Ready to get super pumped about leaving the lab and not thinking about proteomics for a few days? I'll help you get started with
QUAAAAAALLLLIIIIIIIITTTTTYYYYYY COOOOONNNTRRRROOOOOLL PREEEPRIINNNTTS!
Let's start with this great new Primer!
Just like how you have to put primer on right after you sand most things and before you do the actual hard part of applying the paint people will see --- this'll get you ready for the this bit of important misery --
Got a few minutes to read through a summary of 210+ references and a thoughtful perspective of what proteomics does -and doesn't - do well today?
I'm re-reading this paper from last spring and - wow -
2) Makes it so that if I open a document from my desktop and go to save it as a new name it defaults to some imaginary "cloud" thing so I will never ever be able to find it again. You can "other save options" or you can:
Open every one of the MS Office things you use
Go to file
Go allllllllllllllllllllllllllllllllllllll the way down to Options
Go to Save and shut off this toggle that you never turned on in the first place.
You are welcome.
Really really cool study on this here!
You'll note they're using the ESI source, but if you're using the CaptiveSpray you're blasting your solvent directly into your glass capillary, right? I suspect that you'll see something similar when going from 100nL to 2 uL/min!
But then you realize how many samples you have to go back and rerun and you think "maybe I should really truly quit being a mass spectrometrist"? I'm having one of those days.
Quick solution.
If you upgrade your nice TIMSTOF instrument to TIMSControl 5.0 you'll get the thing above that I labeled #1 and - it is AWESOME. You get much better control over your mobilogram windows that can be guided by your real data. Using it right for DDA can give you dramatic gains in number of relevant fragmentation events. Run a file with your dumb mobilogram that you drew freehand. Then load that file into that PASEF precursor region thing and it will load your whole mobilogram average. Then you can draw the smartest possible mobilogram(s) for your actual experiment. It's beautiful and it might be giving me as much at 10-20% more PSMs on some of these samples. I can't wait to try this on TMT (which moves funny in IMS space and TMT and TMTPro are different, see figure 1C and 1D here)
However, you don't get this for free. You'll have two new glitches to deal with. The first one is sort of funny. Do me a favor and open TIMSControl 5.0 if you have it and turn on Stepping. Then turn it off. (That little toggle I marked with 2). Then note what happens to #3.
What it'll do is swap your fragmentation energies. Honestly, sort of harmless, but just funny. You'll find that you trigger the same number of peptides but you don't identify and of them because you hit them with the appropriate level of energy for fully liberate TMT11-plex reporter ions. BOOOM.
The other one is more nefarious and what I feel the most stupid about.
If you load older TIMSControl DDA methods what we find is a dramatic reduction in MS/MS events. If I run the exact same sample with a DDA method from TIMSControl 4.0 back to back with a brand new one with TIMSControl 5.0 while trying to keep everything the same,
TIMSControl 4.0 in 88 minutes ~25,000 MS/MS scans
TIMSControl 5.0 in 88 minutes ~140,000 MS/MS scans
What it looks to me like it is doing, but this isn't my job so I ain't gonna spend more time on it, is incorrectly reading the Target Intensity and Target Threshold values. Like your max target is now your min target.
I thought maybe it would be as simple as skipping a line in the method, but woooooooooooweeeee, the methods files are very very different.
What deserves two "very"s? There is a sum difference in 280 lines of the methods files between the two software builds. So...while I can't say for sure that the intensity and threshold are swapped, I can say FOR SURE, that the method files should probably not be used interchangeably at all.
This isn't meant to be a criticism of the very nice and incredibly small team that is building the software for all of these instruments.
Josip Blonder had one of the biggest effects on my development as a scientist in this field. He's got a fancy emeritus/pseudo-retired status at the NIH now, but apparently he's not just fishing off of Hvar, even if he isn't pulling down the surfaceome these days. He's updated Proteomics for Drug Discovery and it will be out in August!
And when I stumbled on this, I also found this one is coming out just before it!
The first book on single cell proteomics by mass spectrometry!
How cool does that sound? Zoom should be good for a huge number of external participants! You can check it out here if you're interested. https://jhjhm.zoom.us/j/99656763944
Whether you hate this plant or not, this is a really really cool new preprint!
And......700-ish proteins identified. Which is just about the same f'ing numbers I'd get for plasma in 2011 prepping in a FASP filter and using a terrible old brown Eksigent nanoLC and running samples on an LTQ Orbitrap Velos system. Give or take a 100 protein groups. My memory has faded over the last 13 years.
The point is that it's generally pretty easy to see those top few hundred proteins in plasma and it doesn't seem to matter all that much what instrument you use. You're still stuck at the top few hundred.
But we've been seeing people getting 4,000 proteins from plasma recently. Not the completely unproven to have any quantitative accuracy whatsoever aptamer based stuff, but with super smart sample prep and runing on the newest and best instruments in the world.
The magic for LCMS based proteomics of plasma appears to have been solved. You just need ...$1,000 per sample and a TIMSTOF HT or an Orbitrap Astral...... and most of us have none of those things.
What if you don't need them? How would that change EVERYTHING?
You might recognize some of these names as being the most annoying nerds about "quantitative accuracy" and "matrix effects" and a bunch of other annoying things in proteomics. You'll find their most annoying traits in full force in this manuscript. Translation - holy fuck this looks like the real fucking deal.
The prep looks a little annoying, but it sure beats most of the ways we need to get past those 700 plasma proteins (deplete, run 74 offline fractions) and they conveniently automate this magnetic prep with a generally affordable KingFisher robot. All the scripts to automate it are already available and this Github is the fastest way to get you to the actual RAW data.
However, since this is a MAG-NET based method I don't see why you couldn't use other convenient things
I don't have a KingFisher but who hasn't experimented with automation on an OpenTrons and has one in use or being used for pipette tip storage somewhere. Drop in one of these and I don't see why you couldn't do this whole prep.
Are you interested in the fact that it's way way easier to see metabolites in single cells than to quantify proteins? Then you should check out this great -and short - new commentary!
One thing I learned in my short terms running Metabolomics services is that almost no one cares about all of the things that we can see in an untargeted run. When someone wants "Metabolomics" they almost always have one pathway that they actually want. They really want glycolysis or they really want this one set of molecules that are a side effect of the electron transport chain, etc., While running global is by far the easiest, you are always stochastically sampling those convenient metabolites for MS/MS and so the ones they're interested in are often not the ones where you have your highest confidence identifications.
What is really really super helpful to know up front is - what do most people actually want? That way you can build a panel or panel list that someone can walk up and pick off of a menu. I bet people who are good at Metabolomics with years of experience have those.
This group is very clear on what they consider would be the highest priorities for single cell metabolomics!
Some of these are easier than others - I like succinate (we use a heavy in our dilution buffers as one of our internal controls) and I'm cool with CoA metabolism. Nice big molecules that many retain without miserable chromatography). Others are harder, but you'll never find a nicer list of priorities for a rapidly emerging field. 100% recommended!
I recently had a Windows PC perform an unauthorized system update in the middle of a big batch of files and gave up and bought my first ever MacIntosh/Apple computer. Possibly because I grew up in somewhat extreme poverty and possibly because I don't particularly like the color silver, these things always seemed like they weren't for me. But they are advertised as very very fast.
It's easy to be skeptical, though, because now they are making their own chips. They can use whatever benchmarks they want. The benchmarks I care about are processing proteomics data!
TESTING TIME! My Apple M2 Pro with 16GB of RAM and 1TB hard drive vs my closest looking Windows PC - same number of cores, but Passmark thinks it is a little slower.
First experiment, performed between when my elderly dog needed to pee at 3am and when my toddler woke up at 5am. Details are fuzzy....
Tools - SearchGUI with MSAmanda 3.0 and SAGE
Random HeLa digest file from an Exploris 480 with FAIMS. 200ng separated over 120 minutes on an EasySpray 25cm x 75um with a 3cm PepMap trap. Converted to MGF and having around 110,000 MS/MS scans. I might have generated it or downloaded it. Not sure. It was on my hard drive from this review I did a while back. If you want the file the MASSIVE download link is in it.
Search tolerance was set at 10ppm MS1 and 10ppm MS2. I used the Uniprot Swissprot for human (9606) that I downloded this morning. 20k entries and I had SearchGUI add decoys. M+oxidation and static carbamidomethylations (these are the SearchGUI defaults).
Windows PC - pretty freaking fast at 3 min and 25 seconds!
Okay - I should also point out here that SAGE is nuts. But the discrepancy is even more insane on the much new MacIntosh chip. 13.4 seconds to search a file vs 2.9 seconds to search a file??
Then it occurred to me that it's probably not fair to run a file on a MacBook on a battery. I plugged it into the wall, deleted the first output file directory and reran the file.
It is fair to note that the Windows PC I'm currently typing this on doesn't do much data processing these days. It's certainly perfectly suitable and I have zero reasons to think I'm not going to be using it as my primary office PC for several more years. However, for our main PC "server" which has supported as many as 6 users with SpectroNaut, Proteome Discoverer and Compound Discoverer we've been using a Ryzens 9 (I think this is the chip on that box below) - which aren't even all that fast now! The big EPYCs and Threadrippers are pushing 3x this benchmark - but you've got to be ready to drop as much as $10k just on your processor. Good time to be a consumer if you need PC power!
While the expectation is that we'll see real details of these reagents at some thing in Anaheim in June, one core facility is already advertising that they've got it. You might be able to guess which one, but I think it's fair to say that it is really truly real now.
Short of completely redesigning the tags there are only a couple of ways that this would be able to work, right? And I can't think of a way that you do this without requiring an increase in the relative mass resolution necessary to use the tags.
For some people this might not be a big deal, right? There will probably be some drawbacks, like the whole "now I have 32x more albumin to deal with instead of 18x more albumin" but for some of us this reagent could be completely transformative.
32-plex will allow us to switch to using 2 x 384 well plates!
...you could get a 3D printer and convert it into a ....cheap (??) ... robust and sensitive offline fractionator.
The 3D printer converted to a fraction collector is seriously cool. You can get a shockingly high resolution 3D printer for <$500 these days and you won't need super precise movements to put your tube above the right well in a plate. Steal these plans to build your next fraction collector, for sure!
The...use of an HPLC system that costs the same? more? than an HPLC with an integrated fraction collector...likely makes this more of a niche idea. The sensitivity is really good, though, with good recovery of high pH fractions of 5ng of tryptic digest.
If I knew about this, I forgot about it somehow, but it came up on a cool podcast recording and now I have the link. You can check it out here!
B-sides is the new "season" of THE Proteomics Show where we just have guests that we've really wanted to talk to. Henry Rodriguez has been on my list for a long time, even before he held a position in the WHITE HOUSE!
There probably hasn't been a stronger advocate for proteomics - as a science - in the US government and it was super cool to get his side of the stories - like how did CPTAC get started??
For maybe the most creative abstract/TOC graphic you'll see today -
I legitimately laughed out loud when I saw this new paper ASAP. When Ska went mainstream in the late 90s, I was NOT a fan, but it was absolutely everywhere. You couldn't avoid it at parties and I WAS a fan of parties in the 90s.
More importantly, this paper is really super useful! Who doesn't have a protein protein interaction network on their desktop they are saving for a day they have nothing else to do and feel really really smart? There might be 10 on mine that I'm not entirely sure what to do with, but an answer might actually be in there somewhere. We have some compelling and very confusing FANCM enrichment data that I've looked at off and on for months. There is something in there, I have no idea what.
You know those low mass fragment ions your search engine is probably set to ignore - because - let's face it - how useful is it to know there is a protonated lysine fragment there?
This is dynamic and - while it has the dreaded "pip" in this Github, that's something ChatGPT can help you with if you're not sure where to get started.
I presume for Orbitrap spectra you could start talking about ppB mass accuracy after running it through something like this! For me, I just want Orbitrap level accuracy at (affordable) TOF speeds.
Honestly, I don't think this is anything that will come as a surprise to anyone at all. Particularly if you have taken a look an realized how very very old the Trapped Ion Mobility patent is (2008!)
The first came to us out of Australia, by way of
Full text here! Honestly, my first thought was "why not Ultra" but Flex obviously makes sense, since Thermo doesn't have MALDI capabilities. And since Bruker can't get pasef to work with MALDI, Asstral backend makes an awful lot of sense!
Just a few minutes later we had this release suggesting the obvious - that Veridian Dynamics - is involved in this collaboration.
While we haven't heard much from VD recently, they were the first to jump on the expiration of the Orbitrap patent and begin commercial construction of their own hybrid devices!
Given word choice on the Veridian release, I think it's safe to say that one of our leading labs is involved in this!Guess I do have to work out travel to Anaheim afterall....biggest year for $,$$$,$$$ level mass spec labs ever!
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.
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.
