Thursday, April 18, 2024

Is MS Office trying to save all your stuff in some imaginary "Cloud" place?


I had another mandatory Microsoft update thing which 1) enabled their ....sub-performing.... blend of ChatGPT and Bing in the lower right corner of my screen where the button should be to get me back to my desktop... and 

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. 

Wednesday, April 17, 2024

Impact of source conditions and flow rates on CCS in TIMS!


So...front end ion mobility things like TIMS are gas vs electric pull, right? So what happens if you run 5x the flow rate for your LC input? Will that alter the gas pressure enough to change your perception of the collisional cross section (CCS) and thereby change the observed 1/k0 values? 

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! 

2 new "features" in TIMSControl 5.0. Don't load old DDA methods!


You know when you finally solve a mystery that is driving you completely bonkers and you get that relief at first that you solved it.

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. 

Tuesday, April 16, 2024

Two great new proteomics books coming this summer!

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! 

This isn't like preodering a video game. The corporate shmucks can't just cut development and bug testing because they've made enough money for their shareholders. Preodering doesn't change the product, it just means I don't forget to get them. 

Monday, April 15, 2024

Junmin Peng is giving a cool Alzheimer's LCMS talk here on Wednesday - zoom link!


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.

Sunday, April 14, 2024

Single cell proteomics of Arabidopsis root cells!

12 years after leaving a historic Ag university and I'm just almost over my over-exposure to Arabidopsis thaliana (Greek for "scumbag plant" or something). 

Whether you hate this plant or not, this is a really really cool new preprint! 

Personally, my favorite part of this is TMT LABELING WITH AN OPENTRONS! What a nice cheap option for this step that I wouldn't have thought you could do (the OT-2 pipetted don't do a great job with solvent/positive vapor pressures). So while I don't actually have time to spend on this right now, I'm absolutely going to find out how they did this part, even if I don't care about this stupid plant. 😇

Saturday, April 13, 2024

MAG-NET - 4k proteins from plasma with accurate quan without spending $4k/sample!


I just prepped and ran an absolute shipload of plasma samples last week using a lot of the best of today's technology. S-Trap 96-well plates so it's fast and reproducible. EvoTips and EvoSep one so they're clean and - again - ultra reproducible and the newest best diaPASEF on a system with all the updates so it's screaming fast on nice little windows. 

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'll note that this isn't brand new. I wanted to buy a lot of ....some....reagents....before I shared this. I made the mistake with posting some labeled DIA reagents a while back and then had to wait 6 months to get the reagents myself. 

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. 

If I have an issue with this method/preprint at all, it is not from me. It is from criticisms from a super Promising method from a while back that I loved and the criticism that it got from others. It was called Promis-Quan and while it had a couple of assumptions that annoyed people, one was that it was mostly analyzing extracellular vescicles (EVs) and some people thought that was a different thing than plasma proteins. Just relaying what I remember. I guess you could worry that there is criticism that this is the same thing? Don't ask me, I think if it's being circulated in blood we probably want to know about it.

Anyway, that's enough on this. The 4 groups doing single cell proteomics that I'm not actively collaborating with are all waiting on reviewer 3's comments on their manuscripts and I should be tearing these things apart 😇 instead of writing more on this amazing study. Also, they did use nice mass spectrometers, just not the ones you'd expect to see for numbers this high! 

Friday, April 12, 2024

A call for accessible tools to unlock single-cell immunometabolism research!

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!  

Thursday, April 11, 2024

How fast is a Mac M2 chip for proteomic / scientific data processing?


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! 

M2Pro -- 

2 minutes and 5 seconds! WHAT? Not the way I expected that to go. 

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.

Maybe a tiny bit faster, but within the margin of error here? 

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! 

That was a lot of words, for - hot dog - these Mac chips (and SAGE! WTF?) are FAST! 

Monday, April 8, 2024

Is a TOF killing technology on the horizon? TMT32-plex looks official!

We saw a leak to potential investors of one of our field's oldest and most ethical companies stating that a 32-plex was coming. Not a "I SILAC'ed it and TMT'ed it" technology, a real life reagent that would allow you to multiplex 32 samples simultaneously! 

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. 

This is how we multiplex single cells in our lab (from Eberhard and Orsburn, which should be out any day now) using 2 x 96 well plates for 2 conditions. That leaves us with 16 unused wells per plate, which is what we use for LFQ single cell method development. Anything you see out of our group like the acetic acid preprint or our upcoming miserable work with 20um ID columns, is done using the 16 cell left over from the hundreds of plates we have went through the last 3.5 years. 

32-plex will allow us to switch to using 2 x 384 well plates! know what is crazy? This actually almost makes it easier for me to automate. I just have to fabricate a new sample deck that will allow me to stack my plates sideways!