Hello all,

I've been working with Skyline/SkylineRunner to get Skyline running in a quick way for our laboratory.

As part of this process, I have been manually generating decoy peptides and slipping them into our Skyline file, and have run into a problem. For peptides identified closer to the low end of our mass range, ~400 m/z charge 2's, I am getting the following error when decoys are loaded into Skyline:


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System.Reflection.TargetInvocationException: There is an error in XML document (56321, 6). ---> System.InvalidOperationException: There is an error in XML document (56321, 6). ---> System.IndexOutOfRangeException: Precursor isotope [M+2] is outside the isotope distribution -1 to 1.
   at pwiz.Skyline.Model.SequenceMassCalc.GetFragmentMass(String seq, IonType type, Int32 ordinal, Nullable`1 decoyMassShift, Int32 massIndex, IsotopeDistInfo isotopeDists, ExplicitSequenceMods mods) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SequenceUtil.cs:line 1034
   at pwiz.Skyline.Model.SequenceMassCalc.GetFragmentMass(Transition transition, IsotopeDistInfo isotopeDist, ExplicitSequenceMods mods) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SequenceUtil.cs:line 995
   at pwiz.Skyline.Model.DocSettings.SrmSettings.GetFragmentMass(IsotopeLabelType labelType, ExplicitMods mods, Transition transition, IsotopeDistInfo isotopeDist) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\DocSettings\SrmSettings.cs:line 325
   at pwiz.Skyline.Model.SrmDocument.ReadTransitionXml(XmlReader reader, XmlReadContext context, TransitionGroup group, ExplicitMods mods, IsotopeDistInfo isotopeDist) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2763
   at pwiz.Skyline.Model.SrmDocument.ReadTransitionListXml(XmlReader reader, XmlReadContext context, TransitionGroup group, ExplicitMods mods, IsotopeDistInfo isotopeDist) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2713
   at pwiz.Skyline.Model.SrmDocument.ReadTransitionGroupXml(XmlReader reader, XmlReadContext context, Peptide peptide, ExplicitMods mods, DocNodeCustomIon customIon) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2523
   at pwiz.Skyline.Model.SrmDocument.ReadTransitionGroupListXml(XmlReader reader, XmlReadContext context, Peptide peptide, ExplicitMods mods, DocNodeCustomIon customIon) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2459
   at pwiz.Skyline.Model.SrmDocument.ReadPeptideXml(XmlReader reader, XmlReadContext context, PeptideGroup group, Boolean isCustomMolecule) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2282
   at pwiz.Skyline.Model.SrmDocument.ReadPeptideListXml(XmlReader reader, XmlReadContext context, PeptideGroup group) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2201
   at pwiz.Skyline.Model.SrmDocument.ReadPeptideGroupXml(XmlReader reader, XmlReadContext context) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2170
   at pwiz.Skyline.Model.SrmDocument.ReadPeptideGroupListXml(XmlReader reader, XmlReadContext context) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2010
   at pwiz.Skyline.Model.SrmDocument.ReadXml(XmlReader reader) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1961
   at System.Xml.Serialization.XmlSerializationReader.ReadSerializable(IXmlSerializable serializable, Boolean wrappedAny)
   at Microsoft.Xml.Serialization.GeneratedAssembly.XmlSerializationReaderSrmDocument.Read1_srm_settings()
   --- End of inner exception stack trace ---
   at System.Xml.Serialization.XmlSerializer.Deserialize(XmlReader xmlReader, String encodingStyle, XmlDeserializationEvents events)
   at System.Xml.Serialization.XmlSerializer.Deserialize(TextReader textReader)
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass127.<OpenFile>b__125(IProgressMonitor progressMonitor) in c:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 254
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 223
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Util\Util.cs:line 1592
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 171
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 125
   at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in c:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 258
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The way I interpreted the "System.IndexOutOfRangeException: Precursor isotope [M+2] is outside the isotope distribution -1 to 1." follows...

My thought is that the M+2 peak may not be expected to be of interest due to the small peptide having a very low expected signal in the M+2 channel for the naturally occurring C13, but am unsure why Skyline would disallow interrogation of the peak if it was included in the Skyline File-- of course this could be a misinterpretation of what information "isotopeDists.MassIndexToPeakIndex" and "isotopeDists.CountPeaks" are actually returning. Please let me know if there is any obvious issue which is giving rise to this error.

I have included a Skyline file with two decoy peptides appended which both cause the same problem on the M+2 isotope. If any further information will help to narrow down the issue, please let me know and I'd be happy to provide it.

As always-- thank you to all the Skyline team.
All the best,
William