Wednesday, June 12, 2024

Astronaut multiomics week with 44 new papers and open access data everywhere!

 


Does it seem like space and astronaut data is all over the place right now? It is! 

44 new papers just dropped and many are proteomics, transcriptomics, metabolomics/lipidomics of astronauts (both human and otherwise). 

You can check it out (and get access to the data if you want to investigate it yourself - this isn't Space Karen stuff, this is NASA stuff, it's open!)

Sunday, June 9, 2024

Spaceflight changed the skeletal muscle proteomes of 2 astronauts!

 


A common theme in a lot of serious science fiction is often how life from our planet will need to adapt to the challenges of low or zero gravity. Makes sense, right? Astronauts spend a lot of time recovering after time up on the ISS.

Want to actually understand what is changing?!? Of course you do! 

It is a short read and it has some really optimistic statements, like how exercise can help mediate some of the biggest changes in mitochondria.

This is the journals front page today, btw, which gives some insight into how you actually exercise on the ISS? 


I'd never before heard of this journal before scholar alerted me that someone I follow published something new. Digging into this finds that it's not the first entry. There have been multiple proteomics studies in this journal because - I mean....it's not like being in space for 6 months is altering your DNA a whole lot. Those changes are clearly proteomic! 

Another recent paper looked at similar things to this one, but did it in space mice! 


One reason this multi-omics paper is super cool is that it turns out NASA has a whole data repository of data from organisms that have gone to space. This group took phenotypic data with transcriptomic, proteomic and DNA methylation to better understand muscle loss in mice that spent 30 days in space! 


As an aside, a few years ago I volunteered to help analyze proteomics of Arabidopsis that had went to space. The data hadn't been released yet and I needed to get clearance and the paperwork turned into a hassle for everyone and they dropped it. I have been pretty busy, but I would have found the time for that study - purely and completely so I could make the following joke. 

Space Plants! 


Meh. Maybe some day. 

Saturday, June 8, 2024

CLIPPER 2.0 - (Re)Annotate those positional isoforms!

 


Picture this - you decided to do something wacky and actually use one of those FASTA databases that contains a lot of different protein isoforms to see if you could find them! Not just the smallest UniProt database where it's about 100% one open reading frame for every gene for every protein entry. We all know that isn't how biology works at all, but what else do you do? 

Chances are you will can make those peptide hits and do a protein rollup and then have no real way to easily dig through those isoforms you were looking for anyway....yay.....

What if you could take your output from a lot of the common tools and drop them into something that can help you find those isoforms? Check this out! 

Welcome to the party, CLIPPER! I've got some RAS mutations I could use your help with. There are different mutations on each chromosome, too, (super fun) so multiple sites map back to the same single entry and I think you're going to help. 



Friday, June 7, 2024

Searle lab Stellar preprint resolves a lot of questions about the new ion trap!

 


I tried to keep up on what was going on in Anaheim at ASMS, but it's hard to keep up when you're actually there. Not there is almost harder. I ended up with more questions about Stellar than answers, but - yet again - Thermo collaborators dropped a series of preprints throughout the conference.

This one really clarifies what this instrument is and can do


I knew from the architecture (ion trap on the back of what appears to be the phenomenal Altis + QQQ instrument?) that it was going to be crazy sensitive, and the headlines were clearly that this ion trap can hit >100 scans/second. 

What this preprint goes into is how you can use this for both global proteomics and targeted validation. They use the instrument in DDA mode with offline fractions to build a library and evaluate that versus gas phase fractionated libraries with DIA. And then they do the targeting. So...if you had questions, it looks like this instrument can at least do all the normal shotgun proteomics stuff. A lot of us old people had bad experiences with nominal mass instruments way back in the day. I swear, if I put enough LTQ XL MS/MS spectra into a program called Bioworks and ran a Sequest search (around 2009-2011, this was my workflow) I could literally generate proteomics data to support ANY hypothesis you brought my way. There was low accuracy, poorly matched MS/MS spectra for every peptide from every protein.

The first real way of estimating FDR wasn't published by Gygi lab until 2007 (link),  but it didn't get to me in any form I could use until I was running Orbitraps. And who uses target decoy for PSMs today? We all use intelligent deep machine intelligence thingies. It's easy to think that if we had these informatics tools maybe we'd have less fear of nominal mass instruments. Exciting thought and since this truly sounds like an instrument with a load of capabilities at a much lower price point than the other headline grabbing ones today, I bet we'll find out soon! 

Thursday, June 6, 2024

Finally!MALDI-prmPASEF for spatial targeting (preprint says prototype software)!


Wow. Do you have to dig for this one. 

Background: if you aren't paying attention and you get a TIMSTOF Flex that can do ESI - PASEF (DDA) diaPASEF (DIA...duh...) and prmPASEF (targeting with ion mobility and quadrupole and with high resolution fragments!) you might be very confused to find out that none of these features work when you.... turn....on.... the.... MALDI... source. 

What you have to do is figure out the mass of your target (which might change if it picks up a funny matrix adduct) and then it's relative ion mobility and then punch both of those things in to get a TIMS cleaned up MS1...at 35,000 mass resolution....the end result is that you 1) can't scan very fast (because you aren't doing the parallel accumulation bit) AND you can't remove a lot of background. If you're like me it makes you sort of forget you have a MALDI at all.

While rumored for a while, those rumors got a whole lot louder at some meeting in California this week. Not there, I had to really do some preprint digging and - this group has it! And they hid it behind a ton of words that I do not know and 23 other figures. But it's in here! 



To find data from MALDI prm-PASEF you need to go to Supplemental 18 (top figure) and it looks super super legit. Unequivocal, even? I like the word "prototypic" here. 


Wednesday, June 5, 2024

19,000 phosphopeptides by microflow DIA! Is the PTM tipping point finally here?

 


When you think about why to not do DIA you've got maybe 2 reasons right now. Limited multiplexing capabilities - and PTMs.

Obviously the PTM data is there, right? We need the informatics and maybe the methods to grow up. 

Is this great new study signaling the tipping point is already upon us for one annoying PTM

There is a LOT of good work in this paper. Not limited to - wow - that's a lot of IDs for microflow - as well as the use of multiple search tools to get to these data (comparisons of library generation!). That last part is cool because all the spectra used for these deep learning tools for peptides are from Orbitraps. The spectra of other mass analyzers may look a little different. There is a lot to read into this if you're on a SCIEX TOF platform. In any case, a really nice study for multiple reasons including way way way more phosphopeptides than we've ever seen in our lab out of DIA. Maybe it's time to try lookin at some old files again. 

Tuesday, June 4, 2024

Set your Q Exactives up for optimal TMT32/35-plex (90k res?) cycle times!


This week at ASMS there were some reagents on the big stages, including what appears to be either a TMT32-plex or TMT35-plex commercial release. 

While I haven't had time around packing the lab to really dig into the fine details, it appears that a minimum of 90,000 resolution @ m/z of 200 is required to achieve baseline separation of all the tags. 

If you're running on Q Exactives you probably noticed that you do not have a 90,000 resolution setting. On a Classic or Plus you've got 70,000 and 140,000. While you could just run at 140,000 that's a wopping 512 milliseconds per MS/MS scan! 

Running at 90,000 would only be 329 milliseconds, which is an extra MS/MS scan/second. Which would definitely add up across a run. 

Assuming you've got the setup I put up there (70k MS1 and 90k MS2) a top 6 would be 2.2 seconds, rather than 3.6 seconds. 

That's 3,200 MS/MS in 120 min vs 2,000 MS/MS in 120 min.

No, that's not a lot of spectra, but you're 32-plexing! You're offline fractionating and stuff, right? Plus it's TMT, you literally do not care about your chromatographic peak shape.

Also, in one of the slides it looks like they show not quite baseline resolved spectra at a lower resolution which would obviously be faster. 

(Oh yeah, and an HF or HF-X would be about 2x the number of spectra)

How to do this? 

MAXQUANT.LIVE.LIVE.LIVE.LIVE.LIVE (sing it with me, people who plan to retire with a Q Exactive still in their possession!) 

MAXQUANT.LIVE 2.1 is now valid until January 2026!!

If you haven't ever used MaxQuant.Live, I made instrument triggering methods you can download here for the Q Exactive HF(it works the same for Q Exactive Classic, I've never tried Plus) as well as a powerpoint that walks you through the steps. You go into the beta and there are two little paperclips to download the .meth file and the .pptx. 

Monday, June 3, 2024

Near real-time plasma proteomics by LCMS!

 


One of the historic (and largely accurate) criticisms of LCMS proteomics is that it is slow.

Slooow.

Slooooooooooooow.

What parts are slow? 

Sample prep has historically taken 16 hours, though faster higher temperature methods have really taken center stage. We often do 2 hour digestions at 47C but that's after 1 hr of reduction and alkylation and possibly after homogenization or enrichment. Still not fast. 

Also the HPLCs on mass specs are slow. Smart new LCs have improved this a lot, including technologies like dual trap single column HPLC (DTSC).

What if you ran an LCMS system really really fast. And what if your total sample prep was almost as fast? 


What's your argument against LCMS proteomics then? Dynamic range? Okay, valid, but that number is going up by the day. 

Dual trap single column HPLC mass spec (fast) coupled with a new protease that can digest a sample in minutes???