(Loss of) resolving power when importing mzXML files from HRAM QTOF

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(Loss of) resolving power when importing mzXML files from HRAM QTOF anders honore  2020-08-04
 

Hi
Working with small molcules in MS1 from Bruker QTOF instrument. Data is stored as centroided and converted to mzXML (by Bruker converter) prior to upload. Resolution is typically +40,000.
After import resolving power is reduced to <10,000 and otherwise obvious peaks when reviewed e.g. in MZmine2 becomes severely distorted. Appears as though resolution is reduced to <<10,000
Transition Settings: precursor as monotopic, full scan count, peaks 3, precursor mass analyzer TOF, resolving power 50,000
Include all matching scans

Probably error-40 by a novice user - but please advise...

 
 
Nick Shulman responded:  2020-08-04
Can you post a screenshot of what you are seeing? I am not sure that I understand your question.

If you would like, you could send us your Skyline document and your .mzXML file and we could take a look.

In Skyline, you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that .zip file is less than 50MB you can attach it to this support request.
Otherwise, you can upload it here:
https://skyline.ms/files.url

You can also send us your .mzXML file.

Skyline knows how to read Bruker .d raw file directories, so it was probably not necessary to convert them to mzXML. If you would like, you could zip up one of your raw files and send it to us too.
-- Nick
 
anders honore responded:  2020-08-05
Hi Nick

Thanks for reverting very fast. I am certain that lack of my lack of experience/understanding of Skyline comes to play.

Attached the observations that I find challenging compiled in one example with the relevant files. The example is a synthetic mix of standards, where some of the chromatographic profiles for some analytes (SucP, ATP, ADP, AMP, etc) correspond very well with observations when viewed with Bruker DataAnalysis - and some where they are completely off (G6P, F6P, F16P, PEP).
I did not manage to open Bruker .d (from maXis 4G instrument) - so did also include the .d file as zipped. Really curoius to learn how this is feasible

Looking forward to your comments
BR,
Anders
 
Nick Shulman responded:  2020-08-05
Anders,

Thank you for sending us your files.

I see that many of your MS1 chromatograms are spiky, with long straight lines where the chromatogram skips over regions in the retention time.

The reason that this is happening is that Skyline is incorrectly deciding that some of your MS1 scans are intended to be "SIM" scans. For Skyline, a SIM scan is a scan where the mass spectrometer was told to collect MS1 data over a narrow range of m/z values for the purpose of targeting only the molecules in that range. Skyline decides whether a particular scan is a SIM scan by whether the width of the m/z range that was collected is less than or greater than 500 Daltons.

It seems that the way that Skyline is figuring out that m/z range is by looking at the actual data m/z's and intensities in the spectra, so it depends on what the actual observed data was. Skyline is supposed to figure this SIM vs MS1 distinction by looking at some sort of data independent scan metadata information, but I assume that that information is not present because of the fact that this was converted to mzXML.

Because of the fact that Skyline things there are some MS1 scans and some SIM scans, Skyline actually extracts two separate chromatograms for each analyte in your file. Skyline only shows you the SIM chromatograms, so the chromatograms that you are seeing consist of only those spectra that had less data in them.

I also see that when I try to extract chromatograms from your Bruker .d directory directly, I get the error from ProteoWizard: "Error reading BAF files".

I will ask around and see if we can come up with a workaround for you.
-- Nick
 
Brendan MacLean responded:  2020-08-05
Could Anders maybe convert the files to mzML? mzXML is a pretty old format for this purpose. Or why is the conversion necessary at all?
 
matt.chambers42 responded:  2020-08-05
The analysis.baf file in the directory.zip you attached is 0 bytes (empty). Did it get messed up when copying it? Basically I don't see where the raw data is stored in the .d directory without a proper analysis.baf file.
 
anders honore responded:  2020-08-05
Zipped again - and checked. The BAF should now be >0 bytes. Would love if the conversion to mzXML (or mzML) could be omitted
 
Nick Shulman responded:  2020-08-06
Anders,

Skyline has no trouble reading this new raw file directory that you have posted.
In Skyline, you can use the menu item:
File > Import > Results
and use the Import Result Files dialog to navigate to your folder "100 ppm_4_01_18023.d" and Skyline will extract nice looking chromatograms.

-- Nick
 
anders honore responded:  2020-08-07
Fully agree - I might have used incorrect settings as ALL my files now get imported. And the bypassing the mzXML process cancels problems observed. Great help - thanks!