Wednesday, April 17, 2024

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. 

No comments:

Post a Comment