaner data by tacking one of these awesome things on.
And since this is the Proteomics blog and no one has discovered the hundreds of posts I've made on the Metabolomics blog, I'll move fast on this one. The ID-X (which I love) is a Fusion 1 that has been retarded (not in the insensitive way, in the way tractor trailers slow themselves in the Rockies way) do only do metabolomics, unless you've got 2 minutes and 15 seconds and a basic understanding of how to use a Windows computer to unlock it. The new instrument is based on the Eclipse and I bet it takes longer to release the brakes and make it an Eclipse. There is more intelligent acquisition software for both metabolomics and the small molecule biopharma community. All of which does seem really impressive.
But this is something entirely different. The TIMSTOF is exciting because Bruker figured out how to accumulate ions prior to sending them into a TOF. Bruker has had a TOF for years that can hit QE level proteomic coverage, you just crank up your injections 10-fold. With the TIMS acquisition, you could get speed AND sensitivity. And this is where a ZenoTrap is similar.
The new SCIEX instrument can acquire off the quadrupole (and the quad specs are legit. 0.4Da isolation in high res mode! they do know something about making good quads over there) and the ions can accumulate in the ZenoTrap before being fired into the TOF. Numbers from 4 really good speakers with real data? Looks like 5-20x more signal depending on the molecule.
In addition, there is a collision cell that is two 1 tesla magnets opposed. This (somehow! wtf?) induces a very democratic fragmentation similar to ETD and ECD...with very little decrease in scan speed! Birgit Schilling showed some great PTM fragment spectra to back this up. Think about how slow your ETD or ECD fragmentation is. You've got to inject your reagent and then allow the reaction to proceed, THEN scan your fragments. 50-100 ms reaction time is common. Not including the scan acquisition time, you're at tops 10-20 scans/second. Realistically about 5-10, max. This box can get around 100 EAD fragmentation scans.
I'm out of time so I'm going to just leave this here. I didn't catch the Agilent launch live and I will follow up on that one later, but I think it was small molecule IMS focused. And there were some other launches, but with this out of the way -- HALLOWEEN ASMS IN PHILLY should be just research focused, right?!??
I don't know anything about anything, of course, but the final big ASMS hardware reveal (that I don't know about) is at 11am EST and....umm....you may not want to miss it. Whatever this top secret thing is might be the thing I'm most excited about.
Don't be too grossed out about how much they spent on the video editing. There is real substance behind it.
This stats heavy paper takes a look at serum proteome changes correlated with different negative effects of getting older in a population of men 64 and older.
...how are you tossing these awesome outliers...?
I'm not qualified to really evaluate the downstream interpretation, but it seems solid and thoughtful. The upstream, however, was performed by depletion columns and using one of those giant Agilent time of flight instruments that have the ion mobility cell in the middle and the depth of coverage looks solid.I'm assuming they'll follow up with an analysis of elderly females shortly.
I spend a lot of time these days thinking about how large human cells are and what their relative protein contents are. A great reference, of course, is the original Proteomics Ruler paper.
I'll be honest, it never occurred to me to ask questions like.why is a human cheek cell 10x wider than a human red blood cell? This group did.hate arabidopsis, I recommend just looking at the pretty pictures in this study. They find a really interesting system where a protein called KPR4 maintains a constant concentration from a cell before and after it divides. It does this by some relationship with histones that I don't fully understand. Being arabidopsis poeple, they of course mess with this gene and can back up their hypotheses!
If you haven't seen this paper, you should. This study presents compelling evidence of an entirely new class of molecules that we had zero idea were even around. The ramifications of this are at the level of text-book-altering. Glycosylated short RNAs sticking on the surface of cells doing -- presumably -- super important things. The initial study only identifies a few and they appear to be annotated as:
"Non coding RNA" -- which means, they don't appear to make proteins. Why are they there? No idea. Till now. (Kinda).
I just realized that picture above from the PDF cuts off 5 of the author names. Pedro, Benjamin, Alex, Benson and Karim deserve to have their names shown in the PDF. Get your act together, ElfSeverer. Fixed it.
Without running this out forever, these authors did some really innovative labeling and RNA stuff that I'm sure makes sense to RNA people. And these cool molecules disappear when you treat with something that cleaves RNA or cleaves sugars. They also did some top notch LCMS work. You can find all their RNA data on public repositories, but I can't find the LCMS files. It was obviously something that was secondary to their goals.
It is not, however, secondary to my goals. And I bet that there are a whole bunch of people weird enough to read a proteomics blog that don't know that there are great tools out there for looking at RNA data by LCMS. There are, in fact, even SEARCH ENGINES and a couple of them are in formats that you already know how to use.
A couple of things real fast, though.
1) Nucleotides don't like to ionize in positive mode. Neither do glycans. However at a certain size of a molecule you can pretty much stuff a proton on it somewhere, but the signal might not be great. If you haven't calibrated your instrument in negative mode since installation, you might be operating at a handicap.
2) Nucleotides LOVE to fragment. No joke. Check this out.
What it means is that the tools are in the friendly, open, ultrapowerful, free...
Okay. Great study on a super depressing topic. L-DOPA is, in the short term, often a miracle drug for people suffering from Parkinson's. There are some unbelievable videos that you can find pretty easily of someone having symptoms and then -- boom -- they're gone. But the effects don't last forever.
There is a lot of work in this manuscript, some MALDI was done, as well as the creation of new ways of generating controls(?) on a microscale level so they could even do proteomics in the first place. A QE Plus was used for a lot of the proteomics data analysis along with PEAKS and MaxQuant.
What they find? Convincing evidence that L-DOPA can incorporate into the proteome of a human neuron cell line as well as a whole bunch of proteins that get all distorted in their levels of expression (top figure) due to L-DOPA related stresses.
Just a reminder that there is more stretched out early morning probably alcohol free (no judgment, you do you, yo) ASMS hospitality suite stuff starting soon!
Today should be Waters
I don't know nothin' about nothin' but if you have lecture slides on mass spectrometers, you're gonna want to pay attention to this one. I've removed slides from my mass spec physics lecture because of what they're releasing today. I had a bunch of simple equations that show why what they've got isn't possible on this planet (or at least very energetically unfavorable).
Today is also Thermo's! If you get to page 4 of the sign up and you wonder if you'd accidentally clicked the wrong thing, don't worry, you're in the right place. I think this is tied to clues about their hardware releases because the site is called "La Broots".
I put on my investigative journalism hat (that ASMS never lets me wear. No press pass for me. Maybe this year! We'll see!) this morning and discovered --
Wait. Where is the Hospitality Suites week 1 wrapup? There should be one somewhere.
This is a continuation of last week's post on single cell sample prep. I meant to put a link to this paper in and just couldn't find it.
NanoPots are of course super smart and have been in use for years now and the CellenOne can prep a ton of individual cells on single microscope sized slides!
In the preprint from Karl Mechtler's team, they find around a 30% loss in signal from using the 384 well plates. Doesn't seem to stop this group, though, these results (Exploris 480 + FAIMS + ultra low flow nanoLC 3 hour gradients) because these results are fantastic!
They had to synthesize this themselves and it clearly has some limitations but now that someone solved a strategy for it, I bet other smart synthesizers can expand on it.
The tag neutral losses completely leaving behind DIA MS/MS spectra that don't change. (DIA spectra is already complicated, we don't want to multiply the number of fragments by 3 or 10).
I'm not 100% clear on the differences between Bio-ID and APEX (except that the latter is faster) but they're both proximity based labeling techniques. I'm not really clear on them because in order to perform the technique you have to mutate or fuse a protein in your cell. Or...in this case....192 of them. Then under the right conditions your fused protein will biotinylate everything that is close to it. And just those things that are close to it. Violin! (Viola?) You pull down the biotinylated proteins and now you know all the things in the proximity of your protein of interest.
If you don't know how to fuse or mutate a protein you need to use another technique (or find a smart gene editing collaborator).
This group appears to have some people who can do it. And maybe 192 mutants made in a human cell line isn't that big of a deal. I'm going with...it probably is as hard as I think, or it wouldn't have went in that Nature thing.
After the mutations are made you need to do at least 192 experiments (one for each fusion protein) and they did this using an Orbitrap Elite operating in high/low mode using 60 minute gradients. Minor bit of interest for you serious mass spec nerds (weirdos.) is that the instrument didn't leverage ppm level dynamic exclusion widths, the instrument excluded everything within 0.6 Da. On something as simple as a Bio-ID experiment and a gradient of 60 minutes, I doubt that this has any real effect on the data, where in a global experiment it would cause you to completely exclude some coisolating ions of similar m/z.
The downstream analysis was performed using Mascot and Comet through the Trans Proteomic Pipeline. Results from that were sent through SAINT.
What did they get for all that work?
Check it out for yourself at www.humancellmap.org