Wednesday, March 27, 2024

Aftermarket high resolution FAIMS allows separation of MEGADALTON protein complexes!

 


FAIMS gets a bad wrap because most of the commercial systems have a resolving power of something between 5 and 20. They're great systems if you just don't want to fragment (or see) +1 ions and you want your mass spec to only see +2 or +3 ions. Cleans up your spectra so your mass spec doesn't have to work as hard, and everyone is happy at the end.

But....is that a limitation of FAIMS technology itself, or is that what is mass 😁 produced for the general market? Sure sounds like it's the latter. 

In this new study an aftermarket/custom high resolution FAIMS system was coupled to a UHMR (which has an upper mass limit of 80,000 m/z? Is that right? That's huge) and oligomers of antibodies (so a monomer is around 150,000 Da!) were coupled. 

The study is maths heavy and there are a lot of formulas, so I found it hard to get to the effective IMS resolution. However, this 2019 study indicates that the FAIMS is >100 resolution, and I think the two devices are similar

And - get this - you can get this FAIMS system for the front of just about any instrument and they can be custom tuned for small molecules, peptides, or intact proteins. And they're a lot less expensive than the 10 resolution units that you can buy for only certain instruments. 

Tuesday, March 26, 2024

5-plex your data INdependent analysis methods by modifying dimethyl tags!


This has sat with a bookmark on it for quite a while I hoped that I'd remember to ask someone intelligent questions about it. 

Multiplexing DIA sounds like either the best or worst of both worlds. More samples/day but you've increased the complexity of your background so those magical neural network thingies have to think a lot harder. 

There is very little chance I'd consider 2-plexing my DIA. That isn't worth it to me in any way at all. 3-plex? That is enough that I bought reagents so I could eventually try it, but I haven't been anywhere near excited enough to actually do the try part. 

5-plex? That's worth thinking about. 5-plex without fancy expensive labeling kits? That's worth bookmarking.

Disclaimer: I've never dimethyl-labeled. I feel like there is some drawback to it, like the tags shift the retention times just a tiny bit? I forget. Again, meant to do some background research - and didn't.

You can read about it here!



Sunday, March 24, 2024

Multi-study meta-analysis of dog samples with oral diseases!

 



I'm wrapping up a meta-analysis right now that I've bugged just about every proteomic informatics person I know about in one way or another. The insanely beautiful data that I'm working on reanalyzing is from a patient study where they said "you thought CPTAC was thorough? hold my espresso". No joke 33 offline fractions at 2 hrs each on a QE HF on these priceless human samples. The data is perfect for what I'm doing, but during the uploading of data for almost 70 patients, they missed a few here and there. The PI is now retired and the team is dissolved, so I ain't getting patient 36 fraction 32 or patient 51 fraction 7. Whether to keep these patients or not is what I've been bugging people about. 

BTW, this data just provided a spreadsheet of which patient was whom and I find it just fine to work with. 😉. I didn't need a short wave radio or to learn Morse code or anything. 

Crap, that's a joke like 15 people might get, again, right? I need to get out of my house. 

The great people at the Proteomics Standards Initiative came up with a meta-data standard for data in repositories called SRDF and it's a great idea for anyone wanting to automatically pull loads of proteomics data for reanalysis. 

Since my poor sense of direction would never allow me to find the utility closet at HUPO where the PSI meeting is allowed to have their annual meeting, I missed it. When I was told I was supposed to be using this format, Google helpfully told me that it has something to do with condensed short wave radio signals

And....now....we're around to the topic of this post....


These researchers successfully drew conclusions from an extremely wide array of proteomic studies deposited over time for the under-studied disease that they are interested in. 


Including ELISA data - old spot cut out MALDI-TOF peptide mapping stuff and some more modern iTRAQ and TMT work.




However, I do think this is a good example of studies where this hasn't been done and no one will go back and do it later, where some interesting findings were still made. 

SEER Proteograph prepped plasma on TIMSTOF Pro2 and HT!

 

There has been lots of excitement (and some scary good data) out of the SEER proteograph system for plasma proteomics.

Here is some more! 


While the authors clearly intended this to be more of a comparison of two very nice recently released instruments in their lab, as you can probably see from the figure at the top, the proteograph steals the show.

Clearly the 14-bit digitizer and higher capacity TIMS improve identifications, but the plasma precursors go up 3x - 4x when moving the prep to a kit that I have absolutely no idea at all how any of you can afford to use. 

The peptide loading plots are also really cool. I've never considered running over 400ng on the TIMSTOF Flex, and we only use that much when we use the EvoSep which runs closer to microflow than nanoflow levels. 

Friday, March 22, 2024

Registration is open for Cold Spring Harbor Proteomics Course 2024!

 


Do you want to buckle down and spend 2 straight weeks learning proteomics? 

There is probably no better way to make that happen than getting accepted at the Cold Spring Harbor proteomics course. You can apply here

I'm lucky enough to have been able to participate as a guest instructor twice in the past at this amazing workshop. I did have to take it off of my CV, however, because I'm not listed on the website as an instructor. 

The way I've described it to people is "Sunday night it's a bunch of people in a room with a slide deck that says "What is proteomics". Later that week there are 10 people crowded around an instrument monitor at 1:30 am watching phosphopeptides someone taking the course prepped themselves finally start eluting and fragmenting off an instrument someone taking the course is running themself. Then everyone celebrates!" 

It might have changed I was there a long time ago. 

If you do get to go pay very very close attention to the train station map. There are ZERO sidewalks and a windy back road with New York drivers so you and your roller bag may have to rapidly dive off the road with your roller bag if you walk in from the wrong stop. (Story I heard from someone who is very bad at maps). 

Thursday, March 21, 2024

Deep learning - of glycopeptides!?!?!?

 



Okay.....on the surface this was first surprising that the first PTMs we'd see deep learning successfully applied to en masse was going to be glycopeptides. Then I thought....well...the problem is the stupid sugars all have the exact same masses. 

Here is an illustration from an unrelated study for fragmenting the fragments of the fragments to figure out what a glycan chain actually is because the fragments of the fragments of these important glycan chains still have the EXACT SAME MASSES. 


So maybe this isn't the biggest stretch in the world ever (link to this paper and topic of this post). 


Unlike the topic of yesterday's post, this tool is still in the - you need some skill with a computer to actually use it - but if you have such skills you can get DeepGlyco here. It does require installation on GPU for the deep learning magic. 

Wednesday, March 20, 2024

AI assisted proteomics - for everyone -with Ms2ReScore 3.0!

 


MS2ReScore is a great idea! Let's use Artificial Intelligence things to reanalyze our proteomics data based on various features of confidence to create bigger and higher confidence lists. It's free, too!

Then....you see what you're dreading you'll see. "DOCKER"?? "git"?!?  

Great...another tool for bioinformaticians written for bioinformaticians that I could spend the next 3 weeks learning how to use if I didn't have 11 mass spectrometers that are running suboptimally, a grad student who still hasn't returned from Dubai and 3 grant deadlines. (These are only examples, but you get the point, time is limited). Could I just have a Windows installer for some of this magic? 

You can now! 

Not to be missed here is MSAmanda 3.0 which can be installed in PD 3.1 or newer and has stand alone versions for PC, Mac and Linux and can be ran through PeptideShaker. MSAmanda 3.0 by default outputs the features that Ms2ReScore uses to reassign peptide confidence.

When using MS2ReScore you also get cool HTML QC plots along with a bunch more peptides. When compared to MSAmanda + Percolator, it looks like a solid 20% increase on low abundance peptides. The authors use a nice SCP dataset that I'm going to assume came from Erwin Schoof's group and then they do a bunch of analysis that makes it look like these peptides are real and make sense in relation to the other peptides. 

I'm all for more data analysis tools, and I'm even more for them when I can just download them and run them! 



Tuesday, March 19, 2024

PRECISE readout of MEK phosphorylation cascades by top down proteomics!

 


.....wow.... I won't lie. I'm stunned. I didn't think we were here yet.... and, to be fair, maybe we arent,but this group is! 


Bad background by Ben: A whole lot of the central regulatory pathways controlling tons of things in cells are based on some key central phosphorylation cascades. MAP kinase and MTOR are famous ones. They modify proteins by phosphorylating them because the modification can be fast and it reversible. If you go into any oncology centric place there is probably some really really really skilled pathway scientist (or 4, if they can afford them) who can dissect these cascades, probably through western blots and FACs. These people can tell you that if MEK1 S2998 is phosphorylated but T2992 is not - that means something critically important. 

When we do shotgun proteomics, we cut this region into a small piece and peptides phosphorylated once on the same region of that fragment coelute. It's often very hard, if not impossible, to tell which site is phosphorylated - or both. 

Obviously we should do this without digesting them, right? The problem with that is that top down proteomics only really works on small proteins. If MEK1 was 16kDa, it would still be tough, but you could do it. MEK1 is almost 50kDa, though! 

Enter "Individual Ion Mass Spectrometry" IIIMS? (older post explaining what that is here) and - what?? - ETD IIMS? 

I was going to cut in parts of the materials and methods section here, but I don't want to intimidate anyone into thinking this is clearly just a proof of concept. However, I will say that the process to get to these data makes label free single cell proteomics look like a nice fun day (which it is NOT). 

However, we have to start somewhere and being able to confidently localize 4 separate phosphorylation sites on a critically important protein this big - with anything - is a step in the right direction! 

Monday, March 18, 2024

US HUPO 2024 special live episode of THE Proteomics Show is out!

 


No joke at all, this is by far, my favorite episode of the podcast so far. RASR has a great radio voice, asks super smart questions and Dr. Olga Vitek has such a cool perspective and story to share! Such great content that Ben couldn't even ruin it. 100% recommended!