Sunday, December 15, 2019

Exploris mass accuracy at highest speeds? Or is the data being read funny?

I'm skeptical of these results, but I still think they're worth mentioning.

This is an Exploris data dependent experiment. 80ng of commercial HeLa digest on a 60 minute gradient with different resolutions. 120k MS1 and two separate runs (n = 1, look ma' I'm a scientist...)

The 18ppm MS/MS fragment tolerance lines up with what I've seen with various tools for the HF and HF-X. The higher speed.....that's a bit larger of a difference.

The weird part is the fact that in the third run (and I just checked to make sure I wasn't being stupid) I used the internal calibrant (fluoranthene gnarly? radical?) for the MS1 and MS/MS.

Obviously, n=1, but that's pretty weird, right?

This output in MetaMorpheus, but a lot of software does that calibration step these days and I'll check those, but I do have to wonder if there is a scan header thing?

In a couple of tools I've had some weird glitches with Exploris data -- which was fixed by updating my MSFileReader on various computers. I don't have any conclusions here. but I do think it's something worth keeping an eye on!

Also -- please keep in mind that post-acquisition calibration is basing an MS/MS fragment on theoretical. At lower resolution we're seeing more coalescing peaks (they've overlapped and we can't tell them apart) so the error is stuff like that (and mis-assigntments) on top of any drops in mass accuracy.

Remember how the scan headers got swapped around 2012 and old MSFileReaders would sometimes put the pre-MIPs mass in as the monoisotopic mass? That was fun! Not saying that's what is going on, but it has certainly happened before.

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