Peak areas

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Peak areas Michael Cundell  2018-09-10
 

Hi all,

I am using an Orbitrap Fusion Lumos to acquire both DDA and PRM data within a single method. I became confused as to why Skyline v4.1.0.18169 was giving me spikey XIC's (see skyline.emf).

These spikes are not present when I extract the same peptides transitions using Thermo's Freestyle application (Freestyle.jpg). Within freestyle I typically do this by selecting the appropriate PRM filter e.g. "FTMS + c NSI d Full ms2 123.4567@hcd28.00 [120.0000-1500.0000]" and then filling in the transition masses that I am interested in. This essentially ignores the DDA data within my RAW file.

I think the spikes in the Skyline data are coming from the DDA part of the data. The DDA part within the method uses different parameters to the PRM part (eg 240ms fill time vs 120ms fill time respectively). In freestyle if I extract one of the transitions with the filter set to "ms2" (this includes both DDA and PRM data) you can see the source of the spikes in skyline comes from the DDA data (see Freestyle_2.jpg).

Is there a way I can get skyline to ignore the DDA data within my RAW file?

Thanks for your help.

Best,

Michael

 
 
Nick Shulman responded:  2018-09-12
The only way I can think of to do this would be to use msconvert to create a .mzML file that is missing the scans that you want Skyline to ignore.

Here is the list of all of the sorts of "filters" that you can tell msconvert to use:
http://proteowizard.sourceforge.net/tools/filters.html

Here is the main documentation page for msconvert:
http://proteowizard.sourceforge.net/tools/msconvert.html

I do not see anything in there that would tell msconvert to remove the DDA scans and keep the PRM scans, but maybe you see something.
I think you might have to just specify "--filter scanNumber " and then a whole bunch of ranges in square brackets indicating which scans to keep.

-- Nick
 
Michael Cundell responded:  2018-10-17
Hi Nick,

Thank you for your suggestions. For now I stuck to Thermo Freestyle to extract the required peak areas but I will certainly give those a try in the future. Much appreciated and sorry for the slow response.

Best,

Michael