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.

It is likely that your question has already been asked and answered.  Please use the search box in the upper right corner of this screen before posting a new question.

Support is provided by the creators of the software, as time allows, though we hope others will share their experience as the user community is now quite large.

In order to post to the forum, you'll need to sign-in or if you don't yet have an account sign up. Forgot your password? You can reset it using the "(forgot password)" link on the sign-in page.

You can also follow the Skyline support board through email updates after you sign up.

When you post a question, please include the following information:

  • A detailed description of your problem or question, including instructions for re-creating any problem that you are encountering. Screenshots are often helpful.
  • Your operating system, and the version of the software that you are using.
  • Any other information that may help us to answer your question, including whether you are working with proteomics or small molecule data.

If you are including text output from a tool, please attach files to your message, rather than pasting in long text.

If you are including a Skyline document, please use Skyline's File | Share menu item (choose "Complete" if asked), which prepares a single zip file with your document and all the needed supporting files in it. Then upload that file to the Uploads page. If the actual raw data files are needed to illustrate a problem, those will need to be zipped up and uploaded separately.
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Showing: limited to 100 requests
Error occurred running process
(5 responses) smanda 2018-12-16

Hi Brendon,

I have created a library using three search engines (Mascot, X!Tandem and MSGF+) and combined and exported in mzid using peptideshaker. When I import the library into skyline, I get the following error after a while trying to import:

System.IO.IOException: Error occurred running process.
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 139
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 171
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 137

Any help appreciated. I have created similar pipeline with different dataset, earlier and worked fine for me.


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Where to upload raw data file
(1 response) t j f m voermans 2018-12-18

Dear users,

I am struggling to find a way to load my raw data file in skyline. The data file was created bij a QTOF X2 from Waters, but when I choose 'import' --> 'Results' I receive the message that I must enable the full scan settings. But I am unable to find where I can turn on these settings. Even after using google and reading some tutorials.

Hope you can help.



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Only coeluting product ions as quantitative (extended)
(5 responses) Tobi 2018-12-17

Dear Skyline team,

I noticed that setting the "Quantitative" checkbox also works with copy and paste, but no matter if done by copy and paste or by manual clicking, it seems impossible to get the same checkboxes as in the Coeluting column because of constant recalculations overwriting manually set checkboxes (in PRM)

For just a single file one could think of exporting and reimporting a transition list filtered for coeluting product ions, but for a larger experiment, this is not feasible. Is it possible to somehow fix the quantitative column to make it directly susceptible to manual editing (in general just setting "not coeluting" to "not quantitative")? Is there a smarter way to do that?

I know about unchecking integrate all, but it would be much better to have full control over the quantitative state of a transition compared to just removing the target from the target list which impacts all datasets.

The attached file shows what would be the desired output, its just not fully implementable for a whole experiment.

Thank you for all thoughts and ideas on this topic.

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Instrument Platforms
(3 responses) amruta indapurkar 2018-12-14

We have a skyline software and we wanted to add some new instrument platforms to it, so that we can analyze data from multiple instruments. The instruments that we want to add to our software are:

  1. Thermoscientific Q-Exactive High resolution mass spectrometer
  2. Agilent’s 6530 Accurate Mass Q-TOF LCMS

I was wondering if that would be possible. Please let me know. Thank you.

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Build spectral libraries using a large number of DDA files
(7 responses) benoit fatou 2018-12-04

Hi all,

I am trying to analyze DIA data and then extract peak areas for peptides/proteins of interest.
I would like to use a spectral library I generated from a large number of DDA files (about 1300 files).
When I was building it into Skyline, I received a error message saying I have reached the maximal number of spectrum files which is 500.

I am contacting you to know if it possible to increase the capacity to Skyline to analyze a larger number of DDA files for spectral library generation.

Thank you very much for your help !

Best regards,

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Importing Sciex Results from 2 Periods (pos/neg)
(7 responses) dawn dufield 2018-12-10


I have an experiment where I have collected pos and neg data in 1 file in 2 periods. When I try to import that data to review in skyline. It appears to only see the first experiment (pos) and shows no data for the neg ion transitions? Is there a way to be able to look at all the data from the file within skyline?


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Importing several .wiff2 files
(3 responses) jakobsen 2018-12-13

When using Skyline Daily it's not possible to import several raw-flies at a time and some of the fail.
Importing the files one at a time works perfectly fine.

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skyline not recognizing full .mgf name from .dat file
(3 responses) erik.soderblom 2018-12-10

Hi Skyline Team!

I have a situation where I have a .mgf file (created through PD) searched in Mascot with the search results saved as .dat. Sometimes those .dat files don't match (verbatim) the .raw file. Therefore, when if I manually build a spectral library and select those .dat files and then input my raw data... it doesnt associate the actual search results with the actual raw data (and features such as align retention time, etc. is not available).

Since the .mgf file name (which always will be the same as the .raw name) is in the .dat file structure... would it be possible to go into the .dat file and make sure its assocaited with hte correct .raw file... even if the .dat file and the .raw file are not named the exact same thing?

I've attached uploaded a file called ID19225_03.dat to the skyline FTP for an example. Thanks!


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PRM FDR control
jfoe 2018-12-13

Dear skyline team,

I have been experimenting with the advanced peak picking model based on mProphet.
I have an assay with heavy internal reference and I would like to have q values for each peptide.

Now due to our quite tight scheduling for the PRM acquisition I can't imagine for scoring based on second best peaks to be of use.
When generating decoys however, they get +10 precursor mass so the would need another window during acquisition.

Do you think a workflow is possible, where one uses decoy transitions with the PRM data as is?
I think FDR in PRM is an important topic and I would like to put some effort into making this work.


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Default import results folder
(2 responses) dawn dufield 2018-12-11


Can you set a default folder for importing results? I have to navigate in about 15 folder levels every time I try to import results. Can you either set a default location or have it use the most recent?


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What are the dotted-line chromatograms?
(2 responses) graceac9 2018-12-11


I am using Skyline to look at .mzML files from a DIA analysis. Some chromatograms for some peptides are dotted lines and the peak area and retention time graphs are faded. Attached is a screenshot example of what I am referring to.

What do the dotted-lines mean?

Thank you!

  • Grace
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issue downloading the software
(3 responses) sriabhi9 2018-12-10

despite having around 100gb free in my C drive, the error that pops up during the installation says less storage.

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Allow integration of multiple isotopes for high molecular weight fragments (Top-Down PRM)
(4 responses) v delcourt 2018-10-10


With recent analytical chemistry publication, there's something interesting to do with multiplexed top down PRM (

While doing some experiments on my side, I thought this was indeed interesting, but there might be something to add to Skyline to complete data analysis of these experiments. Indeed, in case of high molecular weight (with charge > 1), it's very likely that fragments' most abundant isotope would not be the monoisotopic. However, Skyline seems to integrate only the first one (see capture attached). Maybe this feature is already implemented but I couldn't find it.

By adding all/multiple isotopes to target ions integration, this would increase detected signal intensity which would affect results significantly.

I can send data if needed.
Best regards,

view request
(5 responses) liisa arike 2018-11-30

I am getting a following error when opening older processed Skyline files:
Failure opening C:\Users\xarili\Google Drive\Projects\CR crosslinking\MS562_CR Chymotrp human\MS562_01_25\Crosslinking_human
The file contains an error on line 1897 at column 7.
OK More Info
System.Reflection.TargetInvocationException: There is an error in XML document (1897, 7). ---> System.InvalidOperationException: There is an error in XML document (1897, 7). ---> pwiz.Skyline.Util.AssumptionException: error reading mz values - declared mz value 756.338 does not match calculated value 757.34582457991
   at pwiz.Skyline.Util.Assume.Fail(String error) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1918
   at pwiz.Skyline.Util.Assume.IsTrue(Boolean condition, String error) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1877
   at pwiz.Skyline.Model.Serialization.DocumentReader.ValidateSerializedVsCalculatedProductMz(Nullable`1 declaredProductMz, TransitionDocNode node) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1371
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionXml(XmlReader reader, TransitionGroup group, ExplicitMods mods, IsotopeDistInfo isotopeDist) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1357
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionListXml(XmlReader reader, TransitionGroupDocNode nodeGroup, ExplicitMods mods) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1247
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionGroupXml(XmlReader reader, Peptide peptide, ExplicitMods mods) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1117
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionGroupListXml(XmlReader reader, Peptide peptide, ExplicitMods mods) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1043
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideXml(XmlReader reader, PeptideGroup group, Boolean isCustomMolecule) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 877
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideListXml(XmlReader reader, PeptideGroup group) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 761
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideGroupXml(XmlReader reader) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 732
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideGroupListXml(XmlReader reader) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 573
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadXml(XmlReader reader) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 539
   at pwiz.Skyline.Model.SrmDocument.ReadXml(XmlReader reader) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2022
   at System.Xml.Serialization.XmlSerializationReader.ReadSerializable(IXmlSerializable serializable, Boolean wrappedAny)
   at Microsoft.Xml.Serialization.GeneratedAssembly.XmlSerializationReaderSrmDocument.Read1_srm_settings()
   --- End of inner exception stack trace ---
   at System.Xml.Serialization.XmlSerializer.Deserialize(XmlReader xmlReader, String encodingStyle, XmlDeserializationEvents events)
   at System.Xml.Serialization.XmlSerializer.Deserialize(TextReader textReader)
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass925_0.<OpenFile>b__0(IProgressMonitor progressMonitor) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 301
   at pwiz.Skyline.Util.ProgressWaitBroker.PerformWork(ILongWaitBroker broker) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\UtilUI.cs:line 123
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 232
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1854
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 180
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 132
   at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 295
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PRM relative label free quant
(1 response) adrien schmid 2018-12-07

Dear Skyline team,

I would like to inquire if the scientific community uses a combined PRM approach, where some targeted peptides are monitored along their heavy labelled surrogate peptides for absolute quantification (standard PRM approach), whereas within the same PRM method, a sub population of peptides (of different proteins) are only targeted for monitoring any changes in relative abundance between biological samples? This would resemble a label free (relative) quantification method using MS2 spectra for assessing the changes in relative abundance of a protein.

Would this make sense ?
many thanks for your feedback on this question.
best regards


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Where to find precursor peak area scored script?
(1 response) andyzcq 2018-12-08

I have used Skyline to analyze about 18 SWATH-MS files and finished refinement.
How can I export precursor peak area?

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Chromatograms not imported / visualized for small molecules
(4 responses) fredrik edfors 2018-12-08


Thanks for the awesome software. I usually work with proteomics but got interested in the small molecules tab and I'm exploring some of our metabolomics datasets using Skyline. I'm getting an inconsistent import of chromatograms, and it seems like skyline has imported everything but cannot visualize a random subset of the identified peaks. It will return an area but says that the Chromatogram is unavailable).

Please see the minimal dataset enclosed. I can see 9-Decenoylcarnitine for example, but not a single tracer from Lactic for instance (even thus I get a bar in the peak are graph). It seems like this error also occurs in the middle of a run for a set of my tracers when working with an extensive list of compounds (500+) but could be solved by dividing the set of molecules into a smaller amount. This set is however only covering 21 precursors so I don't know how to explain it.

Cheers, and grateful for any help.


 Hydroxy acids and 
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How to add RT and Analytes Name to Peak labels
(2 responses) dawn dufield 2018-12-04

When we have a method with multiple peptides or small molecules and I want to look at the overall TIC for whole run I shift select the ions and then it labels them on the chromatogram. 2 questions. If the names are too long it puts a .... and truncates the name. Is there a way to expand that name so I can see the whole name. 2) how do I add the ion name and the RT to the chromatogram? When I click on just 1 ion it shows the RT, but when I select multiple ions it changes to the ion name and no RT.


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Small molecule method export
(6 responses) mcarey17 2018-12-03

Hi all,

Relatively new to Skyline but could use a great deal the method export feature. That's the one where based on a transition list/retention time and an existing *.exp file (which is the MAsslynx MS method details file) Skyline creates an MS method file ( *.exp file), which would be otherwise really clumsy with MAsslynx when more than 100 compounds are involved.

Re-create the error:
Open Skyline; Import transition list; Change setting to XEVO-TQS; Export MEthod

Operating system: Windows 7; Skyline version: latest; MAsslynx version: 4.2

Error message, see it on 'error support 3.PNG'
System.IO.IOException: Error no known instrument type found.
ERROR: Error no known instrument type found.
---> System.IO.IOException: Error no known instrument type found.
ERROR: Error no known instrument type found.

System.IO.IOException: Error no known instrument type found.
ERROR: Error no known instrument type found.
---> System.IO.IOException: Error no known instrument type found.
ERROR: Error no known instrument type found.

at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in c:\proj\skyline_4_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 59
at pwiz.Skyline.Util.Extensions.UtilProcess.RunProcess(ProcessStartInfo psi, String stdin, String messagePrefix, IProgressMonitor progress, IProgressStatus& status) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Util\Extensions\UtilProcess.cs:line 45
at pwiz.Skyline.Model.MethodExporter.ExportMethod(String exeName, List1 argv, String fileName, String templateName, Dictionary2 dictTranLists, IProgressMonitor progressMonitor) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 3772
at pwiz.Skyline.Model.WatersMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 3571
at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action1 performWork) in c:\proj\skyline_4_1_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\skyline_4_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1835 at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 176
at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 131 at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action1 performExport) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 1885

 error support 1.PNG  error support 2.PNG  error support 3.PNG 
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Export method to MassLynx 4.2 version
(2 responses) danielacgranato 2018-11-27


I was wondering if the new version of Skyline (v.4.2) allows to export SRM methods to MassLynx version 4.2. Last year I was unable to do so and I received a response that Waters was working on making it possible. As a solution to the problem I have been exporting the method to a computer not connected to the equipment (Xevo TQ-XS) and to an older version of MassLynx (v.4.1). Do you know if it is possible now to export directly to MassLynx 4.2?

Thank you very much. Best, Daniela

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Import modifications to Skyline daily
(6 responses) sandberg 2018-12-06

I try to import a peptide search using Skyline daily. When I build the spectral library using a Mascot DAT file the program cannot find my modifications (Pic1) even though I can see that they are in the imported peptide list (Pic2). If I do the same thing in regular Skyline the modifications are found (Pic3). Is this a bug?

 Pic1.png  Pic2.png  Pic3.png 
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Is L/H ratio corrected for shift in isotope patterns?
(2 responses) Tobi 2018-12-05

Dear skyline team,

reading this nice technical note ( ) brings me to ask if and which corrections skyline applies to SILAC ratios when quantification is not based on the whole isotope cluster but just on the precursor or fragments from isolating just the precursor. (want to make sure i dont accidentially apply correction twice)

The issue is well illustrated in figure 3 of the linked pdf, stating that the shape of the heavy peptide is shifted towards the precursor with the heavy precursor intensity beeing up to 10% higher in a 1:1 light:heavy mix (depending on the type of labeling). Adding up intensities of precursor plus isotopes before calculating the ratio evens out this shift, but this is not happening fully in PRM and quantification on the precursor level.

It would be great getting a short reply.

Best, tobi

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Wrong area plotted into calibration curve
(2 responses) Yasin 2018-12-02


I think I found some bug, although I am not sure about the reason it is happening. I was trying to make a calibration curve, with three replicates per concentration. This worked fine in general but for one molecule (Methionine in the uploaded example) one of the replicates for the concentration of 1000 nM (sample 100_C13_2 in the uploaded example)is plotted with a wrong area into the calibration curve. In the area-replicate comparison all areas of the samples (100_C13_1, 100_C13_2 and 100_C13_3) are almost exactly the same. Just on the calibration curve something went wrong.
Is this really a bug or am I doing something wrong?

Anyway thanks a lot in advance.


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Peptide shows two different peaks with in 2 min RT
(1 response) rajirathore3 2018-12-03

Hello skyline team

I am facing an issue with one of peptide which shows 2 different peaks for same peptide and contains same pattern of fragments. Data has been acquired on Thermo Q-exactive. This peptide elutes with in 2 min of RT and in some of the replicates it shows almost similar intensities and ppm error for both the peaks. This is a glucose modified peptid . Unmodified peptide is also acquired and that does not show this kind of pattern. I have attached spectra of few of the replicates which show only one peaks and other which show 2 peaks with almost similar intensities. I have attached a graph of retention time of replicate comparison. Please help me to resolve the issue

 skyline support.pptx 
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Peak splitting.
(5 responses) Michael Cundell 2018-11-30

Hi all,

When I import with MS1 filtering (Isotope peaks included:Count, Precursor mass analyzer: Orbitrap, Peaks:3, Resolving power: 60,000@200m/z) and MS/MS filtering (Acquisition method: Targeted, Product mass analyzer: Orbitrap, Resolving power: 60,000@200m/z) I get split peaks on some chromatograms (Untitled1.jpg). If i remove MS/MS filtering and set it to none and reimport my data the full peak is restored (Untitled2.jpg). If I switch back to the original settings (MS1 and MS/MS filtering) and reimport, my peak is again split (Untitled3.jpg). I am using skyline Split peaks seem to be rare. I have a large document and most peaks are fine. I think that the split peaks occur on singly charged peptides with m/z >1000 but I would need to look into that. Any advice would be appreciated. The reason I tried turning off MS/MS filtering was that I found an old issue that restored a peak under this scenario ( Am I doing something wrong or is there a bug somewhere in there? Thanks for any advice.



 Untitled-1.jpg  Untitled-2.jpg  Untitled-3.jpg 
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Problem importing modified peptide into library
(5 responses) ronen gabizon 2018-11-28


We are analyzing samples of recombinant protein which were incubated with small molecules that attach covalently to cysteine. The objective is to identify the positions of modification and extract the chromatograms of the labeled peptides for characterization. Essentially I define the molecule's formula in MaxQuant (saved in the modifications.xml file) and define both the molecule and carbamidomethyl as variable modifications. The analysis by MaxQuant clearly identifies peptides containing the molecule as well as their sequence and the exact labeling site (including if there are two cysteines in the same peptide). My objective was to import the peptide search into skyline to build a spectra library and to extract the chromatograms of the carbamidomethyl and molecule-bound peptides. However, the peptides with the bound molecule are never added to the library - only carbamidomethyl peptides are. This is even though MaxQuant clearly identifies the modified peptides and they are defined in the same way.

Is there anything you can recommend to help in this problem?

Details: Windows server R2 enterprise 64 bit

        Skyline 64 bit


I attach the screenshot from skyline. Cysteine containing peptides are added to the library (carbamidomethyl containing) and some are not (molecule-labeled).

I also attach the screen shot from the MaxQuant output showing the identification of the molecule-labeled peptide.

Thank you

 Screen Shot 2018-11-29 at 1.56.27.png  Screen Shot 2018-11-29 at 1.58.png 
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mProphet discrimmination between heavy and light pairs
(7 responses) Fabian 2018-11-21

Dear Skyline team and users,

I have a DIA data set containing peptides with synthetic heavy peptides. In every sample the heavy peptide is well identifiable. The endogeneous light variants are well identifiable in every sample. In such cases I would like to have missing values for the light variant.
To do so, I trained a mProhet model which showed nice discrimmination between forward and decoys (also gausian shaped).
I was surprised to see that skyline kept all integrations in the light variants, although they were sometimes pretty bad.
I figured out, that when I delet a heavy peptide, that in such cases (no heavy counterpart) not all peaks were integrated in the light variant. So in these cases it seemed to work, although not so discrimminative as I hoped, but thats another question...

So it seems to me that the scoring is not done separatedly between H and L?

Kind regards

view request
wiff2 file support
(12 responses) sandberg 2018-11-06

Is it possible to import and process the new wiff2-file format from AB Sciex with version 4.2? The file extension does not appear in the list of importable MS formats and the files do not show up in the import window. Thx.

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Isotopologues at high resolution
(5 responses) Yasin 2018-11-29


I have a question regarding the masses of isotopologues calculated by Skyline for high resolution mass spectrometry. Apparently Skyline is making some kind of average over several isotopologues. Is it somehow possible to calculate isotopologues according to the resolution used as in the envipat package?

Thanks a lot in advance!

view request
Setting sacn numbers range in SIM mode
(2 responses) Vahid F 2018-11-30

Hi, I am wondering if there is a way to set the integration area within certain scan number range? Example: I am interested in peak area in between scan number 1250 to scan number 1274. Is there a way to set this numbers in skyline manually? I am working in SIM mode and data is from QE-HF.


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Protein/peptide extractions from multiple runs
(4 responses) zainab noor 2018-11-28

Dear Skyline Team,

This may be a very basic question but what should I do to see peptides which may be present in any two replicates if not all after I apply the mProphet and q-value cutoff. For example, a peptide which is present in 2 out of three replicates only with 99% FDR. Currently, I am seeing the peptides which are found in all the replicates with 99% FDR after I integrate the peaks using mProphet model.

Thanks for all the help.


view request
MS1attributes of SILAC pairs
Tobi 2018-11-28

Dear Skyline team,

thank you for providing such a helpful software, it means a lot. There are few things in terms of chromatograms and document grid I would wish to ask.

screenshot 1

  1. While DotProductLightToHeavy is identical for both light and heavy of a SILAC pair, the ratio dotp always shows #N/A for the heavy, shouldnt it also have the identical value as the respective light peptide? (for better filtering of results for silac pairs, at least it should not hurt or would it?) Is it possible to filter the documents grid for silac pairs where both isotope patterns of a silac pair fulfill certain filtering criteria (e.g. both of a pair having idotp>0.9), is it essential to use pivot isotope label for this?

  2. The Ratio dotp is always the dotp displayed in the target list (one silac pair) while the DotProductLightToHeavy seems to be the average of all charge states of one peptide (multiple silac pairs in a way). Just want to mention its easy for users to mix them up unintentionally, similar with average mass error and peak apex mass error.

  3. The Coelution feature says: "similar apex and extents as the other transitions within the peak group" but how is that peak group defined (all quantitative transitions? light and heavy silac pair in one group) ?

The coelution was partially explained here, but i dont find the coelution count feature, was it removed? :

screenshot 2
The document grid precursor serves as a link which moves the displayed single chromatogram to the peak, but it stays within one and the same raw file even if the selected precursor was from another file. Is it possible to have a link in the document grid which always directs you to the exact chromatogram you want to see, even across loaded raw files?

not on screenshot
When working with quantitative PRM, does skyline consider if a single specific fragment ion can be theoretically interfered by signals from other fragment ions (with ammonia/ water loss). How is it when applied to a silac pair, also when multiplexing light and heavy fragments into one measurement ( i know this sounds bit weird but its implemented in mascot and i would like to try translating it to skyline)

Sorry to post so much text here but i hope it also helps other users as well. Thank you for your support, its highly appreciated.

Best regards,

 SILAC dotp.JPG  document grid link.png 
view request
Cannot import data!
(1 response) Will Thompson 2018-11-27

After updating to the newest daily I cannot import data. Error is the pwiz dll error reported in message below also. In order to assist in troubleshooting I have attached a skyline file and a raw data file which imported with previous version.

I have confirmed that when I use the 'share' function to save as a Skyline 4.2 document and open in the released version, I can import the data files.

I had to use 7-zip to archive the Waters raw data file (Xevo TQ-S) because it would not upload as a folder substructure.


Will  ID_Kyn-Neuro-KinetexCol-_WAA253_5135_112027_003.raw.7z 
view request
I got error message after update Skyline-daily
juyeon lee 2018-11-27

After updating Skyline-daily, mzML files could not import.
The error message : Could not load file or assembly 'pwiz_data_cli.dll' or one of its dependencies. The specified module could not be found.

view request
Error importing Agilent QQQ data with Skyline-daily
(7 responses) Sarah Michaud 2018-11-26

Hi Skyline team,

I recently upgraded to the newest Skyline-daily and am getting an error when importing Agilent 6490/6495 data. Import fails with the following message: "Could not load file or assembly 'pwiz_data_cli.dll' or one of its dependencies. The specified module could not be found."

I'm able to load the data into the same document or a new document with no problems after downgrading to the previous version of Daily.



view request
Export method on Thermo Altis QQQ
(8 responses) kristin geddes 2018-11-16

I just had a Thermo Altis QQQ installed this week in my lab. I am trying to export a Skyline method and I am getting the error "Error: Failure during initialization. TSQ-EZ Method support may not be installed. Method export for a TSQ should be performed on the TSQ instrument Control computer and requires TSQ version 2.3 or better" (see attached screen shot)
I am on the instrument computer and we are running TSQ version 3. Any tips? I have tried both Skyline and Skyline Daily.



 Export Method Error.docx 
view request
Unable to extract chromatogram of the same ion in MS1 and MS2
Chris Ashwood 2018-11-26

Dear Skyline support,

I hope you are all well. I am using the small molecule side of Skyline (most recent daily version and non-daily versions) and have encountered a limitation of Skyline that is present in both versions. If I set up my Skyline assay to integrate unfragmented precursor in MS2 scans, an extracted ion chromatogram of the precursor [M] is missing in MS1 but an extracted ion chromatogram of the precursor in MS2 is extracted. Other isotopic ions are detected, it's just the monoisotopic ion that is not being extracted.

I currently bypass this limitation by not including the unfragmented precursor in the fragment list but thought I would report it anyway.


view request
Efficiency of iRT in predicting PTM peptide RT
(1 response) Alok 2018-11-24

Hello Skyline team,

I am studying post-transnational modification, particularly acetylation. I wonder how accurate is iRT in predicting retention time of acetylated version of the peptides?

I have used iRT and Biognosys peptides many times for unmodified peptides but I am not sure about accuracy of this approach in trying to predict RT of acetylated peptide?


view request
how to convert chromatogram (LC-ICPMS) from Water empower 3 to skyline
(1 response) binh chudinh 2018-11-22

Dear Sir / Madam
This is Dinh Binh Chu, a lecturer, and researcher from Hanoi University of Science and Technology, Hanoi, Vietnam, also a skyline user. We got an LC ICPMS system from Perkin Elmer and the software for speciation analysis from Water, Empower 3. However, we would like to export our chromatograms from Empower to Skyline. Unfortunately, we were not successful in this task. I would like to ask you how to convert such kind of chromatogram to skyline
thank you so much and looking forward to hearing from you
Best regards
Dinh Binh Chu

p.s. You can reach me by email:

view request
Quick way to import hundreds of targeted proteins to Skyline?
(4 responses) William 2018-11-22

Hi Skyline team,

I wonder if there is a quick way to import hundreds of targeted proteins withoout creat fasta file containing all those targets?


view request
Application download did not succeed.
(1 response) hskim09 2018-11-23
Dear. Skyline software developer.

I have tried to install Skyline, but get the message "Application download did not succeed. Check your network connection, or contact your system administrator or network service provider."
I have tried to run the set-up.exe as Administrator, but I get the same message. Do you know a way of solving this problem?
Thank you in advance.


 error message.log 
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Unable to install MSstats
(2 responses) Frank Zh 2018-11-22

Dear Skyline team,

We tried many times to install MSstats from the tool store in Skyline 4.2 and Skyline 4.2 daily recently but failed every time. The error shows “No internet connection” (see attached screenshot) but our internet is connected. We have installed the R package 3.5.0 and we have the same problem in 2 PCs. Please check what the problem is. Thanks.

Kind regards,

view request
Points Across Peak
(7 responses) Yasin 2018-11-11

Dear Skyline team,

when creating a report template it is possible to add the "points across peak" - column. However, this number only counts the number of data acquisition points. Often it would be nice to filter for peaks with a certain number of points above 0 in between the integration boarders as this would save the time of manually deleting peaks with less then a specific number of (connected) points above 0. Hope that is something you would consider to implement.

Anyway, thank you in advance!


view request
Dashed red lines/number
(2 responses) Michael Cundell 2018-11-21

Super basic GUI question.

I have heavy/light labels. When I select a transition for either label, what do the small vertical dashed red lines that span the x-axis and the corresponding red number refer to in the chromatogram view?

At first I thought that these corresponded to the number of datapoints for which the transition is observed across the peak. However, I have light labels with nearly no signal yet a lot of the red dashed lines. Then I thought ok its just the number of datapoints in the heavy label. However, the number between the heavy and light label is often slightly different for the same transition. I cant seem to rationalise what they mean.



view request
About the export the information of Internal Standard Type from the Skyline file
(1 response) yin lu 2018-10-29

Dear Skyline team,
I'm working on the SRM targeted proteomics data from CPTAC Assay Portal. My goal is to access and export the information in the *.sky file using the SkylineCmd.exe. For now, I could export some column information, such as, Protein Name, Peptide Modified Sequence, Isotope Label Type, Product Charge, Replicate, MultiplicationFactor, Concentration, SampleGroup and so on, by means of creating report template. However, I can't find one attribute named "Internal Standard Type" from there.
I found that I could find an item named "Internal Standard Type". I could find it by opening the *.sky file in Skyline GUI: On the Setting menu and Peptide Setting. How can I export the information of "Internal Standard Type" from *.sky files using the command line tool SkylineCmd.exe?
Thank you.


view request
Number of transitions in the integration report file less than the number of transitions specified in the sky file
(16 responses) pram1707 2018-11-18

I have 619 transitions in the .sky.xml file. When I use this to run my MS data, the resulting report has only 591 transitions. The rest are missing. Why is that?

view request
Theoretical peptide z-ion mass appears to be incorrect
(7 responses) JHamey 2018-11-14

Hi there Skyline team,

I've come across a problem to do with the theoretical mass of z-ions in Skyline. They appear to be consistently off by 1 Da - they are predicted to be 1 Da lighter than they actually are. E.g. a z13 ion is predicted to be 1316.7 Da when it is actually 1317.7 Da. When I searched for threads related to this issue I came across this one (Issue 498):

It appears as if the "fix" that was made to make z-ions 1 Da lighter was done in error. The 1 Da heavier masses for z-ions I am getting from MS-Product match what I see experimentally for a normal peptide (it was noted in that thread that the peptide where the "error" was observed contained an abnormal gamma linkage at the position where the mass shift was thought to be wrong). C-ion masses are perfectly ok. I see this consistently for at least 5 different z-ions in the peptide.

Thank you in advance for your help.

view request
Problem with MS1 tutorial
(1 response) comtewal 2018-11-19

In preparation for the Duke Skyline course I am going over the Skyline tutorials.
On the MS1 tutorial, when I import the xml files and the wiff files according with the instructions, the Phospho (S,T), Phospho (Y), and Ox (M) are not showing on the list (see screen shot attached).
If I try to add these modifications I get an error saying the mod already exist.
Please help

view request
Skyline MS1 Quant of glycopeptides
mstokes 2018-11-19

Hello, we are trying to use Skyline for MS1 quant of peptides with N-linked glyco groups. We are having some trouble building the library from these as we have many hundreds of different added masses, and some identical added masses for structural isomers. Wondering if there is a way to build a library for quant that incorporates the glyco information in addition to peptide backbone sequence. Thanks!

view request
Cannot retrieve application. Authentication error.
(5 responses) aisling donnelly 3 2018-11-16


I had the previous version of Skyline downloaded on to my computer and tried to download the updated version (64-bit 4.2), however it does not seem to be working. I tried to delete the previous version I had of it and re-download the new one but it still won't work. I also tried the unplugged version but the same error came up that said "Cannot retrieve application. Authentication error." It seems to download fine but once it's finished downloading it won't open.

I have attached an image of the error that comes up and the details it shows. I now can't get any version of Skyline on to my computer.

Thanks for your help,

view request
SMILES peptide sequence entry into skyline
(3 responses) faizanzubair2 2018-11-14

Dear Skyline Team,

Thank you for the amazing piece of software that has made a tremendous influence in biomarker research!

I am trying to develop automated MRM methods using skyline for heavily modified peptides (containing methylation, lipid chains, non-natural amino acids). Due to number of these peptides, entering them and modifying them individually in Skyline is very tedious. I have list of sequences in SMILES format-- can those be inputted directly into Skyline? Alternatively, is there a converter that converts SMILES format into skyline compatible sequences?

Thank you so much!


view request
Integration fails in MS-1 when multiple scan ranges are used
(4 responses) v delcourt 2018-11-09

Hi Brendan,

I've been doing some experiments with a splitted MS1 (1 scan for 200-405 and 1 scan for 400-1300 and then DIA) which are separated in scan events in the method editor. Skyline does not seem to integrate it properly. Indeed, it looks like it's trying to look for target ion in the wrong scan range (see capture below) and results in abnormal integration.

Maybe there's something i'm missing. I've been reimporting my DIA scheme, trying to play with MS1 integration parameters but couldn't find the solution.
I guess this issue could be faced in case of BoxCar analysis as well as this is kind-of the same approach (in a non-multiplexed fashion).

Best regards,

view request
Report file
(1 response) zainab noor 2018-11-15

Dear Skyline team,

I have to export the report file from skyline with the peptide and peptide intensity column. Which column should I use to extract the total intensity of each peptide in each replicate? Is it Max Height under Precursor Results?

Kind regards.

view request
skyline full scan settings not enabled
(1 response) sponce1 2018-11-15

Hi Skyline,

Please fix the attached document so I can view the raw data. Whenever I open raw data, it says "skyline full scan settings not enabled". The molecule is UDP-GlcNAc.


view request
No cleavage if variable modification
(3 responses) dawn dufield 2018-11-13

HI Brendan,

I was wondering if there is a way to adjust the cleavage settings on an enzyme or define a new enzyme that would NOT cut when there was a variable modification ... ie if something were to modify lysines could you have skyline no longer cleave at that lysine automatically or do you need to somehow add them all manually? or with partials?

view request
Exclude Oxidized Methionine not working
(1 response) jmeyer 2018-11-14

Dear Skyline Team,

I'm trying to exclude peptides with oxidized Methionine from an import of the pan-human transition list from swathatlas, and the usual regex exlusion isn't working (see attached):


I have set "pick peptides matching" to "Library and Filter", and I tried Edit>refine>advanced>Auto-select all peptides, precursors, transitions.

Am I missing something or is there a bug?

I put the complete share here:

Thanks in advance,

view request
Any plans for a Linux version of Skyline (even with limited capabilities)?
(3 responses) lparsons 2017-08-07
Skyline is a great tool for me, and I am wondering if there are any plans to make it available - even in a somewhat stripped-down form - for Linux. I have read that the vendor specific libraries that make it possible to open vendor native formats are often locked to Windows, but there are other functions that could be helpful to have that might not require them. If I could open an existing Skyline project and do basic editing of the project (for example deleting ions) and basic exporting (to text) that would be really helpful for me.
I don't know the ins and outs of the Skyline .sky file format (amongst other things), so I don't know if that is a request that would be too great to handle or not.

thank you!
view request
The document format version 4.2 is newer than the version 3.73 supported by Skyline (64-bit) .
(2 responses) voellmy 2018-11-12

Hi Skyline team,

Since upgrading to the latest Skyline version (I now have, I am having trouble using SkylineRunner. I get the following error:
"The document format version 4.2 is newer than the version 3.73 supported by Skyline (64-bit) .". What can I do to resolve this ? I've tried saving an older (and functioning) Skyline document with the newest version and this reproduces the error, so I'm fairly certain the version is the issue.

Thanks for your help,

view request
issue loading data
(1 response) joan miro 2018-11-09


I just installed skyline in another computer and I have the following error

System.IO.IOException: Failure reading the data file D:\JoanMiro\Joan_Histones\All_samples_in.skyd.
Array dimensions exceeded supported range. ---> System.OverflowException: Array dimensions exceeded supported range.
at pwiz.Skyline.Model.Results.ChromatogramCache.LoadStructs(Stream stream, IProgressStatus status, IProgressMonitor progressMonitor, RawData& raw, Boolean assumeNegativeChargeInPreV11Caches) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\ChromatogramCache.cs:line 713
at pwiz.Skyline.Model.Results.ChromatogramCache.Load(String cachePath, IProgressStatus status, ILoadMonitor loader, Boolean assumeNegativeChargeInPreV11Caches) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\ChromatogramCache.cs:line 563
at pwiz.Skyline.Model.Results.MeasuredResults.Loader.CheckFinalCache(String cachePath) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\MeasuredResults.cs:line 1325
--- End of inner exception stack trace ---

Can you help me with that?
Many thanks in advance

view request
Cannot get specific trypsin peptide
(1 response) William 2018-11-08

Hi Brendan,
I have difficulty in getting Skyline to display the tryptic peptide of "VLPSWLTAQVATK" from the CYREN protein (Q9BWK5). Cannot sort out what setting is wrong.
Thanks, William 
view request
Chromatograms not displayed in metabolomics SRM analysis
(3 responses) toonstrac 2018-11-08


I am trying to analyze some metabolomics data using Skyline, but each time I upload the data, none of the chromatograms are visible ('Chromatogram information unavailable'). I can see the chromatograms as the files are uploading, but the chromatograms disappear after they are uploaded. I have uploaded a file (CHL_CE_Heavy_CHL_Palmitate_tr4). I am using a Waters Xevo TSQ. Please let me know if you need anything else. Thank you so much for your help.

Best Regards,


view request
Error message when trying to copy and paste phospho peptides from one file into another file
(5 responses) rschoenh 2018-11-08


we're sometimes having trouble copying and pasting phospho peptides from one Skyline file into another Skyline file. Skyline basically doesn't let us do the pasting, but instead we get an error message saying "The modification Phospho (ST) already exists with a different definition." As far as we can tell, we have the same definitions in both Skyline files. Thanks very much in advance for your help!


view request
Specific-modified peptides don't appear
(6 responses) ncampoloh 2018-11-07


I am writing to ask you help about some trouble I'm having in seeing in Skyline some specific modified peptides; it's probably something easy to solve, but after trying many things, I still couldn't get to solve it.
I have MS/MS data of a digested pure protein, acquired by a universal method in an Orbitrap fusion. I have a control condition and two conditions were the enzyme was treated with an oxidizing agent. My intention is to detect the oxidative PTMs that some amino acids undergo after treatment, and have some approach to its levels doing some relative quantification with Skyline (comparing the peak areas of the modified peptides with respect to the unmodified ones).
I used MaxQuant to process each raw file for doing the peptide search, so I imported into Skyline the msms.txt that MaxQuant generated. For the treated conditions, I found the following modifications in many residues: methionine oxidation, tyrosine oxidation, tyrosine nitration and tryptophan nitration. The problem I have is, that when I do all the steps for analyzing the data in Skyline and import the peptide search, the raw file and the fasta sequence of the protein, I don't see all of the peptide variants that were originally detected. In particular, I see peptides modified by methionine and tyrosine oxidation, but not the ones that contained nitrated tyrosine or tryptophan. I included all of these modifications on the peptide settings (I added manually and checked that the mass values of each were the same that MaxQuant used), so that shouldn't be the problem. I tried both doing MS1 filtering and DIA approaches but in none of the cases those peptides appear. I think that the modifications were properly considered, given that when I remove the filtered peptide sequences by clicking the identified protein, all of the peptide possibilites appear, but with no data assigned to them (as if they were not detected in the peptide search).
I've tried including the ambigous mathces when importing the peptide search, including all the matching scans in the retention time filtering, varying the precursor charges, etc. But those peptides that were found won't appear, and it is a shame because the nitrated variants are the ones in which I'm more interested in comparing.
I would really appreciate any help and aid you can give me, because I couldn't get a solution yet and I really want to do some good use of Skyline for this type of analysis! I hope my doubt has been clearly explained.
Many thanks!


view request
Dynamic isolation windows for PRM
(1 response) hober 2018-11-08

I have a minor question/suggestion, depending on if it's already implemented in Skyline or not.

When running PRMs, the early eluting peptides are the ones that experience the biggest drift in retention time. At the same time, most of the peptides that I'm interested in tend to elute closer to the middle of the gradient and be more stable RT-wise.
When setting the retention time window size, this results in a lot of "unused" potential for big windows at the beginning of the run, where the RT-drift is more evident, when using the same window sizes for all isolations.

Would it be possible to export isolation lists with dynamic window sizes?

If not, would it be possible to implement in some future version of Skyline?
You could in such case for example input a window size range that you would like to use and then let the windows be adjusted based on a "concurrent precursors cut-off".

Best regards
/Andreas Hober

view request
some clarifications needed for SILAC
(1 response) zongtao 2018-11-07

hi, skyline team

  1. "filter for document peptides". if i have multiple mzXML files and one mzid file. will this function stop extracting a peptide in mzid file from a specific mzXML file that does not contain identification of this peptide?
  2. "use hign-selectivity extraction". when using orbitrap fusion, what are the differences (parameter criteria) between checking this function and not using it?
  3. "min peptides per protein". what is the definition of peptides here? if a peptide in both heavy and light forms showed their intact form (charge 2 and 3 identified) and Met-oxidized form (charge 2 and 3 identified), will skyline consider this case as one peptide or multiple (how many?)?
  4. "remove duplicate peptide" and "remove repeated peptide". what are the differences between these two?
  5. see attached partial data from report export. for this peptide, the total areas of the first 6 rows are different from the last 6, while the other parameters are all the same. what is your explanation of such scenario?
 New Microsoft Excel Worksheet.xlsx 
view request
issues of peak area under the curve
(1 response) zongtao 2018-11-07

hi, skyline team
see attached figure showing that the the peak area counted is not the real peak area we want. how can we better the extraction method? a minor question is: what is the "12" in the figure, where it is coming from? i noticed that this number is associated with the extracting time window, and this number changes with different peptides.

view request
Strange error message during import of 6490 data
(2 responses) tasso miliotis 2018-11-07

I can import QQQ data acquired with one of our 6490 instrument that is using Masshunter version B.06.00.
However, I can not import QQQ data acquired with another 6490 instrument that happens to have the MassHunter
version B.08.00. The data are in both cases located on the local harddrive. I am using the same Skyline document
and the same acquisition method. I obtain the error message when trying to load data generated from the
B.08.00 instrument. Any ideas to why I obtain the error message below?

At 15:46:
Failed importing results file 'C:\Users\farmtm\Desktop\DATA_Skyline\Plate1_1_38+3HVs_RIA6490\181103_tmi_QC-A_Repl_1.d'.
[MassHunterDataImpl::ctor()] File not found: C:\Users\farmtm\Desktop\DATA_Skyline\Plate1_1_38+3HVs_RIA6490\181103_tmi_QC-A_Repl_1.d\AcqData\MSScan.xsd

Inner exceptions:
Exception type: System.Exception
Error message: [MassHunterDataImpl::ctor()] File not found: C:\Users\farmtm\Desktop\DATA_Skyline\Plate1_1_38+3HVs_RIA6490\181103_tmi_QC-A_Repl_1.d\AcqData\MSScan.xsd
[MassHunterDataImpl::ctor()] File not found: C:\Users\farmtm\Desktop\DATA_Skyline\Plate1_1_38+3HVs_RIA6490\181103_tmi_QC-A_Repl_1.d\AcqData\MSScan.xsd
at filename, MSData result, Int32 runIndex, ReaderConfig config)
at pwiz.ProteowizardWrapper.MsDataFileImpl..ctor(String path, Int32 sampleIndex, LockMassParameters lockmassParameters, Boolean simAsSpectra, Boolean srmAsSpectra, Boolean acceptZeroLengthSpectra, Boolean requireVendorCentroidedMS1, Boolean requireVendorCentroidedMS2, Boolean ignoreZeroIntensityPoints, Int32 preferOnlyMsLevel) in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 142
at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Int32 preferOnlyMsLevel) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 272
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 191

view request
adding lanthanide element as modification
lvyayao90 2018-11-07

Dear support team

I tried to edit a new modification using two lanthanide elements, but i was noticed that "only H O N C S P Se Li F Na S Cl K Ca Fe Ni Cu Zn Br Mo Ag I Au Hg B As Cd Cr Co Mn Mg Si are compatible". I wonder if you can please add lanthanide elements(especially La Eu Tb Ho Tm Yb Lu) as compatible elements in the list, for our team think lanthanide elements are very useful tags in quantitative proteomics.

I'd be grateful for any help.


Lv yayao

view request
Error while removing peaks and then apply to all function
(4 responses) ssharma02 2018-11-06
So while analyzing, I wanted to remove a peak and then use the apply peak to all function to remove peaks form the different samples. An error keep on showing. A screenshot of the error is attached a powerpoint file.
view request
Dwell time setting
sbhosale 2018-11-06

I haven't found any dwell time setting in Skyline. However, I have about 75 targets (180 peptides) comprised of high and low abundant proteins and I'm doing SRM analysis using TSQ Vantage.
I was wondering that is there a way to assign dwell time differentially to high and low abundant proteins (Heavy and light).
Any help is really appreciated.
Thanks, Santosh

view request
MS2-dependent MS1 chromatogram extraction
(2 responses) megamatt 2018-11-05

We use IP2 software to generate mzIdent files. We import those files into skyline to analyze SILAC and other various ratios. The situation: we import two datasets where the peptide/protein of interest is identified by IP2 from its MS2 spectra in run 1 but not in run 2. Our understanding of skyline is that it extracts chromatograms for the peptide in both runs even though it was only identified from one of them. Our question: is there a way to have skyline only extract a chromatogram when either the heavy or light peptide was identified in that run?

view request
Installing MSstats for non-admin user
(1 response) fcsigloch 2018-11-06

I tried to install MSstats on Skyline_v4.1 for a user without administrator rights in Win10. Administrator rights are needed for the installation of R_v3.5 during MSstats installation.

What I tried

  1. Running Skyline_v4.1 as administrator.
  2. Logging in as a user with administrator rights, locating the Skyline installation of the non-admin user and installing MSstats there.


  1. Nothing happens, program is not started.
  2. I located a Skyline executable hidden somewhere in NON-ADMIN.USER/AppData/local/2.0/SOME.WEIRD.FOLDERS/.
    However, the Skyline version is not displayed when clicking on Help/About, so I am not sure, if I found the right executable.
    Nonetheless, I could successfully install MSstats and R_v3.5 here, but when I log back in as the non-admin user, MSstats is not installed.


  • Why can't I choose the installation path for Skyline?
  • Why do I have to re-install R, if I have a running R version? Wouldn't it be easier to search for an existing R installation during MSstats setup?
view request
precursor ion in transition list
(2 responses) zainab noor 2018-11-05
Hi Skyline team,

I am having an issue with peptide transitions. I want to keep only y and b product ions but when I import the transition list after setting up all the parameters in Transition and Peptide settings it still shows the precursor ion (as shown in pic "precursor.JPG"), even if I disable the MS1 filtering settings. I have also shared the screenshots of transition and peptide settings. Could you pl. guide me which setting needs to be changed to filter only b,y ions?

 precursor.JPG  Transition-Library_settings.JPG  Transitions-Filter_settings.JPG  Transition-FullScan_settings.JPG  Peptides-Filter_settings.JPG  Peptides-Library_settings.JPG 
view request
Product ions tracs showed in only early 10 min gradient in Skyline.
(4 responses) andyzcq 2018-11-04

I wanted to use Skyline to process SWATH-MS data, I found Skyline only showed the product ion traces in early 10min gradient but the entire run is about 60min.

How to fix this issue?

The SWATH files and library files in the attachment.

view request
how to remove the peak with 20ppm?
(3 responses) karolina minta 2018-11-01

I have a problem with removing a peak from Skyline, which has above +20 ppm. In the spectrum I see heavy peptide (-2 ppm) and this light (+20ppm), see attachment. Certainly, the one representing the light peptide is not the peak I am looking for. I tried to decrease ion match tolerance in transition settings ->library and re-import the data, but the peak still appears. Do you know how can I remove it from my spectra?
Thank you,

view request
failed importing results files
(1 response) zongtao 2018-11-02

hi, Skyline team
i was using skyline to do my silac preps, after adding the fasta file>finish. the skyline gave me this error, which has never happened before. could you check the attached error message, and let me know how to solve?

 New Microsoft Word Document.docx 
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instrument serial number in report
(1 response) matthew.r.russell 2018-07-10
I am putting together a QC system based on reports produced by skyline for multiple Sciex 6600 instruments. I would like to be able to include an instrument identifier, such as the serial number, in the report so that I can process data files from multiple instruments through skyline simultaneously and identify the instrument of origin in subsequent analysis.

I can’t find such an identifier in the report editor window, I expected it to be grouped under ‘Replicates/Files’. Is it possible to include such an instrument identifier in a report in a way I haven’t found? If it isn’t, please could this feature be added?
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Unreadable codes in exported reports
(8 responses) user 2018-10-28

Dear Skyline team,

In skyline reports, there are unreadable codes in “quantification” of Peptide Ratio Results and “calibration curve” of Peptide Quantification report (see attached screenshots). I have the same problem in 5 PCs in our group, no matter the default language of them is English or Chinese. How to fix?


 calibration curve.PNG  quantification.PNG 
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Removing proteins without unique peptides
(4 responses) Giovanna 2018-10-25

Dear Skyline team,
first of all I apologize in advance for the chance that my questions are stupid, in my defence it's the first time I manage proteomics data.
I'll briefly explain the steps I've followed.

I've built a spectral library by choosing the option ''Import DDA peptide search'', and importing a msms.txt file from Maxquant.
As last setting I've chosen ''remove duplicate peptides'' since I want to keep only proteins identified with unique peptides. I've obtained 1230 proteins. At this point I could see the chromatograms of the precursor ions in my DDA runs, but not their corresponding MSMS transitions. Here it comes the first question: why? I've already selected View -> Transitions -> All.
Then I've tried to import my DIA runs from File -> Import -> Results, but I still couldn't see no transition.
So I've tried from another path: Blank document -> Settings (where I've inserted the peptide and transitions parameters I want for my DIA files, is that correct or does it affect the following import of the library?) -> View -> Spectral library (where I've chosen mine) -> Add all. The library's peptides appeared, but not the matched 1230 proteins I expected. In order to get them I had to import the FASTA again (File -> Import -> FASTA). At this point the proteins were more than 8000. My explanation for this was that I hadn't filtered the duplicate proteins jet (but if I had to do that again why did I have to put a FASTA in building the library and choose which proteins to exclude? If these protein settings and filters are then lost I mean). I imported my DIA runs through File -> Import -> Results and the proteins' number remained the same (reasonable) but it was then finally possible to select the precursor and the transitions and see them in the chromatograms of the DIA runs. I guess I can find into the DIA runs no more proteins than the ones cointained in my library, so I wanted to clean the proteins in order to keep the ones with unique peptides, and I expected to obtain maximum 1230 proteins. I selected Edit -> Refine -> Remove duplicate peptides, but it happened that all the proteins in the left panel appeared without the list of their precursors and transitions, and their number (that was actually reduced) was superior than 1230. What did I do wrong or not understood?
Furthermore, as last questions, idotp is Isotope dot product I think, but what is dotp? Can I get it in the report and, foremost, can I rely on those parameters in order to trust an identification without manually check it? We haven't used standard peptides for iRT normalization, but we are going to.
Thanks for your patience and attention.
Best regards!


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Overlapping MS1 data
Tobi 2018-10-30

Dear Skyline Team,

even though i looked up the DIA tutorial and other forum posts concerning overlapping data i just dont see the right explanation to a question I have, it would be great getting some hints from you.

The question is, can skyline deal with overlapping MS1 data in a way that chromatograms are built without any overlap causing twice the amount of points across the peak?
For example tSIMs measurements will be done from 399-421, 419-441, 439-461 (overlap of 2 m/z) etc…
Can you built MS1 chromatograms as if 1 m/z (half the overlap) would have been cut away from each side of each scan? (400-420,420-440,440-460…) Or is the Isolation Scheme option limited to MS2 data?

It would be great getting in contact with you, and sorry if I missed something in the tutorials.

Best regards, tobi

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Most shortcuts on Start Page do not work (Skyline Daily
(2 responses) Markus 2018-10-25


just a minor issue and not sure whether this was reported before, but I noticed that on the Start Page of Skyline Daily (Windows 10) actually only the shortcut to "Blank Document" works. All others show a description when hovering over them but nothing happens after clicking.


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iRT not detected in samples
(2 responses) clive cota 2018-10-25

Dear Skyline team,

I am doing a DDA analysis, but when I am building spectral library the SCIEX PepCalMix (iRT-C18) are not detected in my samples spiked with Pepcal. I have attached a screen shot of the problem.


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Resize Previous Files Bar on Start Page
(1 response) Tomas Vaisar 2018-10-26

I would like to make a suggestion /request - would it be hard to make the left-side bar on the Skyline start page with the list of previous skyline documents wider or adjustable?
It does not seem to be adjustable at this point and hides part of the file name if it is longer than 32 characters, which I often end up with (I hope there are other folks who like descriptive filenames).



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Further MIDAS Libraries Questions
(5 responses) roman sakson 2018-10-14

Hello Skyline team,

thank you very much for the great piece of software that you are developing and maintaining! In our core facility for proteomics in Heidelberg, Germany, I work with a QTrap 5500 and we often use the Sciex MIDAS workflow. Some time ago, you have implemented more support for this in Skyline, which is great. However, I have two general question:

When I import a wiff-file containing MRM traces as well as MS/MS spectra into Skyline, as it is the case for MIDAS experiments, I see the usual MRM XICs and also vertical blue lines with retention times over them. I believe, that this lines indicate the timepoints at which MS/MS spectra were acquired in my wiff-file. Importantly, there is no "ID" word on top of those lines yet, since no "real" spectral library containing a database results output like a dat-file from Mascot is present in the Skyline document at that stage. However, since there is now a "midas" library created by Skyline automatically, I still get to see all the imported MS/MS spectra under "Library Match" with a more or less long list of all those spectra with a corresponding RT in brackets offered to me as a dropdown menu. I appreciate that but I have realized that there is also the peptide sequence and a charge state displayed on top of the library match, just as how it would look like for a "real" spectral library match (see first screenshot). At that point, Skyline probably just imports the peptide sequence from the target list, since it has no database results available yet, and I don't really know what amino acid sequence corresponds to the MS/MS spectrum. Am I missing something here?

The second question concerns the new tab "Library Runs" which I can find under "Manage Results". I believe, that there is an entry created for each MIDAS-experiment that I import (see second screenshot). While the "midas" library is automatically created by Skyline in the same directory in which the Skyline-file itself lives, the path for the Library Run seems to point to the folder where my original wiff-files are stored. I don't have any particular issues with that but would just like to understand whether my wiff-files are now somehow connected to my Skyline-files? What would happen if I made use of the "Remove corresponding runs" check box?

Kind regards,


 MIDAS_Peptide_Sequence.tif  Corresponding_Runs.tif 
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Skyline stopped accepting Thermo.raw files
(2 responses) waltteri hosia 2018-10-25

Hi, thanks for generally superb software.
A hiccup. It was working OK, until suddenly start to refuse to open thermo files. Opens some old files, but not all. Tried opening and closing Skyline and moving the file from network drive to my PC. Same problem. Error message attached.

 Error opening thermo raw data.txt 
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Intensity difference between skyline and xcalibur
(5 responses) dtan 2018-10-23

Dear Skyline team,

I'm using skyline to extract the intensity of fragment ions from PRM data for small molecules. However, I found that the intensity extracted by skyline was much higher than that extracted by Xcalibur (~20E6 vs 4.99E6). What should I do about this?


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LOD calculation using internal standard
(3 responses) ssaleh 2018-10-18


we spiked a sample matrix with a mixture of internal standard ( 58 labelled peptides) with different range of concentration. We then run the samples in triplicate on TSQ. The idea is to calculate the LOD.
I was wondering what should I use to build the calibration curve? Total area in precursor results (attached figure)? is there a way to do this in skyline or we should export the values and make the curve in Excel?
Could you please refer me to a tutorial in skyline where you did LOD calculation using IS.

Would like to share the skyline document if needed but in private mode.
hope you can help!


 total area in precursor results.png 
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Skyline 4.1 can't open 6500 Sciex MRM wiff data
(4 responses) heyang 2018-10-23

Error message:
The file contain error on line 1 at col 1.

Any suggestion about this?

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(5 responses) danielacgranato 2018-10-17


I have just starting working with Quasar and I am getting an error message from the software. I have attached the document I started working with. I only have standard samples, that I ran in triplicate and I would like to calculate LOD and LOQ.

Can you help me with this?

Thank you very much in advance.
Best, Daniela 
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Exporting data to skyline from UNIFI software and Xevo G2-Xs Q-TOF
(1 response) martin jutras 2018-10-19

We are implementing a metabolomic analytical service with our Waters Xevo-G2-XS QTOF.

However, it is running ONLY with UNIFI for both acquisition and data processing (no mass lynx software…).
Is there a way that Skyline can upload data from UNIFI?
UNIFI is a database, and there are no raw files I guess as in the case for Masslynx.

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Skyline for unknown modifications
(1 response) mprado 2018-10-17

Hi all,

I am wondering if anyone knows if Skyline could be used for the identification of unknown modifications.

For that, I have been running some test to see if I could identify fragment ions without considering the precursor mass, but somehow... I cannot make it work.

Let me explain it with an example. As you can see in the picture attached, I am trying to find several fragment ions of 2 peptides that differs only in one amino acid (the Met at the N-termini): MPLYSVTVK and PLYSVTVK. If in both peptides I only use y1-y7 fragment ions (disregarding the precursor mass), they should be identified at the same retention time. As you can see in the pic attached... This doesn't happen.

For this test I am deactivating the MS1 filtering in Tools>Transition settings>Full-Scan in order to use only the MS2 fragments, but I am not sure if I am missing anything else

Thanks in advance for your help!!

 Locating MS2 fragments.PNG 
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How Skyline Builds Spectral Libraries
(6 responses) manfred.heller 2014-11-07
We are working with DIA acquisitions for targeted protein quantification and try to figure out the easiest way to assemble a complete and best quality spectral library based on different DDA discovery analyses.

The BlibFilter algorithm chooses the best spectrum based on the best score. However, we have spectral libraries produced from different search engines, e.g. a pride.xml export from PeptideShaker with several search engines (X!Tandem, Amanda, msgf+, etc.), MaxQuant, and ProteomeDiscoverer.

How is the best score criteria applied in this case? By trying to build a spectral library using a pride.xml file containing X!Tandem and Amanda data together with MaxQuant, we realized that most of the library came from this pride file but the quality of the fragment spectra was miserable. I also realized that in this case, the "Filtering redundant library" process during the "Building Peptide Search Library" task was not performed, while it was done when using only data coming from one search engine (MaxQuant).

Hence, I guess it is not possible to build a spectral library using data from different search engines, is it?

Alternatively, the BlibFilter chooses the spectrum with the highest TIC. Based on above observation (and others), I am not convinced that this works really well.

Would it be possible instead of choosing the best spectrum, to compile all MS2 spectra belonging to the same precursor (charge and m/z values) to one single spectrum by adding all available intensities? Unfortunately, this might have a drawback, because not all fragment mass values will have the exact same digits after the coma?

I am looking forward to your answer or suggestion for dealing with this issue.

Thanks a lot and best wishes,
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Skyline export of results
(1 response) daisy verswijvel 2018-10-17

I have a problem with my data. When I am exporting my results from the skyline to excel I see duplicates of rows in my excelfile (see attachements). It is not a problem of duplicated or repeated peptides since the rows are identical (the same sample, retention time, area under the curve,....). I think it is because I uploaded my results several times in skyline and this program is saving this files somewhere. Even when I take a new skyline document, the peptides with its results are duplicated in the excel file.How can I efficiently remove previous results/chromatograms/peptide search in Skyline to event this problem.
Kind regards Daisy

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Peak areas
(2 responses) 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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Number of data points per LC peak vs cycle time in scheduled MRM
(5 responses) benoit fatou 2018-10-15

Hi Brendan,
I am currently using a QTRAP 5500 for scheduled MRM experiments on tryptic peptides.
I was wondering if the number of data points per LC peak (see attached file) is correlated with the cycle time settled in the instrument method.
Thanks for your help,

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Different m/z range for precursors and products
(3 responses) 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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Failed importing results dur to "an item with the same key has already been added"
(1 response) yuanys 2018-10-15

To whom it may concerned,
Thank you so much for your time. I change some transitions today (by insert the transition into the skyline for small molecular) and when I re-import the resluts I got the lines below. Please let me know how to solve it.
best wishes

At 15:37:
Failed importing results file 'D:\6500 data\10122018\J1_prelasix.wiff'.
An item with the same key has already been added.

Inner exceptions:
Exception type: System.ArgumentException
Error message: An item with the same key has already been added.
An item with the same key has already been added.
at System.ThrowHelper.ThrowArgumentException(ExceptionResource resource)
at System.Collections.Generic.Dictionary2.Insert(TKey key, TValue value, Boolean add) at pwiz.Skyline.Model.DocNodeChildren..ctor(IEnumerable1 items, IList1 previous) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\DocNodeChildren.cs:line 25 at pwiz.Skyline.Model.DocNodeParent.set_Children(IList1 value) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\DocNode.cs:line 384
at pwiz.Common.SystemUtil.Immutable.ChangeProp[TIm](TIm immutable, SetLambda1 set) in c:\proj\skyline_4_1_x64\pwiz_tools\Shared\Common\SystemUtil\Immutable.cs:line 201 at pwiz.Skyline.Model.DocNodeParent.ChangeChildren(IList1 children, IList1 counts, Int32 indexReplaced) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\DocNode.cs:line 0 at pwiz.Skyline.Model.DocNodeParent.ChangeChildrenChecked(IList1 children, Boolean changeAutoManage) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\DocNode.cs:line 1172
at pwiz.Skyline.Model.Results.PeptideChromDataSets.UpdatePepChildren() in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 805
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Add(PeptideDocNode nodePep, ChromDataSet chromDataSet) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 793
at pwiz.Skyline.Model.Results.ChromCacheBuilder.AddChromDataSet(Boolean isProcessedScans, ChromDataSet chromDataSet, PeptidePrecursorMz peptidePrecursorMz, IDictionary2 dictPeptideChromData, ICollection1 listChromData, ChromFileInfo fileInfo) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 683
at pwiz.Skyline.Model.Results.ChromCacheBuilder.CalcPeptideChromDataSets(ChromDataProvider provider, List1 listMzPrecursors, HashSet1 setInternalStandards) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 514
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 400
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 270

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Support for mixed acquisition modes
(2 responses) Yishai 2017-11-21
We have some data running targeted analysis using the Lumos where we run EThCD PRM in parrallent to other modes (DDA MS3). We also run other mixed modes on the Lumos but are unable to load the data into Skyline. Do you plan on supporting such modes?

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Individual quantification settings for different small molecules
(2 responses) Yasin 2018-10-10

Dear Skyline Team,

I would have a question/request:

Via View -> Peptide Settings -> Quantification I can set different quantification settings in a global manner. However, I often run into the situation where I want to use different Regression Weightings or Normalization Methods for different small molecules. Is there any way to change those setting on a molecule by molecule basis?

Thanks in advance

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Small Molecule MRM extraction with isomers
(6 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 
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