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kevinkovalchik / RawQuant

RawQuant is a Python package for extracting scan meta data and quantification values from Thermo .raw files.
MIT License
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Non-sequentional master scan numbers #5

Closed kevinkovalchik closed 6 years ago

kevinkovalchik commented 6 years ago

While working on issue #1 I've noticed something interesting regarding the master scan index in boxcar experiments using the new version of the Tune software. Typically, the master scan number of any given data-dependent MSn scan will be the preceding MS1 scan. However, in boxcar data files acquired after upgrading to the new Tune software, we are seeing master scan numbers which reference back several MS1 scans and are repeatedly selected for MS2. It's not that the same peak keeps getting picked for MS2 in subsequent MS1 scans, it is actually the same peak from the same MS1 scan keeps triggering MS2 scans. Needless to say, this seems a little unusual and wasteful...

We're still not sure if this is related to boxcar settings specifically, though so far it does correlate with boxcar experiments.

I will upload some examples of the scan indexes later today or tomorrow.

kevinkovalchik commented 6 years ago

Here is a matrix containing RawQuant's parse output on one of these boxcar files. It's tab delimited.

ch_18May2018_HeLa-InSol_dda-ot-box-iso_2_MS2ParseData.txt

The MS1ScanNumber and MS2ScanNumber columns are of interest. Note that after line 13, the MS1 scans 133 and 144 just keep alternating as the master scan number, and it is the same two masses that are being analyzed each time. Precursor mass 1013.1631 from MS1 scan 133 is triggering MS2 scans for more than half a minute after it has disappeared from MS1 scans altogether...

This also seems to be causing some problems with RawQuant's ability to determine the precursor peak width, though we're not really sure if that is a bug at this point.

chrishuges commented 6 years ago

I think this might be due to the latest tune software for the Fusion not accepting individual isolation windows when you are using a multiplex isolation. When I made those methods I imported a method from the old tune where this was possible and so it accepted it, but I don't think it was supposed to.

I am doing some test runs tonight to figure out whether this is the case.

kevinkovalchik commented 6 years ago

Per the discussion in #1, this seems to have been a problem related to overlapping isolation windows.