It does make you wonder if there might be a way to make expensive multiplexing tags that are...I dunno.... actually pure...?
To people in the outside world it probably looks like we've completed hundreds - or at least....dozens.... of studies where single normalish-sized single cells were analyzed by LCMS.
Our list does not have even 2 dozen actually studies. I'm sure that will change at some point.
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