• Sign In
  1. Requests

Discrepancy in MS1 filter between Bruker MaXis raw data and Skyline

support

View Request

view list print
Discrepancy in MS1 filter between Bruker MaXis raw data and Skyline Chris Pynn  2013-03-27 11:35
 
Hi,

I am new to the Skyline software but already a big fan. I am running Skyline version 1.4.0.4421 (with Brukers Compass Xport tool- which all works brilliantly), on a Windows XP operating system.I appear to be having a couple of teething problems with the generated data set though.(The data were recorded on a Bruker MaXis Q-TOF Mass Spectrometer).

1. Skyline sometimes assigns the wrong charge state to a peptide even with good Signal to Noise ratio data.
2. The mass tolerance for the Chromatogram trace in Skyline does not correspond with that in the Bruker Analysis software.With both mass tolerances set to 0.005 Th for example, there are many more chromatographic peaks in the Skyline data (equivalent to setting the mass tolerance to 0.01 Th for the Bruker raw data. Reducing the Mass tolerance in Skyline does not appear to influence the number of identified peaks until a limit is reached when suddenly no chromatographic trace is observed.

If anyone could advise me on this I'd appreciate it.

Thanks again for such a great piece of user friendly software!

Chris Pynn
 
 
Brendan MacLean responded:  2013-03-27 17:19
Hi Chris,
Glad to hear you are enjoying using Skyline in your research. On your issues:

1. Skyline does not assign charge state. I assume you mean it is picking the wrong chromatogram peak for your peptide, and even that the peak it picks looks clearly to you like a peak for precursor of another charge state. Unfortunately, the chromatogram graph you have provided is not zoomed in enough for me to be able to assess what is going on myself. A great deal of our effort continues to go into improving chromatogram peak picking.

2. As "user friendly" as Skyline may be, the "Match tolerance m/z" has to be one of its most frequently misunderstood fields. It was designed to support SRM (and now targeted MS/MS) where m/z values are supplied as instrument parameters, and may be hand edited. In the early days of Skyline, I was seeing a lot of methods with single decimal place precision for Q1 and Q3 in transitions. The "Match tolerance m/z" is employed to match m/z values supplied by the user. For chromatograms extracted from full-scan data, the m/z values are set to the predicted m/z (at float precision). So, the match is currently between the predicted m/z at double precision and at float precision. I would expect the match to work until about 0.0005 for m/z values above 1000 and 0.00005 for m/z values above 1000. This is just an implementation detail (or bug) in Skyline which has nothing to do with instrument accuracy or resolution.

If you want to change the extraction range Skyline uses for extracting signal for chromatograms, then you should focus your efforts on the "Resolving power" fields in the Transition Settings - Full-Scan tab, which control the extraction widths.

This may also be a useful post to read:

https://skyline.gs.washington.edu/labkey/announcements/home/support/thread.view?rowId=3812

It covers some similar misconceptions and points at resources to better understand what Skyline is doing.

Hope this helps. Good luck in your continued investigation of using Skyline for your work.

--Brendan