Import QuanDirect (aka IS-PRM) data to Skyline

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Import QuanDirect (aka IS-PRM) data to Skyline Markus  2019-11-26
 

Hi Skyline team,

we are experimenting with the QuanDirect (aka IS-PRM) method on the Orbitrap Lumos using heavy spike-in peptides and everything seemed to have worked fine so far. When importing the data into Skyline (daily 19.1.1.309) I run into the problem, that Skyline extracts all MS2 scans, i.e. also the ion-trap scans which are only targeted at the trigger y1 ion and which do not contain any other ion information. When combined with the MSMS Orbitrap scans that actually contain the fragment ions of interest this results in sawtooth-like peaks (see picture attached). How do I tell Skyline to only import/extract the hi-res Orbitrap PRM scans?

Many thanks,
Markus

 
 
matt.chambers42 responded:  2019-11-26

Hi Markus,

What's the scan range on the targeted IT scans? Can you attach a screenshot including scan filter of one of them in QualBrowser?

Thanks,
-Matt

 
Markus responded:  2019-11-26

Hi Matt,

thanks for the quick response. The scan range is 180-190 m/z for the peptides with C-terminal R and 150-160 m/z for the peptides with C-terminal K. The collision energy is set very high to produce a strong y1 signal. Once the signal surpasses a certain threshold this is used as a trigger for the "normal" PRM scans on the heavy and the light peptides. I have attached a screenshot that shows the IT scan and the consecutive triggered Orbitrap scan on the same precursor.

The method setup is also explained in a guideline from Thermo:
"Method Setup Guidelines: QuanDirect: A simplified approach to fast and accurate, high throughput targeted MS2 quantitation using internal standards"
You can view it on the Planet Orbitrap website here:
http://planetorbitrap.com/library?t=QTIyNDllODg0OWE1NWYzYw%3D%3D&keywords=A2249#tab:keywords

Many thanks,
Markus

 
matt.chambers42 responded:  2019-12-03

Brendan,

I think scan range is more discriminating here (in general) than mass analyzer. There are 2 issues though:

  1. If it's not already doing so, then Skyline should not take an intensity value from a scan if the m/z it's looking for is outside the scan range.
  2. Skyline should not aggregate intensities from different mass analyzers in the same chromatogram trace.

I can't see how #2 could cause the sawtooth pattern happening for all transitions in Markus's plot (because the scan ranges should only cover 1-3 transitions, y1 and possibly b1/b2). So the plot suggests Skyline IS counting these narrow scan ranges as 0 intensities.

#1 should be relatively easy to fix. #2 is more arbitrary. You'll have to decide whether the current analyzer comboboxes should decide which mass analyzer to look at (with the 'centroid' choice being ambiguous and possibly needing further granularity, e.g. maybe refactor centroiding as a checkbox), or we generate multiple chromatograms for each analyzer type, or something else.