Welcome to the Skyline support forum. If you have a question about using Skyline, or if you encounter a problem, you can post your questions here.

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  • A detailed description of your problem or question, including instructions for re-creating any problem that you are encountering. Screenshots are often helpful.
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Showing: limited to 100 requests
DIA variable isolation width method
(13 responses) nicakruh 2018-09-14

I'm trying to set a DIA acquisition method with variable width isolation windows for a Q Exactive. I've been able to create this in Skyline and export it into XCalibur, but i'm a little confused on how to set the "isolation window" setting, since it would be variable.
Thanks for your help.

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Different m/z range for precursors and products
user 2018-09-20

Dear Skyline team,

Does the m/z range in transition settings-instrument apply to both precursors and products? If I check “dynamic min product”, what will be the range for products? I have some PRM data from QE series, in which precursor m/z range is 400-1000 but product range is dynamic with minimum of 50.


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"Remove peaks" unexpected behavior on multiple injection runs
(2 responses) juapebe 2018-09-19

On multiple injection runs, if I try to delete a peak (by right click > Remove Peak) that will remove the peak from this specific injection, but then I wont be able to use the same option for the other injections in the same run (option doesn't show in the menu). I have a few peptides that are present in all runs (for references) and in some runs they got truncated so i'd like to delete them altogether. THe way it works currently I am not able to remove these peaks completely for calculations.

This does not happen when the peaks are present in only one of the injections.

Is this an expected behavior? Am I misunderstanding something? See screenshot.

 2018-09-19 06.34.10 pm.png 
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Bug when creating library from pdresult file
(4 responses) paul boersema 2018-09-13

I am trying to generate a spectral library from the data I obtained in Proteome Discoverer 2.2 and realized there must be some bug when using the pdresultfile. When I use the msf files I do not see this issue, but with just the msf files I will not have any way of setting a probability cut-off.

What I tried to do in several Skyline versions (4.1, daily) is to make a library and filter for the peptides that I have in the target list. What I noticed, is that, when I use the pdresult file, for some peptides it comes up with a wrong charge state and retention time while others are correct. It even happens that the incorrect charge state results in a m/z that is lower than the lowest m/z that I set for the instrument. For some peptides it picks a correct/existing charge state and some wrong charge states with an incorrect retention time.
When I, however, take the msf files that were used to generate the pdresult file, the charge state and retention times are correct for all peptides.

I would be happy to provide the msf and pdresult files, but they are in the 100’s of MBs, not sure if I can attach them here.

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Bug in Skyline Daily with clicking on tabs in chromatogram view
(1 response) Will Thompson 2018-09-19

Hi Brendan and Team,

I believe we may have discovered a bug in the skyline daily build *.18257. This has been verified for small molecule and peptide documents by more than user. The bug is simply that if you have the chromatograms in tabbed view, it appears you cannot activate one of the tabs by clicking on it. If you use the replicates dropdown, you can view the chromatogram, but then as soon as you click on the tab of an chromatogram, it defaults back to the very first chromatogram in the document. You can still get to the replicate by clicking on the run in the other windows (replicates intensity and retention time views, for instance) but the tabs simply appear to be inactive.



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Problems starting MsStats as external tool from skyline
(3 responses) tobias.kockmann 2014-03-07
Hi Brendan,

I installed MsStats from the tool store and encountered the following problem:

The execution of MsStats is prohibited because cmd is started with a corrupted path. Here is the output from the immediate window:

'\\vmware-host\Shared Folders\Documents\skyline\20140217_tobiasko'
CMD.EXE was started with the above path as the current directory.
UNC paths are not supported. Defaulting to Windows directory.
The system cannot find the path specified.

I realized that this is the same path which gets displayed as soon as I execute the File->Open. The current skyline doc path is different. Opening cmd manually drops me at my home in C:\Users\<myAccount>

I use Skyline x64 2.5.0. on win7 x64 as guest OS in VMware Fusion

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Assay Library import picks wrong transitions
(9 responses) jfoe 2018-09-17

Dear skyline team,

I have a weird issue when trying to import a custom assay library.
I have set up a skyline document with lots of flexibility in terms of transitions to pick which is probably not a great approach but was supposed to do for now.

When I now import my assay library, skyline will generate the spectral library with peak m/z's as given in the assay library.

The document that is created though, will include transitions which are further off the spectral library than should be allowed (tol. set to 0.001).
I have a peptide for example, that has a y10 transition in the assay library.
This y10 transition is also in the spectral library that gets created.
In the actual transition list of the document, I get instead y11 - 71.1.

While this transition is close by, there is really no reason skyline should pick this, as it is too far off and skyline even sees it as y10 in the spectral library.

I use skyline

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Data points per peak
(2 responses) sstoychev 2018-09-18


Is there a way to extract information on data points per peak from a large DIA dataset so can see the data distribution and if on average there are enough data points to peak to perform good quantitation?

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Peak Area Histogram and 2D Histogram
(6 responses) sstoychev 2018-09-17

Hi guys,

When I try to ploy the Peak Area Histogram or 2D Histogram get a message saying "Not enough data". This is from a DIA dataset with over 15,000 peptides integrated and most with q-values below the cut-off of 0.01 but even without the cut-off still can't plot this data.



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mProphet Modeling for MS1 XIC Analysis
aaron robinson 2018-09-17


I was wondering if you can do mProphet modeling for MS1 XIC analysis of DDA data in Skyline.

Is this something that you guys have tried? I don't see why it wouldn't work and it would make MS1 quant a bit better/more accurate.

I've tried it by adding decoy peptides and training a model and it seems like it works ok...have you guys tried this at all/can you give me any feedback on if it is a reasonable way of filtering MS1 quant data...It seems like it would be the preferred way of analysis for MS1 data, especially since you can set a q-value filter for group comparison analysis.


Aaron Robinson

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Skyline-Daily stops working during results importation in update
(3 responses) stsyp 2018-09-17

Hey Skyline

I just did the new update of skyline. During the import of my small molecule data (Waters.raw files) skyline prompts me with the message it stopped working and I have to close the program. The new vendor reader update is probably causing this issue.
I've put three .raw file in attach to see if you can reproduce the problem, also the skyline document itself is included.

Skyline version: (64-bit)
Operating system: Windows 10 enterprise

Steven Vds 
view request
Points Across Peak - inclusion in reports
(4 responses) norelle wildburger 2018-09-14

Skyline 4.1 now allows Points Across Peak to be visualized, however is it possible to include Points Across the Peak/ #scans for pept. as option to include in report files. This would be particularly useful when optimizing dynamic exclusion.

view request
PRM Small molecules
(3 responses) nicolas drouin 2018-09-15

Dear Skyline team, thank you very much for all work !!!!!

I'm working on small molecule and i would to use skyline to treat my MS-MSMS data.
Basicaly i would like to use known MSMS fragments to confirm identification of the precursor ion in addition of its retention time and its exact mass of course.
Just like in your tutorial about PRM, I would like to have on the same chromatogramm the precursor ion and its fragments.

Could you help me?

Please find enclosed one of my .d files from Agilent (6550). Datas were acquired in DDA with 5 most abondant ions. 3 collision energies (10,25,40) were used for the fragmentation of each precursors. In this file is notably presents serine (m/z: 106.0489) and its MSMS spectra (the main intense fragments are 60.0441; 42.0329 )

Thank you for your help



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QuaSAR - multiple calibration curves
Christoph S 2018-09-14

Hi Skyline team,

I have one question regarding QuaSAR to calculate LOD/LLOQs in Skyline. In my case, I am interested in quantifying a couple of proteins and for each protein, a separate calibration curve exist with different concentrations of the internal standard, all of which are saved in one Skyline project. Is it possible to find an easy way to calculate LOD/LLOQs in a single step using QuaSAR? At the moment it seems, that QuaSAR can only handle one set of an internal standard / calibration curve and you have to export them one after each other.



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Selective peak integration based on idotp
(1 response) Fabian 2018-05-29

Dear all,

i would like to have the option to integrate only peaks above a certain idotp threshold.
At the moment, the option [refine_advanced_results] Min idotp: decides if a peptide is kept or not.
I would like to have the same option based on the peaks not on the peptide.

Did I miss sth. or are there workarounds (peak training model did not convinced me).

Best regards


view request
Compare mProphet models
(4 responses) user 2018-09-12

Dear Skyline team,

I manually selected some high-quality data set (in total ca. 1000 spectra, a small part of them are of moderate quality). I want to compare the models trained by reverse decoy, 1:1 shuffle decoy and 1:3 shuffle decoy with/without “use second best peaks” and try to find the best one from the 6 models to analyse low-quality data set.

  1. Even the “intensity” feature is marked in red, it cannot be unchecked. Why? In this case, for a good model, its positive contribution should be high or low?
  2. In the attached 2 examples with and without checking “use second best peaks”, p and q values look similar. How to judge which model is better?
  3. What are the general rules to select a better mProphet model?


 mProphet models.pptx 
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Generating a library from PEAKS Data Error
(5 responses) JRKrieger 2015-12-17
Hi Brandon,

Hope you are well. Thanks for all you great work. I am trying to generate a library with data that I exported from a PEAKS search. The PEAKS data is a EColi database search of Q-Exactive data, on samples digested with chymo, exported as a pep.xml file.

I am using the most recent release of Skyline for this (not the Daily release) and every time I try to build my library, I get an error indicating NO spectra were found for the new library.

This is the error message I see:

System.IO.IOException: ERROR: No spectra were found for the new library.

   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, ProgressStatus& status, TextWriter writer) in c:\proj\skyline_3_5_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 109
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, ProgressStatus& status) in c:\proj\skyline_3_5_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 53
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, ProgressStatus& status, String[]& ambiguous) in c:\proj\skyline_3_5_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 166
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in c:\proj\skyline_3_5_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 128

I have attached the pep.xml file as well as the mzxml file in case that is useful. I am using a cutoff score of 0 as a test to try and pull in everything.

I am not sure if this is an issue with Skyline, or the PEAKS export, but any advice/suggestions you can offer would be great. This worked quite well yesterday with the Skyline Daily version, and a Trypsin export of PEAKS data.


view request
Peak areas
(1 response) 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.



 Skyline.emf  Freestyle.jpg  Freestyle_2.jpg 
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Issue with Bruker bbCID (AIF) analysis for small molecule
(4 responses) christophef 2018-08-27

Dear support,

I am reporting an issue I have with analyzing small molecule data acquired on Bruker Impact II in bbCID mode.
The idea was to evaluate Skyline for small molecule quantification using MS1, AIF or SWATH. MS1 and SWATH data from Bruker works just fine as expected, but I have a curious issue when importing AIF data. It looks to me like the full scan data with low CE and high CE are just inverted in Skyline, meaning the precursor and the product ion are extracted from the high CE and low CE, respectively. I am expecting the other way around.

Could it be an issue with how Skyline is handling Bruker AIF data file or I am just not doing it right?
I am using Skyline 4.1 and tried Skyline Daily as well. Bruker Compass 1.9 is used to control the mass spectrometer acquistion

Please find attached some slides on the issue I have ( example with 1 small molecule transition)
Slide 1: general view of Bruker data analysis bbCID data
Slide 2: Bruker DataAnalysis vs Skyline, wrong scenario
Slide 3: Bruker DataAnalysis vs Skyline, correct scenario in Bruker DataAnalysis
Slide 4: Skyline mass spectra and transitions setting applied.

Do you have an idea where the issue is and if this has something to do with the Bruker bbCID file structure?

Thanks for your help.


 Issue with Bruker AIF data.pptx 
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Peptide settings with multiple enzymes - assay library and SWATH analyses
(6 responses) Jakub Cervenka 2018-09-04

Hi Skyline team,

I would like to ask you how to set multiple enzyme option in Peptide settings (Digestion). We typically use enzyme combination of LysC and Trypsin in our sample preparation workflow. Is it possible to set this combination in Skyline -> cleave C-terminal to K (always) and cleave C-terminal to R unless followed by P?

We use Mascot as a search engine (LysC+Trypsin digestion, 1 allowed missed cleavage, several modifications…) to create assay (spectral) libraries for SRM and SWATH analyses. I read one of your old answer: “Skyline would generate a spectral library from whatever your search tools output as the peptides it detected for your spectra. The enzyme settings in Skyline are completely irrelevant to this. It is all about how you set up your search engine. The library builder trusts the search results completely.“ ( Do digestion settings (enzyme, max missed cleavages and background proteome) have any meaning for our analyses (especially SWATH)? We have always checked Pick peptides matching: Library and Filter (Peptides Settings -> Library).

Thank you for your help!


view request
(6 responses) henrik molina 2018-09-07

Dear SkyLine (Daily) team
I am analyzing SMALL MOLECULES using high res/high mass acq MS1 data. I generally use ONLY the mono isotopic signal but will like to add M+1. I have a rather long list of molecules (200) where I would like to add M+1. Is there an easy way (not one by one) to add M+1 for all my SMALL MOLECULES?
Cheers, h

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Incorrect retention time window
(6 responses) rocketjishao 2018-09-06

Dear Skyline developers,

I have used Skyline for about one year, but I am still confused about the incorrect rentention time windows that Skyline occasionally gives me.
As you can see in the attachment. The retention time window is cut by Skyline, resulting in a loss of peptide information.
I have ever tried modifying the parameters in peptide settings and transition settings, but the problem still could not be solved, which is really headaching.
Could you please give me some suggestions to resolve the issue?
Thanks a lot.

 incorrect retention time window.pptx 
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Raw files storage path changes or raw data archivial storage
(1 response) ailaria 2018-09-07

Hi Skylien team,

In our lab (Markus Aebi Lab, ETH Zürich) we have two issues with Skyline files as they are based on raw data that apparently have to be in the same storage location (path) as the skyline file...
Issue/question 1) If I change the storage path of my raw data from a location to another after having loaded the skyline files, I cannot open the skyline files again anymore, as the raw data are not found. Is there a way to get around it? like changing manually in the skyline file the "path" of the raw data before opening them again?
Issue/question 2) As we are running into storage problems (because of the huge amount of raw data), we wondered whether skyline read zipped raw data?

Thank you very much for your help, hope I could formulate the questions clear enough...
All the best,
Ilaria Affolter

view request
Publishing Skyline document in Panorama error (small molecule spectral library)
(4 responses) Chris Ashwood 2018-09-05
Dear Skyline Team,

I hope you are all well. When I attempt to publish a Skyline (daily) assay containing a small molecule spectral library via Panorama, I encounter an error:
Failed attempting to create sharing file W:\Sanitized\
Unable to cast object of type 'pwiz.Skyline.Model.Lib.BlibData.DbRefSpectraPeakAnnotations' to type 'pwiz.Skyline.Model.Lib.BlibData.DbRefSpectraPeakAnnotationsRedundant'.
OK More Info
System.Reflection.TargetInvocationException: Unable to cast object of type 'pwiz.Skyline.Model.Lib.BlibData.DbRefSpectraPeakAnnotations' to type 'pwiz.Skyline.Model.Lib.BlibData.DbRefSpectraPeakAnnotationsRedundant'. ---> System.InvalidCastException: Unable to cast object of type 'pwiz.Skyline.Model.Lib.BlibData.DbRefSpectraPeakAnnotations' to type 'pwiz.Skyline.Model.Lib.BlibData.DbRefSpectraPeakAnnotationsRedundant'.
   at (Object , GetterCallback )
   at NHibernate.Tuple.Entity.PocoEntityTuplizer.GetPropertyValuesToInsert(Object entity, IDictionary mergeMap, ISessionImplementor session)
   at NHibernate.Event.Default.AbstractSaveEventListener.PerformSaveOrReplicate(Object entity, EntityKey key, IEntityPersister persister, Boolean useIdentityColumn, Object anything, IEventSource source, Boolean requiresImmediateIdAccess)
   at NHibernate.Event.Default.AbstractSaveEventListener.SaveWithGeneratedId(Object entity, String entityName, Object anything, IEventSource source, Boolean requiresImmediateIdAccess)
   at NHibernate.Event.Default.DefaultSaveOrUpdateEventListener.SaveWithGeneratedOrRequestedId(SaveOrUpdateEvent event)
   at NHibernate.Event.Default.DefaultSaveOrUpdateEventListener.EntityIsTransient(SaveOrUpdateEvent event)
   at NHibernate.Event.Default.DefaultSaveOrUpdateEventListener.OnSaveOrUpdate(SaveOrUpdateEvent event)
   at NHibernate.Impl.SessionImpl.FireSaveOrUpdate(SaveOrUpdateEvent event)
   at NHibernate.Impl.SessionImpl.SaveOrUpdate(String entityName, Object obj)
   at NHibernate.Engine.Cascade.CascadeToOne(Object parent, Object child, IType type, CascadeStyle style, Object anything, Boolean isCascadeDeleteEnabled)
   at NHibernate.Engine.Cascade.CascadeCollectionElements(Object parent, Object child, CollectionType collectionType, CascadeStyle style, IType elemType, Object anything, Boolean isCascadeDeleteEnabled)
   at NHibernate.Engine.Cascade.CascadeCollection(Object parent, Object child, CascadeStyle style, Object anything, CollectionType type)
   at NHibernate.Engine.Cascade.CascadeAssociation(Object parent, Object child, IType type, CascadeStyle style, Object anything, Boolean isCascadeDeleteEnabled)
   at NHibernate.Engine.Cascade.CascadeOn(IEntityPersister persister, Object parent, Object anything)
   at NHibernate.Event.Default.AbstractSaveEventListener.CascadeAfterSave(IEventSource source, IEntityPersister persister, Object entity, Object anything)
   at NHibernate.Event.Default.AbstractSaveEventListener.PerformSaveOrReplicate(Object entity, EntityKey key, IEntityPersister persister, Boolean useIdentityColumn, Object anything, IEventSource source, Boolean requiresImmediateIdAccess)
   at NHibernate.Event.Default.AbstractSaveEventListener.SaveWithGeneratedId(Object entity, String entityName, Object anything, IEventSource source, Boolean requiresImmediateIdAccess)
   at NHibernate.Event.Default.DefaultSaveOrUpdateEventListener.SaveWithGeneratedOrRequestedId(SaveOrUpdateEvent event)
   at NHibernate.Event.Default.DefaultSaveOrUpdateEventListener.EntityIsTransient(SaveOrUpdateEvent event)
   at NHibernate.Event.Default.DefaultSaveOrUpdateEventListener.OnSaveOrUpdate(SaveOrUpdateEvent event)
   at NHibernate.Impl.SessionImpl.FireSave(SaveOrUpdateEvent event)
   at NHibernate.Impl.SessionImpl.Save(Object obj)
   at pwiz.Skyline.Model.Lib.BlibData.BlibDb.SaveRedundantSpectra(ISession sessionRedundant, IEnumerable`1 redundantSpectraIds, IDictionary`2 dictFiles, DbRefSpectra refSpectra, Library library, ISet`1 savedSpectraIds) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BlibData\BlibDb.cs:line 602
   at pwiz.Skyline.Model.Lib.BlibData.BlibDb.MinimizeLibrary(BiblioSpecLiteSpec librarySpec, Library library, SrmDocument document, IDictionary`2 smallMoleculeConversionMap) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BlibData\BlibDb.cs:line 501
   at pwiz.Skyline.Model.Lib.BlibData.BlibDb.MinimizeLibrariesHelper(SrmDocument document, String pathDirectory, String nameModifier, IDictionary`2 smallMoleculeConversionInfo, Dictionary`2 dictOldNameToNew, IProgressMonitor progressMonitor) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BlibData\BlibDb.cs:line 1026
   at pwiz.Skyline.Model.SrmDocumentSharing.ShareMinimal(ZipFileShare zip) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocumentSharing.cs:line 287
   at pwiz.Skyline.Model.SrmDocumentSharing.Share(IProgressMonitor progressMonitor) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocumentSharing.cs:line 187
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 228
   --- 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 1846
   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 176
   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 131
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass16a.<ShareDocument>b__167() in c:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1140
   at pwiz.Skyline.Util.Helpers.Try[TEx](Action action, Int32 loopCount, Int32 milliseconds) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Util\Util.cs:line 1818
   at pwiz.Skyline.SkylineWindow.ShareDocument(String fileDest, ShareType shareType) in c:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1143

I do not encounter this issue when I instead Share the document in a complete format, so I'm guessing it is specific to the Panorama import. This is a sensitive dataset so I can email it if you would like the Skyline assay files.

view request
Flexible transition import
(3 responses) jfoe 2018-09-04

Dear skyline team,

I am working with peptides and I have a need for very flexible transition lists including internal fragments, loss of water etc. in different combinations.
So I am trying to use the assay library / transition list import.

Wator Loss and Amonia Loss as configurable in skyline has been very helpful.
I can't seem to make import of internal fragments work though.

I could give chemical composition etc. if needed.

Is there a way to do this?
I would also supply my own Annotations so it's fine if skyline loses track of y, b ions etc.

I use skyline 4.1.
Help would be much appreciated as skyline has some great features for interactive work!

view request
missing Detection Q values
(2 responses) joli 2018-09-04

Hi Skyline Team,

I'm trying to use the MSstats "design sample size" function on my DIA proteomics dataset to do a power analysis and I keep getting this following error:

"Some DetectionQValue have no value. Please check "Result Grid" in View.
Error in runDSS() :

Can't finish analysis."

I checked the Document Settings Grid and exported the csv file and found that more than half of the peptides have #N/A Detection Q values. I am not sure how to troubleshoot this problem. I am running Skyline (64-bit) version and I've uploaded my Skyline document.

Thanks in advance for your help!


 detectionqvalue error message.txt 
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Issue with shared metabolite document
(1 response) Rob Goode 2018-08-30

I've created some parent ion Skyline documents using either metabolites or peptides, but when I share them with collaborators only the peptide-based ones open. The metabolite-based documents fail to open with the following errors:

System.Reflection.TargetInvocationException: There is an error in XML document (217, 8). ---> System.InvalidOperationException: There is an error in XML document (217, 8). ---> System.InvalidOperationException: Failed parsing adduct description "[M+HH]M+2O[31]"

System.Reflection.TargetInvocationException: There is an error in XML document (364, 10). ---> System.InvalidOperationException: There is an error in XML document (364, 10). ---> pwiz.Skyline.Util.AssumptionException: error reading mz values - declared mz value 592.167741 does not match calculated value 592.671653727269

I've recreated the metabolite documents as (heavily modified) peptide based ones and they work fine. Wondering if this is a common issue or a niche case, as my collaborator is running skyline on Mac in a emulator?

Thanks Rob

view request
Quantitative proteomics using Skyline
nakaokah2 2018-09-04
Dear Skyline Team,

I am a quite novice in mass-spec field,
but I plan to do label-free (relative) quantification of entire proteomes between different strains of bacteria (S. aureus).
If I correctly understand, MS1 Filtering using DDA data should work for our purpose.
But I have also noticed a paper that describes DIA-based quantitative proteomics for the species of our interest (They do not use Skyline, though).
Because a spectral library for DIA analyses are provided in the work,
I am wondering if just running DIA experiments might be a better choice than DDA/MS1-filtering method.

Which is a recommended strategy in terms of
> quantification abilities such as accuracy, precision, and reproducibility (Note that we are going to use Q-Exactive Plus).
> ease of data analyses with current version of Skyline software.

I greatly appreciate any suggestions/comments on the issue.

Best regards,

view request
Schedueld SRM data analysis problem
(2 responses) kallo gergo 2018-08-15

Dear Skyline team!

I have a problem with scheduled data analysis in Skyline and I would like to ask your advice. I am analyzing small molecules in scheduled SRM mode in an ABSciex 4000QTRAP instrument. My samples are derivatized and I monitor the end product of the reaction. After the CID I check the derivatization fragment so the Q3 is the same for all transitions. I am using stable isotope-labeled compounds and because of the different isotopic labels the m/z of several parent ions is the same. I can register the signal for all of my transitions but the problem comes when I want to analyse the data in skyline. For those molecules which have exactly the same transition but has different retention time I only see data for the first retention time window in skyline. In that case I cannot integrate those peaks which elute at another retention time. I tried to find some solutions in the options of the skyline but I cannot fix this problem.
Maybe I just missed something, could you please help me to fix this problem?

Yous sincerely,
GergÅ‘ Kalló
University of Debrecen
Department of Biochemistry and Molecular Biology

view request
Library creation ms-amanda
(1 response) Brett Phinney 2018-08-31

hi everyone, has anyone been able to create a library from ms-amanda run through PD 2.2?

When I try the msf files or pdresult files I get an error about there being no q-values (I use the default ms-amanda optimized elucidator for the q-value calculation .

If I export the mzid from PD 2.2 I get a weird error invalid cvParam

Any ideas?



 amanda error.PNG  ms-amanada.PNG 
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How to analyze Label-free SRM data ?
(1 response) VJ 2018-09-02

Hi, Brendan et al.,

We run our samples (with replicates) in Agilent QqQ, then import .d file to Skyline. Then we go through all the parameters for manually exploring, correct and refine the picked SRM chromatograms. Finally, export a custom-defined report as .csv.

From here onwards how can I analyze the data? We do Normalization across all peptides for each sample and then scaling each peptide across all the sample. Do you recommend this procedure?
(Raw data example attached; this is how my exported data looks)

I want to know, how you all doing the data analysis after exporting the data to excel, importantly, because for the same protein peptide behavior is different (diff due to flying?)
for e.g. Protein "A", have peptide; a1, a2, a3, Protein "B" have peptide b1, b2, But when each peptide peak area is different; a1=100, a2=800, a3=2000, how can we average our peptides to a protein?

How to compare, Wildtype vs mutants.

I'd appreciate good example tutorial if available. Also, if you are using R Scripts, would love to have a copy to see?

Thank you for considering my request and looking forward to hearing from you ASAP.


view request
Retention time alignment - endogenous retention time standard
(4 responses) rle041 2018-07-18

Dear Skyline team,

We are currently working on generating a spectral library to use for DIA in cerebrospinal fluid. We have run three DDA runs prior to our DIA runs, and I have searched the data in proteome discoverer and imported it as a library in Skyline through the File > import > Peptide search workflow for MS1 filtering (no iRT standards specified). From these samples, I have picked 9 peptides to use as a retention time calculator (endogenous peptides that are high abundant and in every sample, and that are dispersed over the LC-gradient).

However, when I create my RT calculator on these 9 peptides, and want to add the other entries from my spectral library, only 4 points are used for regression.

It seems the peptides that are eluting first are the ones that fail to align, but in the regression model these peptides are "missing" even though they are picked from the very same files. Why is this so? Are these 4 points enough to do an alignment to the other IDs in my spectral library?
I would also like to do this for a bigger library that have been run on a previous column using the same workflow, is this a good workflow to do this?

I have attached screenshots of the regression and the coefficients,

Best regards,


 RT_align_SpectralLib_Coefficients.png  RT_align_SpectralLib.png 
view request
PRM for small molecule
(1 response) abhinav kumar 2018-08-29
I have a forthcoming project for small molecule targeted quantitation on Thermo Q-Exactive using PRM method. I was going through your tutorial on Skyline for small molecule but it was for SRM/MRM. Could you please help me with any example of PRM for small molecules on Q-Exactive mass spec.
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how to make sure the skyline only picks up peak in 10ppm
(1 response) dengjingjing01 2018-08-29


I am using Q Exactive to do PRM running. After result importing, I found in the default setting, sometimes skyline picks up precursors at 30ppm. Although there are some fragments matched to the library, I don't think that is my peptide since the precursor error is too big. Is there any setting I can narrow the precursor to 10ppm? I am using the public library, so I could not use the retention time as a criteria to filter out false positive matches.



view request
Can not see mass acq of extracted MS signals in chromatogram window
(3 responses) henrik molina 2018-08-29

We are on most recent version of Skyline Daily. After the recent update we can no longer see the mass accuracy on the extracted LC-MS signal - only RT is displayed. Pls let us know how to get mass acq back.

Cheers, h

view request
spectral library creation problem
(4 responses) egulyas 2018-08-28

Dear Brendan,

I would like to use Skyline(64-bit, for quantitative
proteomic analysis. I have managed to create a spectral library
already several times, but now I am unable. I use ProteinProspector
for DDA database search and generated pep.xml files, but the Skyline
said: "ERROR: 180802_03.pep.xml(line452): The .pep.xml file is not
from one of the recognized sources".
Could you suggest what should I change?

I attach the pep.xml file.

Thanks for your help in advance,

Eva Hunyadi-Gulyas

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Surrogate Standard display
(1 response) blaine roberts 2018-08-18

I am using a Surrogate standard and have set it up in the document grid etc. and can export the values as well, but is there a way to display the normalized to surrogate standard peak area for the analyte? The Normalised option only have total, heavy and maximum when I right click on the peak area graph. Maybe I have just missed it some where.



view request
Thermo Raw files import speed
(4 responses) evgeny onishchenko 2018-08-22

Dear Skyline Team,

We are testing our new cluster to make it perform optimally for various tasks including Skyline (Skyline Daly). To this end we have compared import time of the same raw
DIA Thermo files stored either on high-speed network or on the cluster’s local SSD and, second, did the same test with a single desktop (raw files on local SSD vs high-speed network, less cores and less RAM than on the cluster).
We observed that the import time was basically independent in the processing power and the amount of available RAM, but was dramatically dependent on where
the files are accessed from. In both tests local SSD performed ~10 x faster than the high-speed network (~ 3 min vs 30 min).
My question is what would be the best way to increase Skyline performance with cluster and take advantage of its high processing power?
Would it be possible to run Skyline in a way that it relies on RAM when processing the raw files?

Thanks so much in advance for your help and advise!

view request
(5 responses) sstoychev 2018-08-22

Am I correct that correction for multiple testing for group comparisons within Skyline is done using the Bonferoni method? I assume this is because most MRM/SRM experiments will have a small number of "tests" to correct hence Bonferoni does not result in a too strict of a cut-off?

In the case of DIA experiments Benajmini-Hocjburg would be the preferred method for multiple testing correction. I think this is the method used by the external MSStats tool? Is it possible to add Benajmini-Hocjburg as an option to group comparisons within Skyline or is this too much work to do?

view request
(3 responses) brown459 2018-08-23

I am trying to set up a skyline method that I can use to port data to panorama so that I can share data with a collaborator.

I should describe what I am attempting to do.

I have a collaborator that has some samples and a control. I am trying to generate a DIA project that I can quantify the peptides found in my previous DDA searches (Using DIA to increase my coverage). Then I want to be able to share this data with my collaborator. She is interested in the relative abundances of the different proteins/peptides in the different samples.

I have so far made a skyline document that has the concatenated DDA runs (from all files) and am in the process of acquiring the DIA data from those same samples. I am of the opinion that the DIA will give me better confidence and quantification overlap.

I am not sure if I am setting my skyline document up wrong/or my panorama page up wrong— I would love any help!

I have attached the method that I was planning on using. And am in the process of running the DIA experiment.

I have already reached out to Vagisha and she mentioned that I should also post here.

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Modification not resolved
(4 responses) Gereon 2018-08-22

Dear support team,

I tried to quantify a few petides with the DDA with MS1 filtering worklflow (Skyline Here I would like to quantify also two variable cysteine modifications (NEM and its heavy labeled variant NEMD5). As for some peptides there might be also combinations possible I tried to define both as structural (variable) modifications. The NEM modification is resolved pretty well, but the heavy one (NEMD5) is not recognized by skyline althought the corresponding peptide identification is present in library. Here, it is written that the modification (C[+130.07632] ) cannot be resolved. The theoretical shift is +130.079062.
The data (aquired by a QExactive plus) has been searched by Maxquant (version I already tried to rename the modification both in skyline, the msms.txt and modifications.xml, but this did not solve the issue.
I'd be grateful for any help.



 screenshot1.tif  screenshot2.tif  screenshot3.tif  analysis 
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Group Comparison
ashokprabhus007 2018-08-23

Dear Skyline Team,

please briefly explain what is the difference between the 'Group Comparisons' tools available in Skyline and the external MSStats. For a DIA discovery experiment what is appropriate - peptide or protein level group comparison. I get more associated protein identifications at the peptide level group comparisons and almost a 4th when I do it at the protein level.

Thanking you,

Best ergards,

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How to set up explicit retention time window?
(4 responses) kang-yu peng 2018-08-21

Dear Skyline Team,

Thank you for developing such a wonderful software and offering it for free.

I just started using Skyline two weeks ago to do targeted lipidomics. I have searched a few small molecule tutorials, but could not find the input format for the explicit retention time window column. I will need to pick up several (like 3 in some samples) peaks within a selected time range, so the retention time window will be handy. Please let me know how that can be done, or point to the right tutorial to look at for me.

Best Regards


view request
Integration chosen from light or heavy peptide
(4 responses) cilento 2018-08-22


Greetings from the lab of Dr. Jing Zhang here at UW. I am new to SRM quantification and have a question regarding differences found between my analysis and another member of the lab. We have a panel of peptides in which heavy synthetic peptides have been spiked in to aid in the light detection (pretty standard). In the targets that worked well (peaks clearly detectable in both light and heavy, well above background, and all transitions are detected good), we get identical results, however, in the peptides which are close to detection limit in the light, we found that our values are very different if the integration is dragged from the heavy peptide or the light peptide to guide the other corresponding partner. If dragged from the light partner, peak area replicate comparison demonstrates all transitions detected quite readily (even if some are close to background noise), however if dragged from the heavy partner, often Skyline seems to use some kind of algorithm to determine that some transitions in the light may be background (as they don't show up in in the peak area replicate comparison).
Mainly, I am trying to determine which approach is best for generating a "real" signal in the light- forcing all lights to generate transition signals (which may perhaps be noise), or use the heavy to guide that light signal which may in turn not detect transitions at times (determined somehow by the software). Either way, signal to noise criteria will be applied to the peptides, but advice on this starting point would be very helpful.
Thank you!


view request
Skyline DIA workflow and MSstats external tool
(2 responses) ashokprabhus007 2018-08-21
Dear Skyline Team,

1. I have just started working with SWATH analysis. I wanted to check the biological variation in my system. Hence, I isolated proteins from the roots of 6 plants of the same cultivar and age and subjected them to DIA (80 variable window, TTOF 6600). I am using a spectral library prepared from the DDA runs of the same sample pools injected 6 times. Please let me know if I can use the peak area histograms as an indicator of the biological variation. I am getting a median of 19.4% (Below 20%: 52.3%) for the products and for the precursors it is 33.5% (Below 20%: 28.2%). Which one should I consider? Does it mean that only 28.2% of my proteins can be reliably quantified at the protein level?

2. I also did another experiment where I isolated proteins from 3 healthy and 3 diseased roots as a pilot study and did both a DDA and DIA analysis, as above. Control MSstats as an external tool: I could successfully install the tool but I am not able to do the group comparison using MSstats (Skyline's group comparison works). What should be my MSstats input settings in Skyline? Should I use MS1 or MS2 for group comparison? I get the following error.

""C:\Program Files\R\R-3.5.0\bin\R.exe" -f "C:\Users\u15407218\AppData\Local\Apps\2.0\QJGPLL6E.O5C\LTQ26TN4.44Z\skyl..tion_e4141a2a22107248_0004.0001_8a31a35b7206059e\Tools\MSstats-3.13.6\MSStatsGC.r" --slave --args "C:\Users\u15407218\AppData\Local\Temp\MSstats_Group_Comparison_MSstats_Input.csv" ControlvsInfected 1 FALSE FALSE FALSE -1 1

 ** Loading the required statistical software packages in R .....


 ** Reading the data for MSstats.....

** iRT proteins/peptides are removed.

** Peptides, that are used in more than one proteins, are removed.

** Truncated peaks are replaced with NA.

Error in SkylinetoMSstatsFormat(raw, removeProtein_with1Feature = TRUE, :

  ** Please check precursors information. If your experiment is DIA, please remove the precursors. If your experiments is DDA, please check the precursor information.

  Can't finish analysis."

Regarding determination of sample size, I tried the analysis using Skyline external tool option and once again I get an error message. What should be my MSstats input settings in Skyline? Following error:

""C:\Program Files\R\R-3.5.0\bin\R.exe" -f "C:\Users\u15407218\AppData\Local\Apps\2.0\QJGPLL6E.O5C\LTQ26TN4.44Z\skyl..tion_e4141a2a22107248_0004.0001_8a31a35b7206059e\Tools\MSstats-3.13.6\MSStatsDSS.r" --slave --args "C:\Users\u15407218\AppData\Local\Temp\MSstats_Design_Sample_Size_MSstats_Input.csv" 1 TRUE 0.80 0.05 1.25 1.75 FALSE FALSE FALSE
 ** Loading the required statistical software packages in R .....
 ** Reading the data for MSstats.....
** Peptides, that are used in more than one proteins, are removed.
Error in `$<`(`*tmp*`, "Intensity", value = numeric(0)) :
  replacement has 0 rows, data has 129474

 Can't finish analysis."

Thanking you,
Best regards,
view request
Issue with Apply Peak to Subsequent
(2 responses) segri 2018-08-20

When I right-click and choose "Apply Peak to Subsequent," all my samples undergo peak boundary refinement. In essence, selecting this function is having the same effect as the "Apply Peak to All" option.

view request
Integrate fixed window
(1 response) meowcat 2018-08-21


is there a way to set Skyline to not perform any peak detection, but just integrate from a fixed time t1 to t2? I have a few cases where this is a relevant use case.

I know I can do that manually for a substance (small molecule transition or I guess also peptide) by setting a peak window and then doing "Apply to All". However, to do this for 20-100 substances, for every batch I analyze, is very time-consuming.

The transition properties "Explicit Retention Time" and "Explicit Retention Time Window" are apparently not for this, right? They will just choose where to look for a peak. Maybe one could add a checkbox "use fixed window integration" for each transition.

Thanks for the great work, by the way!

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Precursor extraction for small molecules
(8 responses) Bing 2018-08-13

Dear Skyline team,

I have a question about how Skyline extracting fragments. For lipids, sometimes precursor mass still appear in MS2 spectral. When I try to extract the precursor from MS2, it failed.
In attachment, please find the screenshots for lipid analysis as example. On the left side of the figure1, it is a measurement contains full scan and PRM. On the right side, it is a measurement only contains PRM. As you can see, there is no fragment as “precursor” for measurement 2. But I checked the raw file with Xcalibur, the precursor mass (885.5491) showed in MS2 spectral (Figure 2).

It seems the precursor only can be extracted from MS1 level. Is it possible to extract precursor from MS2?

Thank you very much!


 Figure1 precursor extraction.jpg  Figure2 MS2.jpg 
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Skyline crashes: Regression - Score to run
(6 responses) gpimienta 2018-08-15

I am all of the sudden having trouble with Skyline every time I try running a Regression - Score to run calculation. Skyline crashes. I have uninstalled and re-installed it. I also have the latest version.
Skyline seems to be having trouble interacting with Windows according to the message given. Problem event name: AppHangB1.
Any help with this?

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how do I delete the results imported in skyline
(1 response) lourdes thevanayagam 2018-08-16

Hello Brendan,
How do I delete the results imported in skyline
Because if I need to compare the peak areas from a set of chromatograms and some of them if I want to exclude and the ones I don't need them still showing up the peak comparison, because the data still uploaded.

Thanks for your help

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Skyline transition settings for DIA with Waters MSe SONAR
(2 responses) christophef 2018-08-15

Dear Skyline users,

Can someone help me with understanding and choosing the right settings to process Waters DIA data acquired in MSe with SONAR option?
So far, I was always using Acquisition method: DIA with Isolation scheme: All Ions, when using Waters MSe data.

I have recently added SONAR acquisition mode: Quadrupole scan, 400-900 m/z with 50 Da windows, which means according to Waters terminology, 200 bins/500 Da * 50 = 20 bins.

I was wondering if I need to process this data as SWATH (with windows of 20Da) or still as All ions?
The "SWATH" approach does not seem to work in extracting MSMS data.

Any recommendation or confirmation about the isolation scheme to chose in SKYLINE?

Thanks in advance for your help

Christophe Folly

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Search for specific records in "targets" list
(2 responses) evgeny onishchenko 2018-08-15

Dear Skyline team,

I am using Skyline 4.1. After processing DIA Results with large DDA library (~ 1000 proteins) I get summary in a “Targets” window. Now I am interested in browsing the chromatograms for some specific proteins among these 1000 targets (e.g. YPL210C and YER151C). Is there any way to filter this targets list in this window with some kind of “Find” function to quickly allocate these records? Now I have to manually scroll through the whole list to find them.

Thank a lot in advance for your help!

view request
train mProphet models for peak picking only using stable-isotope labled standard
(3 responses) a. schroeder 2018-08-10
Dear Skyline Team,

I am new to Skyline and my question is of general nature:
Is it possible to train mProphet models for peak picking using only the stable-isotope labled standards and ignoring the light targets?

We are using PRM data consisting of hundrets of sample-sets, each set with the same peptides but in varying concentrations.
Spiked-in to each sample-set are standaridized amounts of stable-isotope labled standards for each peptide.
Our idea is to train a model on the stable-isotope labled standards (which should be comparable across the sample-sets) and subsequently apply it to the light targets.

The size of our data-sets doesn't allow us to measure decoy peptides in parallel, can we use the "second best peak" option when using only the stable-isotope labled standards?

Thank you very much for your effort!
view request
error while building library from msms.txt
(6 responses) zeyusun 2018-08-14

Dear Skyline team

Since I updated the Skyline to version, I have issue to build a library using an msms.txt file generated by MQ (version on QE and QE-HFX raw data, I get the error message looks like this:

System.Exception: Failed trying to build the redundant library K:\rabbit\prm\GGG.redundant.blib.

I have put the mqpar.xml to the same folder of msms.txt. I have also tried different datasets, which produced the same error.

Any advice will be much appreciated.

Kind regards


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No transition ions found
(4 responses) as3e15 2018-08-13

Dear Skyline team,

I am doing an MRM experiment and am having trouble with one of my peptides.

I have created a spectral library from my samples and used it to identify peptides and transitions. I have ran a dilution series to get a calibration curve and its has worked on all peptides except one. it seems skyline cant find any transition ions for a specific peptide.

If it was just in my heavy i would think that perhaps there is just no transitions present but there isn't any in my light either. the light is the same sample i used to create my spectral library so i would expect to see it.

i have the transitions settings set to targeted acquisition. i have product ion selection = from ion 1 To last ion (i thought this would help but it didn't)

why cant skyline find the transitions?

i have attached a screenshot to help.

Thank you in advance,


 no transitions.png 
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Small Molecule MRM extraction with isomers
(3 responses) wbarshop 2018-08-13

Hello Skyline team,

I have run into an issue where transitions which are acquired in our method are not properly being associated to the small molecule inside Skyline. This results in "empty" transition data.

More long-winded:
I have been working on a project where we are quantifying small molecules which exist as multiple isoforms and are separable chromatographically.

We optimized collision energies via direct infusion, and started by utilizing the observable fragments and their empirical masses. Of course, when we switch from isoform to isoform for this process on our QQQ, the masses reported back vary a little, even if the fragments being monitored are the same.

My suspicion [based on very little] is that when we have multiple small molecules where transitions exist with the same (or very similar) m/z values for both the precursors and fragments, the matching behavior of Skyline (via the "Method match tolerance m/z" setting) may be getting tripped up and leaving out data.

I have included a simplified Skyline document with four analytes. All share the same precursor m/z, several of them share all transitions (often within the limit of the method match tolerance m/z setting). In this case, you can see the "Deoxycholic acid" analyte loses its transition extraction (despite skyline having the exact m/z values stated by the instrument method-- though I know some values like monoisotopic m/z and average m/z seem to be float representations and not precise).

In this Skyline file, you'll notice that Deoxycholic acid fails to extract its fragment ions. If you modify the small molecule and remove the explicit retention time, you'll see that we extract the peaks from the "3b12a" molecule, as they are within the method match tolerance.

Even, though, if I drop the method match tolerance to be very narrow, I cannot seem to recover the extraction of the proper MRM data for "Deoxycholic Acid."

I am included here the simplified skyline file, along with the same RAW file which is imported into the sky file. Please let me know if you need any additional information or data, I will be happy to provide.


 2018-08-13  2018-08-03-microflow-16min-30ms-SRM-NEAT.raw 
view request
(1 response) user 2018-08-12

Dear Skyline developers,

I’d like to use some high-quality data set to train a mProphet and directly use this model (do not train anymore) to analyse another low-quality data set (from same experimental condition). Does Skyline support this? I tried but failed. Do I have to add decoy peptides as well for the low-quality data set?

In addition, in the trained model (see attached), the feature “intensity” has negative weight and positive contribution. It is marked in red but cannot be unchecked. How to deal with such feature?

Kind regards,

view request
Issue with install of Skyline version 2.6 instructions but 4.1 available only
(1 response) yadira cantres 2018-07-10

we are in the process of using your paper Multiplexed Peptide Analysis using Data Independent Acquisition and Skyline on our Thermo Q Exactive Plus with Easy-nLC 1200. This paper references version 2.6
At this time the current version is Skyline 4.1. When following your paper I got stuck on Step 18 in Thermo Xcalibur Instrument setup as it asks me to import Targeted MS2, and the closest option is Targeted-SIM / dd-MS2. we are unsure if this is the appropriate option and if so should the SIM values match the dd-MS2 values or be left at factory settings.

Do you have an update reference paper, updated manual, or any suggestions otherwise.

Please see attached screen shot for step 18 hang up on Targeted MS2 option.

Thank you for your help.

 UPR Skyline install issue July 10 2018.docx 
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CE equation for Sciex TripleTOF 6600
William 2018-08-12

Hi Bredan,
We plan to do PRM on some phophopeptides with Sciex TripleTOF. Unfortunately, Skyline doesn't have Sciex TripleTOF in the instrument list. I wonder if there's any equation I can put in?


view request
Selecting for a structural modification
(6 responses) sstoychev 2018-08-09


What will be the best way of selecting for a structural modification. For example, I'm interested in seeing how all N-terminal acetylated peptides are behaving in the samples. I know how to select these in Skyline (Peptide settings - Modifications) but how would you remove all other peptide sequences i.e. other than N-terminal peptide. Ideally I would also want to see ratio of acetylated vs non-acetylated for same peptide if possible, if not looking at levels of acetylation between samples will be sufficient.

Perhaps the Edit - Exclude peptides (not matching expression) will allow for selection of N-terminal acetylated peptides only?


view request
Handle low quality library
(1 response) user 2018-07-31

Dear Skyline developers,

I find in some cases, even the library of the “best replicate” is not good enough and thus dotp values might not be accurate. If I want to replace a few low quality spectra for some peptides in a library with high quality library spectra from PeptidesAtlas etc., does Skyline support this? Can Skyline remain the old RT in this case?


view request
enable all parameters in mProphet model
(2 responses) poliakh 2018-08-10

Hello Skyline team,
I have a question about mProphet model. Sometimes mProphet does not count with several parameters(library intensity dot-product, precursor-product shape score, intensity etc). Is it possible to enable all features? I use Skyline Daily.
See examples in an attachment.
Thanks and have a nice day,

 mProphet features.pdf 
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Peak picking
(2 responses) Yasin 2018-08-02

Dear Skyline Team,

I hope you can help me with some questions.

I have a targeted method for about 100 different small molecules (high resolution instrument) and would like to screen for each of them for about 20 different adducts. For most of those compounds I know already the retention time. Via the explicit retention time column in the transition list I can now force skyline to pick the peak closest to this retention time.

So I have the following questions:
However, how can I prevent skyline from picking a peak with a different retention time if no peak with the expected RT is present?
And also: Is it possible to prevent skyline from picking spikes (consisting of only 1 to 3 data points as peaks?
Moreover, it would be nice to be able to tell skyline to just integrate from e.g. 14.4 to 14.6 ignoring if there are spikes, a peak or nothing at all in that range.

Thank you a lot in advance!


view request
Skyline's Statistical Analysis Function
(1 response) johnny perez 2018-08-10

Quick question about the statistical data generated by Skyline. When analysis unscheduled data for example, how does Skyline determine the integration window for each peptide? I know that the window can be modified by the user, but what is the default setting set by the software? (2-4) times the FWHM?

view request
Loss of adjusted peak bondaries after copy paste
(1 response) stsyp 2018-08-09

Hey Skyline

After I've imported data and adjusted the selected peaks/peak boundaries of the targeted small molecules, I wanted to copy all of these so I can rearrange them to certain criteria (according to retention time window, according to abundance, according to peak shape,...). So one target molecule can be in two or more groups.
I prefer not to make already the groups while importing the transition list as this would mean I have to manually adjust the selected peaks in every group again.
Is there a known workaround I can apply/ is there a setting that I forgot to enable?
There is short ppt presentation to make clear what I mean.

Steven Vds

Skyline version: (64-bit)
Operating system: Windows 10 enterprise

view request
Cannot correctly install MSstats to Skyline
(4 responses) natbat 2018-08-07


I need MSstats for data analysis, but whatever I do to install it does not go correctly.

At first, MSstats was installed more or less, but when I tried to run QC, it gave the error message that
pwiz.Skyline.Model.Tools.ToolExecutionException: Error running the installed tool MSstats\QC. It seems to be missing a file. Please reinstall the tool and try again. ---> System.IO.FileNotFoundException: Could not load file or assembly 'file:///C:\Users\natbat\AppData\Local\Apps\2.0\KN6PMERL.PTM\1K88DWB8.VY9\skyl..tion_e4141a2a22107248_0003.0006_46da8c86f5903517\Tools\MSstats_external_3.6.1\MSStatArgsCollector.dll' or one of its dependencies. The system cannot find the file specified.
at System.Reflection.RuntimeAssembly._nLoad(AssemblyName fileName, String codeBase, Evidence assemblySecurity, RuntimeAssembly locationHint, StackCrawlMark& stackMark, IntPtr pPrivHostBinder, Boolean throwOnFileNotFound, Boolean forIntrospection, Boolean suppressSecurityChecks)
at System.Reflection.RuntimeAssembly.InternalLoadAssemblyName(AssemblyName assemblyRef, Evidence assemblySecurity, RuntimeAssembly reqAssembly, StackCrawlMark& stackMark, IntPtr pPrivHostBinder, Boolean throwOnFileNotFound, Boolean forIntrospection, Boolean suppressSecurityChecks)
at System.Reflection.RuntimeAssembly.InternalLoadFrom(String assemblyFile, Evidence securityEvidence, Byte[] hashValue, AssemblyHashAlgorithm hashAlgorithm, Boolean forIntrospection, Boolean suppressSecurityChecks, StackCrawlMark& stackMark)
at System.Reflection.Assembly.LoadFrom(String assemblyFile)

I tried to re-install MSstats. I went Tools --> Tool store --> MSstats --> Reinstall but it seems nothing happend. I am not even sure that oit was really un-installed and installed again. there was no messages confirming that.

Then I went to Tools --> External tools --> Menu contents, and Removed MSstats related tool. After that I went again to Tools --> Tool store --> MSstats, and pressed Install.
Something was installed. MSstats\Data processing, group compatrison and Design samle size appeared. But the QC option was missing.

Then I went to MacCoss lab site --> External tools and downloaded the zip file. However, when I try to extract files, it shows problems with encripting the files.

I do not understand what else can I do.
Please help, I need this tool for data analysis.


view request
Request to allow Skyline to handle 'special characters' in files
(1 response) Tim McCubbin 2018-08-09

Dear Skyline team,

Apologies for the somewhat silly request but we have been sent raw files for analysis a number of times by people who insist on using the micro symbol in the file names (e.g. to specify the concentration or mass of protein in the sample or volume injected into the MS). This appears to create issues for Skyline, i.e. DDA files can't be imported via Import -> peptide search. We are also currently experiencing difficulties with DIA files with the micro symbol in the name; which import okay but peak quantification appears to fail; although we are yet to really demonstrate that this issue is caused by the 'micro' character. Simply renaming the files is not sufficient; it appears that raw files 'know' what they were originally called. We are looking into possible ways around this and would appreciate any suggestions if you already have some, such as converting the raw file to a file format we can possibly view and edit; but I thought I would raise this as a possible request as others might run into the same issue.

Obviously this is just poor file naming practice but once created, it does not appear to be trivial to correct.

Many thanks,


view request
Merging spectral libraries in Skyline
(6 responses) sstoychev 2018-08-01

hi guys,

I would like to find out what would be the preferred method of merging spectral libraries using Skyline? I see that under the "Peptide setting - Libraries" one can select more than one library. If this is done would the redundant peptides between the selected peptides be removed or will get duplication?

Also, the recommended way of importing proteins and matching to the spectral library (for SWATH experiment) is via the "File - Import - Fasta" option. However this can also be done by the "View - Spectral Libraries - Associate Proteins" once the background proteome has been set under peptide settings. Using this "associate proteins" option I always get more proteins imported into Skyline. Why is this this the case?

view request
error exporting a method - invalid XML
(2 responses) laf228 2018-08-07


I am trying to export a method for an SRM experiment and I've gotten an error message saying "invalid XML, mass list errors". Is there any troubleshooting that you can recommend? I'm not sure if the problem is with the skyline file or if it's some sort of compatibility problem with the template file. It may not be relevant, but I'm attempting SRM on peptides without discovery data/a spectral library.

Please let me know if you have any suggestions or insight.


view request
Skyline cannot build a library from MaxQuant msms.txt because it cannot understand the fixed modification
(3 responses) luzjpaulo 2018-08-07

Dear Skyline Team

Skyline cannot build a library from MaxQuant msms.txt because it cannot understand the fixed modification.

I had successfully built libraries from maxquant before, but now skyline display an error message saying "unknown modification in mqpar file. Add a modifications.xlm file to the same directory as msms.txt which contains this modification". However such "unkown modification" is a fixed modification that I am using, and maxquant does not create a fixedmodifications.xml. I think skyline cannot understand the new version of maxquant.

 mqpar.xml  msms - Copia.txt 
view request
public library for skyline
(7 responses) dengjingjing01 2018-07-30


I am doing a PRM project and have successfully imported GPM database. I am happy to find there are spectrum match for some of the peptides, but I find for some peptide which has a spectrum data in GPM library when I search in manually on line, somehow shows no match in the skyline. I understand some peptide in the library is TMT or iTRAQ labeled, but the one I was looking for is not labeled. Could you please help me with me? I am really confusing since all the criteria (peptide length, enzyme, miss cleavage site) seems to be fine.


view request
Feature request: Monitoring [M-1] ion for small molecules
(3 responses) Chris Ashwood 2018-08-08

Hi Skyline Team,

I'm working in the small molecule space and have noticed that sometimes, when using a wide extraction window (30ppm), my "new" targets are off-by-one errors of existing targets (e.g the monoisotopic peak for one target is within 10ppm of the first C13 isotope of another small molecule). This occurs due to my library-building DDA run performing MS2 on the 1st C13 isotope, and despite attempts at correcting this using Proteowizard's precursorRefine filter, there are still some uncorrected precursor masses that could mistaken for real targets.

In this case, the ability to integrate or include the [M-1] ion in peak integration would provide a way to quickly evaluate the off-by-one error with the peak area window in Skyline, rather than inspecting the target in full-scan view for each suspect peak. I've observed that idotp with a count of 3 is not a sufficient parameter for mitigating the off-by-one error (0.91 dotp value for both the target and off-by-one target, attached picture). I don't know if anyone else would value this feature, as this issue could be significantly reduced at the hardware/method level by running at higher-resolution or centroiding the data prior to analysis with Skyline but thought I would suggest it, in case it was seen to be a useful feature.


view request
Adding internal protein as iRT into spectral library
(1 response) sstoychev 2018-08-07


I've set up a new iRT std based on an abundant protein in our samples. I now want to add this iRT std to the spectral library so can perform SWATH based extraction later. What will be the best way to do this? Usually can select the iRT std when building the library but of course this iRT is not present in the drop down list. So would the way to do this be by building the library first than going into the iRT calculator (Peptide settings - Predictor), selecting the new iRT calculator and adding the library to it? Would this than normalize the RT in the library?

view request
Working with isoforms on Skyline
(5 responses) danielacgranato 2018-07-31

We have started working with protein isoforms. We are interested in monitoring by SRM peptides that are exclusive and common to each isoform, so that we can evaluate the expression of each isoform in different cell. The protein of interest has 7 isoforms and we would like to know how can we use Skyline to edit the document. Currently we are using the option of ´´duplicated peptides´´ to maintain only exclusive peptides. Is there an easier way to create the document?

Thanks in advance. Best, Daniela

view request
Error Loading Data Files
(1 response) Kendra Adams 2018-08-07

Hi All,
I have been having an issue with my Skyline document, for some reason the data isn’t loading and I receive the attached error message upon opening the document with Skyline Daily It also tells me I cannot save the document under a new name because it is not “fully loaded”. The data that is imported into the document is on a server and I’m wondering if that is an issue, however, I’ve been using this data and document successfully for a month or two without any issues and it suddenly seems to have stopped working! I am working with a Waters TQ-S using a targeted small molecule method.
Thanks in advance!

 Skyline Error 080218.pptx 
view request
Quickly Delete Peptides with Missing mProphet Features
(9 responses) phillip ihmor 2018-02-19
Hey Skyline team,

I'm running a DIA workflow and have >150k peptides in my spectral library. I am using Skyline I want to employ the mProphet reintegration to filter for well identified peptides. When setting up the reintegration model, several key metrics are grayed out as not 100% of the peptides have values in that metric. Now, I understand that the mProphet model cannot deal with missing values, so one has to exclude them. To loose 1k peptides in a panel of 150 is no issue. The thing is just that I only know one way to delete them: click on the binoculars of each grayed out feature score, double click on each peptide in the "Find Result" list, press delete and repeat until the list is finished.
I can do that for 20 peptides, but not for >1k peptides?! Is there any way to do that quicker?
Is edit -> refine -> advanced the right place for that? I can't find an option.

I believe this issue became a problem when switching to 4.1. Before that, the mProphet model seemed to be more robust/applicable to more peptides? But maybe I just have a wrong setting somewhere. The library intensity dot product for instance seems only to be calculated, when more than 2 transitions are available. I've found the new feature in transition settings -> library -> pick minimum product ions, but that is just for the selection of the library, not in the DIA data, right? There seem to be peptides that just miss some transitions due to random / noisy data, which then then screws up the whole mProphet model? That seems quite drastic. Am I missing a setting here?

Furthermore, it would be tremendously helpful if you could give me some rule of thumb on the expected number of identified peptides. With an unfractionated library of 40k peptides, I can identify about 12k peptides at a qvalue < 0.01. Is that expected? I found one statement by George Rosenberger for openSWATH, that he identifies 70-80% of all peptides of an unfractionated spectral library.
I know, there are a million reasons, why my number might be low, but some ballpark figure could help me to gauge my analysis. In most papers, I find the number of positively identified peptides, but not the size of the spectral library. That seems to be kind of crucial... In the Navarro paper, you get >30k peptides, but that is from a deep fractionated spectral library. With my 150k library and current limited features in the mProphet model, I get next to no peptide to pass the qvalue<0.01 threshold.
My DIA run was over a 90min gradient on a Q-Exactive.

Thanks for all the hard work. I can't imagine how much work it must entail to build something like skyline...

 2018-02-19 2 transitions lead to missing dotp.png  2018-02-19 search for peptides with missing features.png 
view request
Library building cut-off score
(1 response) sstoychev 2018-08-05


I would like to understand better the cut-off score used during library building. For example when importing Protein Pilot data a score of 0.99 is used which I think corresponds to 99% confidence in Protein Pilot? Depending on the search space and /or number of LCMS runs searched the 99% (0.99) cut-off can include a different number of false positives i.e. a small search space/ dataset could have fewer than 1% FP while a large search space/ dataset could have higher than 1% FP. So rather than using the 0.99 cut-off would not be better to use the FDR analysis output from Protein Pilot since that is independent of search space and dataset size? Since Skyline can not read this FDR output maybe could look up the 1%FDR and match to the corresponding % confidence score from Protein pilot?

How would the same work with Max Quant data? I think the msms input file for Slyline is already 1% FDR filtered by MQ so can import all entries i.e. cut-off score can be left blank?

view request
Retention time shift
(10 responses) yulun 2017-04-28
Hi all,
I run some standards (metabolite) in different runs and would like to analyze by Skyline. Please see the attachment. The most right panel is the correct peak identified (~13.5 min) by the Skyline. However, the most left (1.5 min) and the middle (22.8 min) ones return the wrong retention time. I know the Skyline did the good job to pick the highest peak. My question is: Is there any possible way from setting to get rid of these 2 peaks identification since it is out of rt windows (0.2 min for rt window)?
Thank you
view request
Conversion to mz5 or mzml in centroid
(11 responses) sstoychev 2018-06-16

hi guys,

In webinar 14 on DIA data you mention that for Sciex files use of centroided data is best. Can you share the settings for MS Convert to get a SWATH wiff file converted to centroided mz5 or mzml?

Thank you.

view request
Fail to import masslynx
(1 response) RGB 2018-07-31

Hi, I am attempting to import masslynx data from a Waters synapt Qtof instrument into skyline and getting the following error. My colleague is having the exact same error message come up with her Thermo raw files from her QEHF. I have already performed the requested software updates. Any tips?

 skyline error 073118.pptx 
view request
Problems with building BiblioSpec 2.0 on w10-64bit system
(2 responses) MarcoM 2018-07-29


I'm trying to build BiblioSpec 2.0 on a windos 10 64bit system. I'm following the instructions but I'm encountering problems with some missing file (but I'm not sure of this, I'm not an expert of compiling C source files). I attached the build.log file of my last attempt. Can you help me fixing the problems or just send an executable?

Thank you

view request
ms stats question for skyline PRM
haolchem 2018-07-31

Dear Skyline team,
I have a question about using MS stats to process PRM experiments in skyline. My proteomics sample is SILAC labeled with heavy lysine. So I need to use the light to heavy ratio for quantification. I have successfully set up the PRM experiment with targeted list in skyline. But I think the MS stats is using the raw intensity instead of the ratio to generate all the plots like profile plot and condition plots, counting all the light and heavy peptides as peptides belong to the protein. As shown in the screenshot, I tried to adjust the MSStats Document Grid to include the ratio and delete the intensity, but the MSstats won't work on the new document grid, showing the error message. Can you help me with this problem?

Thanks a lot!

 Screen Shot 2018-07-31 at 9.53.55 AM.png 
view request
Can I use Skyline if....
(1 response) tnapolitano 2018-07-30

Can I use skyline if I have only mass data from MS analysis of a crude venom duct?

I have two data sets: one is transcriptomic data of mature venom peptide sequences from marine snails, and the other data set is only masses from crude marine snail venom extracts. Can I use skyline to compare these two data sets? If not, any suggestions?

view request
(2 responses) Yasin 2018-07-27


I was wondering if it is possible to set a baseline for peak integration and if not if that is something that will be implemtened?

Thanks a lot in advance,

view request
MS1 Quant with t-SIM partially solved, still questions on skyline
(3 responses) Tobi 2018-06-22

Dear the whole Skyline Crew,

hope you had a good time in San Diego, I sadly could not join, so it would be nice if I can please ask you something here.

We want to do absolute peptide quantification, at first without any MS2 data or library (I read the older forum posts on this topic, hope I didnt miss anything). We measured 5 BSA Peptides of a simple solution without any background or standard with both a t-SIM only and a Full Scan t-SIM aquisition on thermo q exactive hf. Adding the peptide target list and loading the thermo raw files into skyline works fine and we get the t-SIM MS1 data.

The Problem is to get the Full Scan MS1 data with skyline from thermo raw files, which contain both t-SIM and Full Scan MS1 data (only t-SIM data gets displayed). A solution we have for now is to use MSConvert and filter the msLevel 1 into an mzXML file. When loading the mzXML file we do see the Full scan data, which is nice, but it takes a lot of time and resources.

Is there a way to selectivley display both the t-SIM and the Full Scan MS1 data with skyline from the original raw file directly? Or do you know any other workaround, like making a exact copy of the raw file just without the t-SIM data?

The second small issue, could you please help me with an opinion on if to use the high-selectivity extraction feature (halving the extraction width), especially for high resolution t-SIM data? At least for FullScans on samples with complex background, the area to background ratio gets improved, but the detected m/z changes slightly. For now, I am just not sure if the accuracy of the picked peak remains as high as without high-selectivity extraction.

For the end, I would just mention the observation, that displayed peak area replicate comparisons are sometimes shown as a line graph even if bar graph type is selected, just for your information.

Skyline seems pretty nice and I wish you continued succes with it in the future.

Best regards,
view request
DIA upload problem
(9 responses) Tomas Vaisar 2018-07-26

Skyline team,
I ran into bizarre issue with uploading of DIA data into Skyline. Used DIA to check set of synthetic peptides and for one of them I could not get the upload work correctly. I can see the precursor and the MSMS in the Xcalibur QualBrowser and see correct fragment ions there as well, but after upload into Skyline I cannot get the same. The precursor happens to fall exactly on the edge of DIA window.
I attach the Skyline document and the RAW files here for your reference.
It is the peptide SVS from sample A10c (the EFG peptide is to show that other data uploaded fine).

view request
Edit Precursor/Transitions after loading Assay Library
(1 response) sam beard 2018-07-26


Skyline is fabulous, great work.

I am having trouble editing my Skyline documents to show the desired transitions in the targets pane. I begin by uploading an Assay Library (csv output from iSwathX tool), this populates the targets view with proteins, peptides and transitions. I then import my DIA data. The problem is I am unable to alter the number of transitions shown, and I cannot remove the precursor ions at all (and therefore cannot run MSstats).

Settings are:
transitions>Filter>Ion types>y,b "autoselect all matching transitions"
transitions>LIbrary>"if a library is avail, pick most intense"
transitions>Library>pick 5 product/5 min product--"from filtered product ions"

Altering these settings usually adjusts the transitions shown (eg "pick 3 product ions" shows three in targets, setting ion types to y,b only removes precursors). But in my documents originating from an assay library import, changing these settings has no effect. I have tried everything I could think of. I found I had to delete the targets, then manually input new targets via Edit>Insert>Peptides. I can then adjust transitions.

Am I missing something, is there another way to edit the target list?


view request
Fluorine modification
(1 response) nicakruh 2018-07-26

I am trying to monitor peptides from a recombinant protein in which a fluorinated tryptophan was incorporated. I am trying to add this modification to the peptide settings, however, the mass calculation doesn't appear to take the loss of H into account and is adding 19+ instead of 18+ to the peptide. How can this be corrected?

view request
iRT issues
(2 responses) user 2018-07-23

Dear Skyline team,

I have read iRT tutorial and webinar 7 etc., but I still have some doubts about iRT to be confirmed:

  1. Can we manually calculate iRT scores of all target peptides and create the .irtdb files and then directly import them into Skyline? If yes, how to create the .irtdb files by ourselves? Which software can edit .irtdb files?
  2. What is the easy way to merge many different .irtdb files (to get the average iRT values)? Seems we cannot directly import many .irtdb files at the same time, we need to merge one by one?
  3. When include iRT standards, Skyline use the iRT scores in the library to match iRT scores in each RAW data file?
  4. BiblioSpec library format maintains separate retention times for separate mass spec runs, but it cannot maintain iRT scores for separate mass spec runs?
  5. If iRT scores cannot be maintained in the merged library, for a merged library from many DDA runs to analyze many DIA RAW data, we have to use the same set of standards and the same RT-iRT equation in the merged library but we can perform separate linear regression for different RAW files? In this case, the iRT scores in the merged library should better be the average ones?
  6. For non-linear gradient, I noticed that you suggested using LOWESS regression. I am using a segmented gradient (0 min, 3%B; 4 min, 7%B; 204 min,22%B; 244 min, 35%B; 248 min, 90%B; 260 min, 90%B; 264 min, 3%B;280 min, 3%B) but seems linear regression still worked well (see attached graph, using 31 CiRT standards)? Do you think it is better to use a large number of standards to do linear regression for segmented gradient instead of 10-20 ones? If use LOWESS regression, how many standards should be included (e.g., for a 3h segmented gradient)?


 LOWESS vs. Linear regression.PNG 
view request
OpenMS converted pepXML
(2 responses) leon bichmann 2018-07-20


I would like to import a pepXML database search result using the Comet + Percolator combination into Skyline to generate a spectral library.

As I used OpenMS for the database search pipeline I had to convert the resulting idXML to pepXML.

Unfortunately, loading the pepXML seems to cause an error that I dont get rid of:

System.IO.IOException: ERROR: ...pepXML(line 11): The .pep.xml file is not from one of the recognized sources

I hope you have an idea how I could fix this problem to pipe my pipeline results into skyline.

The files are deposited in the file sharing folder for skyline support:
170407_AM_BD-ZH12_Spleen_W_10%DDA#1_400-650mz_msms38.idXML_merged_perc_fdr_filtered.pepXML (merged replicate IDs)

Thanks and best regards,

view request
Problems with peak scoring and export of DIA-data
(1 response) Jos Philipp 2018-07-22

Dear Skyline team,

thanks for developing skyline and your consistent support.
I use Skyline (version, 64-bit) for analyzing a DIA data set of 15 .raw files (Thermo) by a library of 300 .raw files (both samples and library ran with iRT's). The settings I used are pretty much the same as they are recommended in the two DIA-Webinars.
For my analysis, I have adjusted the iRT and used shuffled decoys implemented in skyline, then I tried to improve the peak scoring by using mProphet.
However, in the outcome not all options are available (see the figure attached) that may can lead to an improvement of the peak scoring.
I experienced while exporting protein list following group comparison that the lists hardly differ between different treatments (from 10.500 down to 500) although I used for all the same file. Is there any default filtering criteria (hidden) which reduces the number of exported protein lists?
Thank you for your comments and suggestions.

Best, Jos

view request
Two integration/importation issues
(3 responses) avollaro 2018-07-23

Hi there,

I am using Skyline 4.1.018169 for metabolomics data, and I had a couple issues.

First, while looking at acetylcholine, Skyline displayed a different retention time window for one sample "run 16" of 50. In addition, it looks like the .raw file imported transition 148->87 m/z, but did not import transition 148->60 m/z data for run 16. Please see the attached PowerPoint file "Skyline AC Issue.pptx". I included a view of the .raw chromatograms in MassLynx, which have the correct retention window and a peak for the run 16 148->60 m/z transition.

Second, Skyline frequently misses the integration of a peak for a single transition. In the attached example for run 37 of 50, the gamma-aminobutyrate 104->87 m/z transition is not integrated, but the peak is shaded as if integration was performed. The second transition 104->86 m/z for run 37 has been integrated successfully, and all prior/subsequent runs for this transition were integrated successfully. However, instances of missed integration happened for 9 separate metabolites in this data set It is frustrating to have to redo the integration of several peaks throughout large data sets. Is there any solution for this? I have already tried using "Apply Peak to All," but this reintegrates all the peaks and usually leads to more corrections of improper integration (many times Skyline does not capture the entire peak area).

Best Regards,
Alyssa Vollaro

 Skyline AC Issue.pptx  Skyline Integration Issue.pptx 
view request
Missing mass ions
(2 responses) jessica fairhall 2018-07-20

Hi there,

I was wondering if anyone has had any issues skyline completely dropping chromatographs for certain ionization masses? and if you know how to fix it?

Attached is a screenshot of replicate runs for the same compound, you can see that in the first run the M+H does not show at all, however it does in the second run. I have spot checked my data using Agilent's MassHunter and the M+H definitively is there +/- 0.0003 m/z. I have my mass accuracy set to 20ppm, so I don't see a reason why Skyline would not include it. Some samples are completely missing both M+H and +Na with zero background.

Thanks in advance for your help,


view request
No Enzyme for MS1 filtering
(3 responses) CD 2018-07-04


I would like to do MS1 filtering on some non-protease produced peptides and am following the MS1 full-scan filtering tutorial. It's going well but when I import a FASTA file, it insists I select an enzyme for in silico digestion. I'm currently using a workaround by uploading my peptides of interest as separate protein entries in the FASTA file and then making a custom enzyme that doesn't cleave any of my peptides. It seems to be sort of working but is there a more elegant solution for this? I understand that a no enzyme 'cleave anywhere' search would be computationally expensive but is there a no enzyme 'cleave nowhere' option available?

FYI, I'm on Skyline 64 bit


view request
Select and show peaks within a specfic retention time window
(1 response) amy nguyen 2018-07-18


I an trying to selection and show a peak at 4.5 min on my chromatogram. I also have a peak at 4.2 mins where I dont want to be shown on the chromatogram. Is there a way to selection peaks on this was with croping the retention time window.


view request
Overlay two peptides with all there transitions in one graph
(1 response) amy nguyen 2018-07-18


I want to include two peptides and there transitions in one chromatogram, how would i do this?


view request
Error building Spectral library from mzid generated by Peptide Shaker
(4 responses) sstoychev 2018-07-17

Hi guys,

I'm getting the following error whilst buiding a spectral library from an mzid file generated by Peptide Shaker:

System.IO.IOException: ERROR: Could not find spectrum file HILIC_50mM 0X0.007FEDP-1022BC-HILIC[.MGF|.mzXML|.mzML] in current directory,.

at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in c:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 59
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in c:\proj\pwiz_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 171
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 128

The spectra file "HILIC_50mM.mzml is stored in the same folder as the mzid file so not sure what "current directory" means in this case

view request
Incomplete MS1 XIC in case of additional ms/ms filtering
(4 responses) Fabian 2018-05-08

Dear all,

I,ve encountered an issue when trying to load MS1 and MS2 XICs into skyline.
The method is dd with an inclusion list on a Q-Exactive HF.
I would like to see the complete MS1 XIC, therefore I set "Include all matching scans" in "Transittion settings" in the "Full Scan" tab.
This works well in case no MS/MS filtering is included, however setting "MS/MS filtering" to "target" to include the ms2 informations, the first and last ms2 are used as the borders for ms1 and ms2 XICs.
This is rather unpleasant for my purpose and seems to influence ms1-based quantification as well, as one might interprete from the attached figure

In case of only 1 ms2 is present, the ms1 trace is complete, but the ms2 XIC looks pretty odd.

Many thanks in advance


 MS1XIC_DLGEEHFK_487_7325_MS1_only.PNG  MS1_and_MS2_XIC_DLGEEHFK_487_7325_MS1_only.PNG  Only_one_MS2_CCTESLVNR_569_7526.PNG 
view request
Spectral library building from MaxQuant msms.txts
(3 responses) danielz 2018-07-13

Dear Skyline team,

foremost thank you very much for providing such a tool to the community.
I have a suggestion on the spectral library building from MaxQuant output files (that I hope was not raised too often before):

MaxQuant does a lot of peak processing on the original raw scan, i.e. it tries to decharge multiply charged fragment ions if also a 1+ version of the fragment is detected. This does basically no harm to 2+ precursors, but for precursors with z>= 3+ a lot of times it means that the spectrum that is stored in the MaxQuant .res files as well as all fragment m/z information that is written as annotated peaks to the msms.txt file does not represent what the raw scan looked like.
In an extreme example, after building a library from the msms.txt and there is a theoretical 3+ peptide, there is now an y12(1+) fragment at 1201 m/z ion in the spectral library which is the base peak for that spectrum. If Skyline now trys to extract that transition from the PRM/SRM/MRM raw file you will have tough luck, because that fragment ion is actually present at 600 mz as y12(2+). Ergo: Low dotp, lower confidence, lower signal.

As the decharging of spectra cannot be disabled in MQ, my current workarounds are a) use Mascot/Sequest/EngineXYZ where you can control this parameter (by then potentially imparing the DDA data search through noisier and more complex MS2 spectra). Or b) you extract the raw scans yourself, manually or using some implementation of the ThermoRAWFile Reader library.
Spectronaut handles this by talking the spectrum number and rawfile name from the IDs in the msms.txt and goes back to the raw file (if path provided) to extract the raw scan, annotates the scan and stores that it in the spectral library.

My question is a) are you aware of this, b) is this recognized as a problem and c) are there plans to integrate such functionality or is the direct import of spectral libraries from MaxQuant files anyway only used by a low proportion of users, because most spectral libs come from different tools/sources?


view request
Problem of viewing CE optimization result for some transitions (small molecules)
(4 responses) William 2018-07-02

Hi Brendan,
I have imported the CE optimization result to Skyline (small molecules) and encountered some strange issues. I can see CE optimization results for most transitions except two (dG-A and G-dA), both with precursor of 634.28. Could you please have a look what's wrong with the file or settings? I checked it in Xcalibur and seemed ok (different CE steps were there).

 Pos CE 
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