Showing: limited to 100 requests
Colour coding of Peptides and transitions
(2 responses) nr412 2019-05-22

Dear All,

I am new to skyline and want to check if there is any way to export the colour coded peptide and transition information while exporting out the data?

view request
Peptide library building from MaxQuant.
(2 responses) luzjpaulo 2019-05-20

I created a library from MaxQuant using the latest Skyline-daily update but I found out an issue. Skyline-daily gives me very bad peptide spectrum matches while Skyline does not have this problem.

 msms.txt  modifications.xml  mqpar.xml  SugarCane_histones.fasta 
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Exporting p, q, or e values in Peptide Quantification Report?
(1 response) lparsons 2019-04-24

I imported MaxQuant results into Skyline, and now I would like to export a Peptide Quantification report. I've found all the fields that I'm looking for except for the peptide score (from MaxQuant) itself. Is there a way to get this?

In this case I have 6 samples, so I would expect there could be 6 p, q, or e values for each of them, which would be fine. Or do I have to do a different report in order to get these?

thank you

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Questions on baseline intensity and peak calling
(1 response) lparsons 2019-05-07

I am using a method similar to these two papers ( that combines MS1-only scanning with directed feature ID.

Briefly, our process starts by running two samples in triplicate MS1-only scans. We now use OpenMS for feature detection and consensus mapping to produce a list of peaks of interest that are selected as a result of group t-tests (3 runs of sample A vs 3 runs sample B). These features are then used to produce an inclusion list for the instrument to then do a directed DDA where features are pre-selected for MSMS. The MSMS data then is run through MaxQuant, and the MaxQuant data is fed into Skyline. In Skyline we then omit the raw MSMS data, instead using the raw MS1-only data in its place so that the peak intensities from the MS1-only data will be used for the MSMS features.

The results of this generally look pretty good. However, there are some features that come out of this with vastly different intensities from Skyline than what they had in OpenMS. In particular, there are some features where we know that sample B should not show any intensity while sample A should. On some of these samples, Skyline reports very low intensity (exported as "Total Area MS1" in Peptide Quantification report) where we expect none and yet on others Skyline reports very high intensities (up to 10^6 or more) when we expect none.

This leads me to a couple questions

  1. Is there a way to find out what Skyline sees for baseline intensity at a given time in a given file? I would expect this could be helpful for trying to figure out if the lower intensity peaks are just baseline AUC.
  2. Does Skyline do any kind of baseline correction for Total Area MS1? I didn't see a parameter for it though I may have missed one along the way.
  3. Is there a parameter I can change that will set the tolerances (RT, M/Z) for peak calling? I am wondering if the peaks might be called too generously here and the intensity is coming from neighboring unrelated peaks.

I also looked at the dot products of these features to see if that could lend some insight. When I plot median dot product vs median B intensity - for the peaks where I expect to see zero intensity from B but am getting positive "Total Area MS1" for B - I don't see a strong correlation though there is a wide distribution of median Total Area MS1 in the range of median dot products > .085 and < 1.

thank you

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Integration threshold
lzhangok 2019-05-22

I load 200 MRMs into Skyline and do the process, cannot find threshold parameters, so in the end, some of the metabolites have green dot indicating positive detection, where many others have not green dots indicating no detection at all. But I can confirm with the vendor Quant tool that many "not detected" metabolites can have good peaks and be detected. Hope to see your suggestions.

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Isotopes via Insert Small Molecule Transition List
(9 responses) Fabian 2019-05-16

Dear all,

I have a list of small molecules containing various numbers of Isotopes.
For exporting and re-importing of this list I setup a suited document grid with the correct order for:
Edit - Insert - Transition List - Molecules.
However, trying to reimport the list gives me only the monoisotopic precursor.
The option I have is to tell skyline the product m/z which differs between the Isotopes.
This results in the error attached.

Kind regards


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Retention Time Calibration for DIA-data
(1 response) yb77618 2019-05-17

Hi Skyline Team:

I have DIA-MS data and pre-built assay library, so I want to use Skyline-Daily for data analysis.

The assay library was loaded successfully, 11 iRT-peptides were chosen as standard for retention time calibration.

However, I found out the criteria for DIA-MS data loading was so stringent, 80% peptides were needed with a correlation of 0.99 in regression.

Is that possible for us to change the requirements for calibration? Like to calibrate by hands or lower the R-square value?

(I want to remove some iRT-peptides for better correlation in the regression, an alert popped up, see attached image)

Thank you in advance for answering my questions.


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Thermo Altis Collision Energy Opt
(11 responses) avizrosenberg 2019-05-21

Ran several peptides with collision energy optimization on Thermo Altis. When imported to skyline I am only seeing peaks for a single transition per peptide rather than all the transitions that Ive optimized (I can see the peptides and transitions in Xclaiber).

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Issue with Recognizing User Created Modification
(23 responses) dwwilk02 2016-05-27

We were trying to get Skyline-Daily (on Windows 64-bit) to create a library from a final_fragment.csv file from a Waters Synapt G2-Si MSe experiment (csv file is attached). The file has the modification GEE(Q) (an addition of H6C4O2 to Q residues) that was created in-house for a collaborator. We get the following error when loading csv files with this mod:

System.IO.IOException: ERROR: The modification 'GEE (Q)' on line 489 is not recognized.
ERROR: reading file GEE_Test_Mouapi_Pink01_IA_final_fragment.csv

   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 109
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status) in c:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 53
   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 130

I've tried several things to get the mod to work (adding the mod to Skyline, changing a current mod with H6C4O2 addition to include Q, adjusting the addition for one of the default mods at Q), but so far have had no luck. I can load the file if I delete the GEE mods, but then I miss the library matches for modified peptides. Is there anything that we could try to get Skyline to recognize the mod? Thanks for any help you can provide,

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Small Molecule Zero Peak
bwidner 2019-05-16

Hello Skyline Team!
I am measuring small molecules, and I am looking for a way to get "zero" in the Transition Results table for peaks that are non-existent. At present, I am not quantifying and don't have a good sense of LOD. If I had a sense of LOD, I would handle this in post-processing. Without quantification, I am trying to look at presence/absence or fold change. Often when there is no real peak, but there is some noise, the noise is selected. I was getting around this problem by manually drawing a baseline somewhere else, hoping it would put zero in the table. This worked, but sometimes I draw a manual baseline on a peak that looks small and without zooming in I can't tell if it is a small peak or just a single scan.

Is there a way to screen out peaks that are only 1 or 2 scans? I saw the "Points Across Peak" parameter, but when I manually draw the baseline there are often quite a few scans, so that will not work. It would have to account for the number of scans above the baseline, somehow. Does such a parameter exist? I saw elsewhere on the forums the suggestion to use the FWHM, but this is also driven by how wide the base of the peak is, which for manual integration varies randomly.

Thanks in advance!

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Export method to MassLynx 4.2 version
(3 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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creating peptide library from uniprot database human
(4 responses) sponce1 2019-05-13

i downloaded all of the file formats (fasta, XML, ts, etc.) available from uniprot human reviewed:yes

and couldn't get them to load in the Peptide settings -> Building Library -> Input Files -> error
"fasta.gz is not a valid library input file.

I've attached my skyline file, a dataset and the fasta file. please make it work!

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MS1 Quantification of Precursor Ions
brown459 2019-05-15


The problem: We have generated a database of glycopeptide identifications using a DDA analysis. Each sample/acquisition (i.e. run 1 through run 26) is a high pH reverse phase chromatography fraction. Therefore, not every acquisition contains each of the glycopeptides identified in the database. In fact each glycopeptide should only be observed in a few runs. We want to quantify the abundance of individual glycopeptides in the identified fraction (run) and the surrounding runs.

Is there a way to use Skyline to very simply, export XIC's for a series (we define) of amino acids modified with variable glycoforms (~300). We have the m/z, charge, RT, fraction, elemental composition. We also have a .blib that is composed of EThcD spectra.

I have approached this problem from both the proteomics and the molecule interfaces and have encountered similar erroneous peak matchings. Because we do not identify every species in each fraction, we are finding that in fractions where a glycopeptide is not observed, skyline ends up quantifying an incorrect precursor MS1 that is near it in RT.

Molecules doesn't seem to restrict IDs based on the explicit retention time window. Proteomics may be the only way to go, using MS1 quant and really reducing the RT window? This however, seems to take a long time as there are many possible combinations of modifications.

Are there any other work arounds that I am not thinking of?



view request
combined MS1 PRM no product ion chromatograms
(8 responses) mohammad abukhalaf 2019-05-10

Hi guys

We are running an alternating MS1 PRM scan strategy on a QEPlus. We built a library from previous DDA and PRM measurements of our targets and also imported the FASTA of our target proteins. That all looks fine. However there is no import of product ion chromatograms. We tried adjusting many of the transition settings, i.e. Transition settings->Full scan->MSMS Filtering->Acquisition Method->Target or none, various settings on Transition setting->Filter->Product ions->From To and so forth but no use. We also widened all of the m/z windows. We are using the newest version 4.2.0. Everything to no avail. Any ideas?

Thanks a lot
Mohammad and Wolfgang

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RT prediction not shown for all peptides/samples
(2 responses) ssaleh 2019-05-13

Hi Skyline team,

I recently run around 20 samples in quadruplicate on TSQ vantage (to monitor 12 peptides using MRM mode) and I am currently analyzing the data. When trying to upload the results into skyline I had the following error "unable to finish importing chromatograms because RT predictor linear regression failed". To solve this problem I had to switch off the RT predictor, this way the import was successful.
I then switched on the RT predictor , however the RT prediction was missing in a lot of peptides and samples in a random way (please check attached ppt ). Any idea what could be the reason?
Is there a way to share the in a private mode?

Many thanks in advance!

 RT prediction.pptx 
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Try to set different collision energy for different daughter transitions of a compound
(1 response) yufeng li18 2019-05-13

Hello there,

We were trying to export a method to MassLynx, which each compound has 3 to 4 daughter transitions with different collision energy. When we inserted a transition list in the Skyline, the format would look like in the picture 1 and the exported method in the MassLynx had the same collision energy (always be the collision energy for the first daughter transition in the list) for every daughter transition. We noticed the collision energy can only be modified in the precursor tab (662.1018 in the picture). In order to have correct collision energy in the MassLynx, we had to manually edited the in Skyline (shown in picture 2 ) and changed the collision energy for each precursor accordingly.

Do you have an ultimate way that can export a method from the Skyline with each daughter transitions having the correct collision in the MassLynx? I really appreciate if you could help

Many thanks

 1.jpg  2.jpg 
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Webinars Visualization
(2 responses) GioMag 2019-05-09

Dear all,

I wish to watch some webinars (#17 in particular), unfortunately videos automatically stop the playback after few minutes.
This happens with all browsers I have tested (Chrome, Edge, Safari..) and on different computers.
How can I solve this issue?


 Issue Webinar #17.bmp 
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Library build error (dat file)
(15 responses) Jason Held 2019-02-14

I'm trying to build a library from a large mascot .dat file (2.2 GB) and getting: ERROR unable to read filesize of (my filename).dat.

More info pasted below. Error screenshot attached.


System.IO.IOException: ERROR: Unable to read filesize of F011061_Cys_NewRat_PlusRED_0p0232.dat.

Command-line: C:\Users\lab_held.pcl-held1\AppData\Local\Apps\2.0\NRM38HKN.2B5\OM37C28G.N7K\skyl..tion_e4141a2a22107248_0004.0002_ed14b445d33623de\BlibBuild -s -A -H -o -c 0.9768 -i GloCys055_Cys_PlusRED_Mascot_0p023 "C:\Skyline\SpectralLibraries\GloCys055_Cys_PlusRED_Mascot_0p023.redundant.blib"
Working directory: S:\Users\Jason\GloCys055_131231\Cys
Standard input:

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 60
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 178
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 144

 Screen Shot 2019-02-14 at 9.55.01 AM.png 
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Dimer peptide
(2 responses) chanchalmrt 2019-05-09

I want to make a dimer peptide of two monomers each with 9 aa (eg. LVCIALRKK-NH2) linked by a single disulphide bond between the third cys of each monomer. Is that possible with skylink?

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small molecules: normalized retention time (iRT)
(4 responses) alessio maiolica15118 2018-07-02

Dear Skyline support team,
I am interested in using the iRT functionality for small molecule experiments.
It seems that this is not yet implemented in the current version of the software.

Are you planning some work on this? if yes would you please let me know when would you expect to have it released?
thank you all for your great work.
best regads,

view request
Include fragment ion annotation when "Copy Data" from Library Match tab
jonasbecker 2019-05-08

Dear Skyline Team,

I'm trying to compare spectra from peptides recorded with different fragmentation methods as well as measured from synthetic pools and out of samples.

Is there any possibility to add fragment ion information displayed in the graph to the data which I get by using the "Copy Data" interaction within the Library Match tab? Unfortunately I only get m/z values and intensities, which I than would have to annotate manually.

Or is there any possibility to get this information from a Custom Report for a single spectrum? This one could combine subsequently with the data copied from the Library Match tab to also include unmatched peaks.

I'm using PEAKS Studio X for my search, export data from there and import into Skyline 4.2.0 (64-bit).

Thanks for your help in advance. Best,

view request
Viewing Products from DDA
bwidner 2019-05-07

Hello Skyline team!

I am trying to develop a quantitative method using DDA data, and I would like to use the product ions to confirm the identity of the parent. I will quantify from the parent. I find the way skyline draws the product "chromatogram" by connecting scans that are really far apart to be sort of confusing, since I can't tell the retention time of the MS/MS scan without clicking on it. Is there a way to view the MS/MS scans as individual sticks or dots or something, instead of as a chromatogram with the scans connected?


view request
Targeted MS/MS importing - cutting off ID'd peaks
(4 responses) kvancott2 2019-02-12

I successfully built a spectral library from DDA data and then imported a list of proteins for an MRM-HR method using a Sciex 6600 QTOF.

I imported another DDA data file into my Skyline file. In the Transition Settings, I used "include all matching scans" option for Retention Time FIltering.

After importing the data file, the scan extraction width seems to be all over the place. Some peptides it is only a minute wide, some are 10 minutes wide, some are 20 minutes, and some even higher.

What is most frustrating, though, is that quite often the matching/ID'd peaks are cut-off in Skyline. I'm attaching a powerpoint file that shows an example of Skyline cutting off the scan of a yeast ADH peptide (YYVDTSK) that was spiked into the samples.

I've tried re-setting the Retention Time filtering, re-saving the file, re-importing the raw data file, but it doesn't change. I've tried adjusting the Retention Time filtering to 5, 10, 15, or 20 minutes - it doesn't seem to make any difference what the setting is.

I even tried importing one of the raw data files that was used to make the spectral library - same thing. Multiple peptides that are ID'd are cut off by Skyline. In your Targeted MSMS tutorial, it says on page 31 that the entire gradient should be covered in the chromatograms. But I'm not seeing that. Not sure what I'm missing.

 KVC-Feb-12-2019 - MS1 filtering problem.pptx 
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Failure opening Skyline Session
(3 responses) aline doerrbaum 2019-05-06

Dear Skyline Team,
I cannot load my Skyline session anymore.
The following error occurs: "Failure opening "C:[...]. Exception has been thrown by the target of an invocation." (also see screenshot attached).
We put a lot of work into checking and integrating the peaks already, so it would be great if there was a chance to rescue it.
An older version of the session, that I saved under a different name before curating the data, can be loaded without problems.
I'm using Skyline and Windows10.
Do you have an idea how to fix it?
Many thanks in advance!

view request
No precursor ion chromatograms found
(15 responses) bwidner 2019-04-30


I am new to Skyline and trying to do something very simple, but I cannot get it to work. When I import the transition list with just the precursor information, everything works. I see the peaks as expected. However, when I add the product information, I get the "No Precursor ion chromatogram found" message. I double checked the product m/z and looked through the tutorials, and still I am quite befuddled!

The data is from a Thermo Fisher Fusion Lumos orbitrap. I have attached the skyline files and the raw file I am working with.


 test4.skyd  test4.skyl 
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Import of report template
(1 response) sstoychev 2019-05-06


Would like to import the attached report template, how is this done?

Also, is it possible to export a Skyline library in .csv format?



view request
Spectral library essential in Skyline for the generation of optimum MRM method
(3 responses) shobhabr20 2019-05-03


Is spectral library essential in Skyline (Peptide Setting-> Library) for the generation of optimum MRM method for a peptide.

Please clarify.


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Calculate the confidence score and q-value for searched peptides
(7 responses) sunbergsoon 2019-04-30


I am doing SWATH analysis using Skyline. We filtered the searched peptides based on dot-product which above 0.8.
Recently we also tried the mProphet model and using the second best peak to calculate the q-value to do more confident
filtering. However, the results are not so good and the Q-q plot shows a bad shape. So how can I use Skyline to do more
confident filtering on SWATH analysis? Can I try other method like using openSWATH model?


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Getting The operation has timed out.
(3 responses) kartik kundgol 2019-05-05

Hi Brendan,

I am using SkylineTool.dll in my application. Unfortunately not able to establish the connection between Skyline and my application.

Getting "The operation has timed out exception" in the SkylineToolClient constructor.
toolClient = new SkylineToolClient("$(SkylineConnection)","Test Interface");

Can you please guide the solution.

Thanks & regards,
Kartik K

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Modified Sequence Full Names - apparent bug
(1 response) Nathan Manes 2019-04-29

There is an apparent bug in the current version of Skyline 64bit (v4.2.0.19072). When exporting a report that includes precursor "Modified Sequence Full Names", sometimes a mod will be missing. Out of 321 peptides that I imported (Edit --> Insert --> Peptides) into a new document, two had this error (in both, cys carbam was missing in the heavy form; strangely it is present in the light form). Inside the attached ZIP (Modified Sequence Full Names - apparent is the Skyline file with the 2 peptides and the resulting exported report (transition list) with the incorrect "Modified Sequence Full Names" values (heavy rows are incorrect; light rows are correct). I didn't check if anything else is incorrect.

 Modified Sequence Full Names - apparent 
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Small molecule method export
(8 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 
view request
Failure imorting mzXML
(3 responses) cfarnsworth 2019-05-02

I get the following message when trying to import mzXML files. Note only 6 of 24 files in total failed.
Thanks, Charles

Failed importing results file 'D:\IMAC Keezer\180418_28574_CF_x.mzXML'.
Object reference not set to an instance of an object.

Inner exceptions:
Exception type: System.NullReferenceException
Error message: Object reference not set to an instance of an object.
Object reference not set to an instance of an object.
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.get_PercentComplete() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 806
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.UpdatePercentComplete() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 599
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.GetChromatogram(Int32 id, Target modifiedSequence, Color peptideColor, ChromExtra& extra, TimeIntensities& timeIntensities) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 587
at pwiz.Skyline.Model.Results.ChromData.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\ChromData.cs:line 77
at pwiz.Skyline.Model.Results.ChromDataSet.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 233
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Load(ChromDataProvider provider) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 144
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 452
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 263

view request
Failure to import MS1 filtered data
(1 response) sstoychev 2019-05-02


I'm trying to import precursor ion data from DDA runs but Skyline freezes during the import. Tried importing .wiff or .mzml as well as one-at-a-time vs individually but issue persists. Attached is the Skyline document as well as LCMS data so can see if you can reproduce the issue on your side.



view request
Shimadzu Triple Q data in Skyline
(5 responses) t j f m voermans 2019-04-29


In the article: "The Skyline ecosystem: Informatics for quantitative mass spectrometry proteomics" it is stated that skyline supports SRM files from Shimadzu (.gpd). However, the only files I have are .lcd files (see attachment). This file is of a scan+ measurement done on a Shimadzu LCMS-8045.

Any way to import these in Skyline as well? (When I use the normal way of importing data via File - Import - Results, I get a message that there is no usable data found.

Thank you.


 Test file.lcd 
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Unable to generate calibration curves
(3 responses) o mannion5 2019-05-01

I followed the tutorial for generating a calibration curve but after following all the steps I get a blank calibration curve with the message "Error: All of the external standards are missing one or more peaks. The selected replicate has missing or truncated transitions." I don't understand this error message as all of my standards have clear well-defined peaks. Has anyone else encountered this problem ? Am I missing something?

view request
Collision Energy on QExactive
(3 responses) lisa.j.zimmerman 2013-10-23
Hi Brendan
Has something changed in the way Skyline calculates the collision energy for an inclusion list for the Q Exactive? Previously the values were different depending upon the peptide but now the value is 27 for all peptides?
Also, how is the collision energy calculated for the QEXactive since there is no drop-down option for this….it simply defaults to the Thermo TSQ Vantage?
view request
Issues with downloading Skyline
(1 response) ddoza 2019-04-30


I am currently having trouble downloading Skyline 4.2 64 bit on my PC that is running Windows 7 Professional 64 bit. I get an error message "An error occurred trying to download ''." whenever I attempt to download skyline. Restarting the PC or attempting to download Skyline under a different user does not help. I have attached the setup log file. Can you please help?

Many thanks,

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System.IO.IOException: ERROR: Illegal character p found in sequence .....
(5 responses) jason a macgurn 2019-04-15


I am using skyline with MS1 full scan filtering on data acquired from a Q Exactive and searched on MaxQuant. I am using Skyline to inspect peptides (especially phosphopeptides) and validate quantification from SILAC labeling.

I had been using a very nice workflow (based on the MS1 full scan filtering tutorial) that was perfect for our application. But recently, I had some computer problems which forced me to install the latest versions of both MaxQuant and Skyline, and now whenever I try to set up the filtering on my data I receive the following error:

"ERROR: Illegal character p found in sequence AAGpSGESpTPER (line 13)"
(detailed error info in the attached text file)

I only get this error when I attempt to filter data generated with my new version of MaxQuant - if I filter analysis from the old version, there are no errors.

My interpretation is that Skyline doesn't like something about the way MaxQuant is formatting the msms.txt output file (phosphopeptides specifically?), so it chokes on it. I figure I either need to re-format the msms.txt output file or adjust some setting for the input on Skyline. Has anyone else encountered this problem? Is there a fix or workaround?
Thanks in advance for any help / suggestions - I am eager to get back to using Skyline for analysis of some of my data!

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save pivot in Report template .skyr
(2 responses) sarah lennon 2019-04-25

Hi Skyline team,

I have got an additional question. I would like to share a report template as a .skyr with collaborators. Is there a way to keep the pivot applied to the report in the skyr?
I was able to save it on my own Skyline installation using : 'Remember current layout' but this information seems to be lost each time I export the .skyr and import it back.

Thank you very much for your help,



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Any plans for a Linux version of Skyline (even with limited capabilities)?
(4 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!
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some peptides show incomplete peaks
(6 responses) lvyayao90 2019-04-23

Hi Skyline team
When I imported my results into the Skyline, some peptides show incomplete peaks, I pasted one in the word file below. Can you please explain it for me and is there anything I can do to avoid this?
Thank you very much!


 an example.docx 
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Retention time difference in library when using PDresult vs. msf file
(3 responses) roberthardt 2019-04-23

Dear Skyline-Team,

I today created a spectral library from ProteomeDiscoverer 2.3 results (Mascot search engine) once using the .PDresult and once using the .msf file(s). I got quite similar results but interestingly, there is a clear retention time shift/offset when using the PDresult file. This becomes immediately apparent when looking at MS1-chromatograms, where the blue vertical peptide-ID lines are clearly "missing" the correct MS1-peak in the case of the PDresults-library.



 ID-times_from_PDresult.PNG  ID-times_from_msf.PNG  example_spectra_from_pdresult.PNG  example_spectra_from_msf.PNG 
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Error importing Bruker xml
(4 responses) fcsigloch 2019-04-23


I tried to import a Bruker XML file of a BSA test run and got the following error message. Conversion of the same file with msconvert BSAAnalysisResults.xml gave the same error.

At 10:53:
Failed importing results file 'BSAAnalysisResults.xml'.
[SpectrumList_BTDX::HandlerCompound] Unexpected element name: fullscan

Inner exceptions:
Exception type: System.Exception
Error message: [SpectrumList_BTDX::HandlerCompound] Unexpected element name: fullscan
[SpectrumList_BTDX::HandlerCompound] Unexpected element name: fullscan
   bei pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, Boolean getBinaryData)
   bei pwiz.ProteowizardWrapper.MsDataFileImpl.HasSrmSpectraInList(SpectrumList spectrumList) in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:Zeile 778.
   bei pwiz.ProteowizardWrapper.MsDataFileImpl.get_SpectrumList() in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:Zeile 475.
   bei pwiz.ProteowizardWrapper.MsDataFileImpl.get_HasSrmSpectra() in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:Zeile 742.
   bei pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:Zeile 194.

Bruker also exports mgf file format. This file could be successfully converted by msconvert BSAAnalysisResult.mgf and also searched with
java -Xmx12000M -jar r:\MSGFPlus\MSGFPlus.jar -s BSAAnalysisResult.mgf -d BSA.fasta -t 0.4Da -inst 0 -addFeatures 1 -tda 1 -e 1 -mod Mods_unlabelled.txt.
I can read in the resulting mzid file as "Peptide Search" into Skyline. When I try to read the mzML as "Results", I get another error:

At 11:04:
No scans in BSAAnalysisResults.mzML match the current filter settings.

Inner exceptions:
Exception type: pwiz.Skyline.Model.Results.NoFullScanDataException
Error message: No scans in R:\BRAIN\Testdateien_AnkeBachert\ProteinAnalysisResults_17.53-39.99.mzML match the current filter settings.
No scans in R:\BRAIN\Testdateien_AnkeBachert\ProteinAnalysisResults_17.53-39.99.mzML match the current filter settings.
   bei pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatogramsLocked() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:Zeile 363.
   bei pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatograms() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:Zeile 215.
   bei pwiz.Skyline.Model.Results.SpectraChromDataProvider.<SetRequestOrder>b__45_0() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:Zeile 556.

 BSAAnalysisResults.mzML  BSAAnalysisResults.mgf  BSAAnalysisResults.xml 
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SkylineDailyRunner.exe error via Symphony
(2 responses) sarah lennon 2019-04-24

Hi Skyline team,

I am trying to use the SkylineDailyRunner.exe via Symphony (a Waters server application allowing the automation of tasks). This used to work with SkylineDailyRunner.exe downloaded the 7th of Sept 2017. It does not work anymore with today's version. I got this error back :

"Unhandled Exception: System.IO.IOException: The handle is invalid.
at System.IO.__Error.WinIOError(Int32 errorCode, String maybeFullPath)
at System.Console.GetBufferInfo(Boolean throwOnNoConsole, Boolean& succeeded)
at System.Console.get_BufferWidth()
at`1 args)
at SkylineRunner.Program.Main(String[] args)"

Do you know what it means ?
Worth mentioning as well that the exact same commands from a terminal works fine.

Thank you very much for your help !



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SD Rat Plasma
(3 responses) Richard Lam 2019-04-23


I would like to predict the peptides of SD rat plasma after trypsin digestion. Can Skyline help? If yes, may I know how to do it? If not what do you recommend? Thanks!



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MS1 filtering - library peptide
(5 responses) mmr6q 2016-02-17
Sorry for the continuing questions.
I generated a spectral library using a Mascot .dat file, which has a high-score match to an 8-residue peptide that contains residue numbers 2-9 in from the protein n-terminus (methionine). When I import the corresponding .raw file, that yielded the .dat file, that peptide is not displayed below the parent protein, despite the peptide settings of 6 AA minimum peptide length and exclude 1 n-terminal AA. This peptide is found in the spectral library explorer, but when I add it from the explorer it appears as a "library" peptide and is not associated with the parent protein (and no RIC is plotted). In addition, this peptide is not included in the long list of possible tryptic peptides shown upon right clicking on the protein in the Targets list. Thank you for any advice you can provide to have that peptide included with the other peptides of that same protein.
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Do we need to add the iRT standards to each fractions when building the library?
(4 responses) zzhang9 2019-01-21

Hi there,

I am new to DIA and I have a question about the iRT values. When we building the library, we usually fractionate the sample and run each fraction individually in DDA mode. However, when we do database search using PD or MaxQuant, we only get one file. How could they use the iRT peptides' retention times in each fraction to calculate the iRT values? Thank you!


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peptide count from document grid plus pivot not counted correctly
(2 responses) sas28 2019-04-18

Hi skyline team,
I am exporting total sum area for each protein using your document grid with the pivot function. As an addition I want to know how many peptides per protein were used for the summed area. I have noticed that there are several options for total area in the drop down menu and I went for Proteins -> Peptides -> Precursors -> Precursor results -> total area which gave the same result as protein -> peptides -> peptide results -> quantification -> normalized area. However when adding peptides as count in the pivot function, it doesn't accurately reflect the peptides that are actually in the Skyline document. For example for iRT peptides which i have got 10 of, it reports 18 in the final exported summed area document. Other examples are only 1 off and sometimes the peptide count is correct. What am i doing wrong? Many thanks.

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Retention Time of the Unknown
(1 response) qaporter 2019-04-18

Hi, I have a question about the tutorial.
What does the tutorial Skyline "iRT Retention Time Prediction" mean when it come to predicted retention time, if the time is already unknown? But, if the retention time is not known, then what material is needed to predicted retention time, if the retention time is unknown? Because I don't understand how it predicted retention time, if the retention time was to be known, but if it unknown, then what material do I need and this is for small molecule.

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Retention Time of the Unknown
(9 responses) qaporter 2019-03-15

Hi, everyone. Does skyline allow you to enter chromatography and can skyline predict unknown retention time based on line with no standard for small molecules, because I read the tutorial on IRT Retention Time but, that was based on peptide, so I want to know if you can do that with small molecules?

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(3 responses) jmr 2019-04-18

I am trying to set up skyline for small molecules and starting by going through the tutorial. On tutorial it shows skyline adapted to small molecule user interface, but on my version of skyline it does not have the button to select small molecules. Do I have the correct version of Skyline daily loaded? see attached.

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Vertical blue ID lines without any library match
(4 responses) roberthardt 2019-04-17

Dear Skyline-team,

for some transitions in my SRM-experiment I see dark-blue vertical peptide ID lines while there is no library spectrum in my spectral library. Also the marking "ID" is missing from those lines. I selected to show only matching peptide ID times.

The library was created from MIDAS runs on a QTrap6500+ searched against customized database with Mascot.

Any ideas?



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Cannot Import Top5 Data from a QExactive
(1 response) khader awwad 2019-04-18

Hi all,

I analyzed a antibody digestion with a Top5 method on QExactive. In Skyline I generated the peptide list. I wanted to import the files in skyline, but I get the error message that chromatograms cannot be loaded before full scan data are enabled.

I have attached the error message from skyline.

The questions are:
Is the import of Top5 data not possible in skyline?
Or where can I enable full scan data in skyline?

Thank you in advance for your help.



 Skyline error 2.JPG 
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Chromatogram unavailable issue
(4 responses) Bo An 2019-04-15

Hi everyone,

I encountered a "chromatogram unavailable issue" with Sciex MS2 data, I have two transitions for the targeted peptide, if I only select one transition in Skyline method, the chrom is ok for both; but if I included both transitions in the Skyline method, only one chromatogram will be displayed, the other one will not be recognized, have any body encountered this issue before?

Many thanks,

 1.PNG  2.PNG 
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Isobaric labeling in small molecules
(2 responses) Juho 2019-04-16

Dear all,

I would like to go trough results of isobarically labeled small molecules (TMT/iTRAQ etc) that were combined for smaller number of samples and measured using scheduled PRM. Now the individual reporter ions present individual samples or standards with different concentrations and I would like to set the analyte concentration based on product m/z and replicate. For the quantification I try to set concentrations from the document grid individually, but skyline prefills all same analyte concentrations of the same replicate and same precursor regardless of different product m/z.

Is there a way to fill individual product m/z of the same precursor m/z and same replicate with different concentration for quantification with Skyline or should I just export integrated peaks and do this in a spreadsheet?

Many thanks,

view request
Export of Scan Indexes
(2 responses) jfoe 2019-04-16

Dear Skyline Team,

I have been exporting skyline chromatograms to perform some diagnostics with scan header information.
For export of the chromatograms I use the Report Objects:
Proteins/Peptides/Precursors/Transitions/Transition Results/Chromatogram/Raw Data/...
.../Raw Times
.../Raw Intensities

When automating this with a lot of files etc, it turns out that matching diagnostic data via scan retention times can get quite messy due to loss of precision on export.
You also store a Vector of ScanIndexes with Chromatograms. Could you maybe add an option to export those?

Best regards,

view request
Importing pepXML, mzid, or mzTab from OpenMS in Skyline
(2 responses) lparsons 2019-04-16

I am attempting to import Peptide Search results into Skyline ( that were obtained in OpenMS (in this case using the Comet search engine). The default output from OpenMS is idXML, which is not supported in Skyline. I looked at the options in File -> Import -> Peptide Search and found three possible formats for this though each returned a different error:

pepXML : "file is not from one of the recognized sources"
mzid : "No spectra found for the new library"
mzTab : "No acceptable score type found"

I checked to make sure these files are reasonable in size; they are all 10-25 MB with 34,000 - 271,000 lines. If I instead continue to work with the idXML file in OpenMS I see plenty of hits as well. If I use the same spectra (in their original raw format) and the same database but instead run it through MaxQuant, I can import the msms.txt file without a problem.

My goal is to import the Peptide Search results and then load a series of MS1-only spectra on top of the results so that peptide quantification can be done using the MS1 spectral data and mapped to the MSMS data of the search results. This works fine if I use the MaxQuant results here instead, though I'm looking to use the Comet (OpenMS) results at this time.

thank you

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Unclear Peaks in Skyline for Heavy Labelled PRMs
(5 responses) sa825 2019-03-24

Operating System: Windows


I am looking at the detection of specific proteins using their peptides in heavy labelled (Oxygen 18) plasma samples on the Thermo Q exacitive. I manually generated a peptide list using Uniprot and pasted it into Skyline. Then I exported this list as an isolation list (Non-scheduled) and searched for them on the Thermo. Some of the peptides were detected from this run but at times the detection of the peptide and its heavy labelled counterpart did not match up as expected in Skyline.

To overcome this, I removed the peptides that were not detected and re-exported the Peptide list as a "Scheduled" Isolation list and searched for them in the sample on the Thermo. This produced a second set of data but this second set of data appears very rough with no clarity in the detection of the peptide or its heavy labelled counterpart.

I have attached 2 images of the detection of the same peptide in the Non-scheduled and Scheduled runs and I was hoping you could tell me why this is the case and how to overcome it?

Thank you,


 Non-scheduled.jpeg  Scheduled.jpeg 
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File text/Free text column into document grid?
(1 response) waltteri hosia 2019-04-16

Hello. Is there a way to copy paste file descriptions from instrument sample lists to document grid? In other words is there a free text column to choose somewhere in edit document grid views? I have tried to look but have not found.
We use forward numbering as file name, and actual sample description, i.e. what was injected, is written to "file text" (waters) or "sample ID" (Thermo) fields, and this info does not come into Skyline. Then with larger document grids it gets hard to follow what's what when one only has this file number-name.

BR, Waltteri

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Unexpected Argument
(1 response) kartik kundgol 2019-04-15

Hi Brendan,

I am using SkylineRunner.exe and was exiting because of the below command,


Can you please guide me regarding this.

Please find the attachment.

And what is the major difference between SkyLineRunner and SkyLineCmd

Thanks & regards,
Kartik K

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calibration curve with spiked ISTD for multiple peptides
(8 responses) DeWi 2019-03-29

Hey there,

I'm quite new in working with skyline.
I've got an issue or I didn't get how it works with skyline, however:
I tried to create my calibration curves with spiked ISTD (5nM) of 18 pepiteds ( à 3 Transitions) simultaneously.
So, I already found the option to define the calibration points of only one peptide in the menu View-> document grid-> replicates.
And the other option to define the spiked-in concentration of the ISTD for all peptides in the documentation grid -> Peptide Quantification.
But I have not found a solution where I can definde both (and for all peptides instead of just one, because every peptide has a slightly different concentration in each calibration point).

Is this possible to do both with skyline?
Maybe you can help me.

Thank you!


view request
Import a precursor isotopes transition list
(10 responses) davidz 2019-04-09

Hi there,

Is there a way for me import a transition list with precursor ion, precursor isotopic ions, retention time, and ranking, here is what I have:

but I keep on getting this error:
"The product m/z 34 is out of range for the instrument settings, in the peptide sequence VSDFGLTKEASSTQDTGKLPVK. Check the instrument tab in the Transition Settings."

I take the format is a little off, but I'm using the format from an exported transition list from previous experiment.


view request
Shimadzu Library file not loading
(3 responses) Alan Barnes 2019-04-08

Dear Skyline Team,

I am having trouble importing a Shimadzu Library file. I have followed the following path is this correct?
Settings > Peptide Settings > Library > Edit LIst > Add > Browse

I have found mlb file format is not read. 
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NIST mps phosphopeptide library issue
(1 response) anne-christine uldry 2019-04-03

Dear Skyline developers,

We've just had a little issue with one of the NIST msp libraries, the human phosphopeptide spectral library human_hcd_labelfree_phospho_selected_passed.msp, which is deposited for instance here:

When we loaded it into Skyline via Peptide settings/Library, we noted that no phosphorylation (or any other usual modifications) was marked anywhere upon inspection of the library in View/Spectral Library, although the msp file loaded without warnings or error messages.

Upon inspection of the msp file, I noted that the format of the modifications in the phospho peptide msp was different to that, say, of hsa20140422.msp, which I found here
and which seems to exhibit modifications as expected when viewed as spectral library inside Skyline.

In the former the mods are marked thus


while in the latter they are marked as, for instance,


So I managed to solve my problem of reading the NIST phospho msp in Skyline simply by changing all the "(" of Mods into "/" and the ")" to nothing; that seems to work, in the sense that I now do see the relevant modifications in the spectral library, what I see in the file appears on the spectra, and there were no warnings or error messages while loading.

I am not familiar with the msp format, and cannot tell which of the two Mods formats I saw is the standard.

My questions to you however are

  1. Do you see any issues with my simple "sed" solution, or could they be other surprises with this (perhaps not very standard) human_hcd_labelfree_phospho_selected_passed.msp file ?

  2. Will Skyline be able in some future version to read this kind of msp format?

Many thanks in advance for your reply.

Kind regards,
Anne-Christine Uldry

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Paste of peptides with PTMs misses some peptides
(3 responses) Nathan Manes 2019-04-11

A new bug seems to have popped up in v4.2.0.19072. If a text list of peptides with PTMs is copy-pasted into Skyline such as:
IADPEHDHTGFLTEY[Phospho (Y)]VATR[Label:13C(6)15N(4) (R)]
then only the first peptide is in the resulting list of peptide targets. If the second peptide is pasted again, it appears. Note that Edit ---> Insert ---> Peptides works correctly.

view request
Change Peak Integration Parameters
viragsagikiss 2019-04-12

Apologies if this question has been answered before and i missed finding it.

Using the small molecule CE optimization approach (skyline-daily version). The analyte peak wasn't picked up automatically in some of the repeats so i wanted to manually adjust integration parameters.

I receive an error message "failure attempting to modify the document, Duplicate or out of order peak in transition Ion [xxx]". As seen on attached screen.

Do you have more information what this error refers for? I have run the CE optimization method with a transition list that i manually transformed from the one i exported from Skyline (on a SCIEX QTRAP, the one i exported would give me errors, but i used the same CE values). Import the results files as you detail in the tutorial, so it would create a new file CE optimization. And the CE steps appear with different colours. Here no 5 different method file, only 1, but i injected 5 times to test repeats.

Thank you and let me know if you need more information.


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Difficulties creating a spectral library
(3 responses) t j f m voermans 2019-04-08


I have been trying to create a spectral library for several days now, but whatever I do I can't seem to get things right. What I did was, I downloaded a SpectraST file for Human Serum from Peptide Atlas (see picture: PeptideAtlas) and for Human from NIST (see picture: NIST). I have added the files I got as well for clarification.

I am sure I forget something obvious, but I can't seem to find it.


view request
Dilution factor per replicate
(1 response) waltteri hosia 2019-04-10

Hello the awesome Skyline team!

I am doing some impurity quantification, peptides. If I have made a calibration curve from pure target protein digest with ISTD concentration of, 400 µg/ml, and injected 10µl, and I have measured unknown series, but for few technical reason I had ISTD concentration of 100 µg/ml and injection volume of 3µl, then after merging files to same skyline document, how can I get quantification's of the unknown series? When trying to apply concentration multiplier in document grid, it applies automatically to all peptides with same sequence, i.e. also to calibration curve injections. Seems not be possible to apply it to unknown samples only?

view request
Modifications for this peptide do not match current document settings
(6 responses) michael plank 2019-04-05


I built a library in Skyline from a .pep.xml I exported from Proteome Discoverer 2.0 using SequestHT as search engine. (The MS-run was on a standard phospho-peptide and I exported only a single PSM. I also exported the .mzML from the same PSM).
The library build succeeded, but when I go to View - Spectral libraries I get 'Modifications for this peptide do not match current document settings.' and it is complaining about a S[+79.9799] modification. (Due to this problem I cannot add the library spectrum to the document.)

The monoisotopic mass for phosphorylation is 79.966331 (79.9799 is the average mass). I checked that this is configured correctly in PD.
The .pep.xml has a line:
2. Dynamic Modification: (...) DeltaMass="79.96633" DeltaAverageMass="79.97990"

(This is on Skyline 64-bit

Can you please help me to get around this problem?


view request
Small Molecule Untargeted Exploration of MS2
(1 response) bwidner 2019-04-09


I am new to Skyline and interested in using it for small molecules for targeted and untargeted analysis. Is there a way to click on a MS1 scan and see the MS2s that were collected for that scan? I looked through the tutorials, and I couldn't find this. I only found mention of MS2s for targeted analysis.


view request
List of peaks with the highest dotp value under the Find Results window
(4 responses) wen ding 2019-04-08

Hi Brian,
I would like to have 550. 2928++ (dotp 0.91) listed in the Find Results window instead of 550.2928 (dotp0.52) (please see attachments). I don't understand why Skyline highlighted the peak 550.2928++(dotp 0.52) at 17.3 min instead of the dopt 0.91 peak at 27.6 min.
How do I make Skyline highlight the peak with the highest dopt peak and have that peak listed in the Find Results window?

Thanks in advance!


 dotp question.rtf 
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Any way of overlapping chromatogram base peak with XIC of peptides in "targets" list?
Edu Zar 2019-04-08


Is there any way to overlap the MS1 XIC of selected peptides with the chromatogram base peak?
Beeing able to compare te XIC of selected peptides with the base peak would be very useful in order to quickly check if the peptides of a specific protein are the most intense peptides in the chromatogram.



view request
Fragment ion XIC intensity
(1 response) stephen swatkoski 2019-04-05


Thank you for the great support you provide. Your responses to my previous posts have been very helpful. I hope this is the last question I have for awhile. I am analyzing PRM data and noticed that when I click on a point along a fragment ion XIC the intensity of the fragment is different than the intensity of the fragment ion in the raw ms/ms data. This is observed consistently across multiple peptides and fragments of those peptides. The XIC intensity at a given point along the curve always appears to be higher that the fragment ion displayed in the ms/ms spectrum. Can you explain why this is the case? Please see attached example. The intensity at the apex of the XIC for y10 is about 2.7e6. However, the intensity of y10 in the raw ms/ms spectrum at that same time point is lower (7.2e5).

Thank you,

view request
Custom SSL format not correctly defined
(3 responses) Mike S 2019-04-04


Just a heads up that the custom ssl format described here: input and output file formats is not followed, at least not in Skyline 64-bit build

For instance, that page says the header should be "file" and BlibBuilder says it needs to be "filename". Also, that page says "retention-time" is optional, but BlibBuilder says the column "RT" is required.


view request
MSX data processing is prohibitively slow
(2 responses) Tim McCubbin 2019-03-31

Dear Skyline team,

We are presently attempting to assess the benefits of using multiplexing to assist in the label free quantification of complex samples, trialing this on a HeLa protein standard. The tutorial on how to do this was clear and simple to follow and we have acquired our MSX scans. However, the import of these result files into Skyline is incredibly slow. While we expected it to be slow given the tutorial said upfront that one user reported a 20 hour import time for a 200 MB file several years ago, a single result file is importing at a rate of ~2% per day (our files are just shy of 200 MB each). Because we expected it to be slow, we used slightly less windows than in the tutorial for our trial (4 windows of width 6m/z). Data is from a Q-Exactive HF-X and our library size is quite large as we are doing untargeted proteomics, so roughly 55,000 peptides and 680,000 transitions. We are currently running version of Skyline on a Windows 10 machine with 64GB RAM and a 6 core hyper-threaded Xeon processor. I thought our computer was of reasonable specs so was wondering if you had a sense of why the import is slow; are we simply not meant to be doing untargeted proteomics with MSX or is it likely we have messed up a setting? Please let me know if you need any files or more information.

Many thanks,


view request
Unsupported score in search output file generated from Peptideshaker and several different search engine output
(9 responses) weixiandeng 2019-02-11
Hi Skyline team,

I was trying to build spectrum library through Peptikdeshaker output which is a mzID file, however, it gives me an error report showed below.

Then I switch to comet raw output(pep.xml), tide-search and MSGF+(mzid), they were all given the same error.

Then I tried these files on both Skyline 4.2 and Skyline Daily, still same error.

Can you please help me figure out the problem?


ERROR: .mzid file contains an unsupported score type

OK More Info
System.IO.IOException: ERROR: .mzid file contains an unsupported score type

   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 59
   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
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FIA of 3500 small molecules and scheduled method
(3 responses) a.petretto 2019-03-31

Dear developers
I have to inject in my QE 3500 compounds in FIA mode to collect the parent (MS1) and daughter ions (MS2). The final goal is to build a metabolic library. Now the best way to develop the project is to have 3500 different methods each of them have a dedicated compound, that is very hard to make manually.
I'm wondering if it is possible using skyline to export a scheduled method giving a template file with the same condition for HPLC e QE with the exception of monitored m/z for the different compounds.
Can anyone help me to avoid me to do manually :)
all the best

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library match spectrum
(1 response) stephen swatkoski 2019-04-02


When analyzing PRM data, is there a way to determine which replicate the library match spectrum came from? Also, is there a way to determine the exact scan number of the library match?


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How to assign report template name in Command-line argument
(4 responses) guoguodigital 2019-03-21

Hi There:
I'm trying to use Command-line interface to export a specified report:

C:\skylinedaily\SkylineCmd.exe --in=D:\ --report-name=Main\Name_of_report --report-file=D:\Name_of_report.csv --report-format=CSV --report-conflict-resolution=overwrite

But it always returned wit this error:
Error: The report M does not exist. If it has spaces in its name, use "double quotes" around the entire list of command parameters.

It seems like the name of report is in correct. But how Can I tell the software to use a specific report template in a specific folder?


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Thermo Altis method
(1 response) avizrosenberg 2019-04-03

When I try to export a method for a TSQ Alatis I get the following error.

System.IO.IOException: ERROR: Registry key (Software\Thermo Instruments\TNG\TSQAltis) not found. TSQAltis is not installed on this machine.

Any thoughts?

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Planned Functionality for Skyline Runner?
(4 responses) hogan 2019-04-02


First let me say thank you for all the work you do to keep this awesome software platform up and running and constantly making improvements!

I am not aware of a way to use skyline runner to upload a template file (.csv table) into the Document Grid for assigning analyte concentration and sample type to the imported replicates. So far I have been doing that manually every time I pull in a new batch of files from my run sequence (AbSciEx 4000 qTrap w/Analyst 1.6.2). I am attempting to set up a small molecule quantitative analysis workflow in skyline and was hoping to be able to automate most of the data processing using skyline runner.

Is this a planned functionality in the future?


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Precursor Ions
(5 responses) Richard Lam 2019-03-28


I was trying to use Skyline to predict the fragments ion of a known peptide.
I used the 'Import Peptide List' function and with the Transition Settings as shown in the attachment.
After the MRMs were generated in Skyline, I exported the MRM parameters as .csv by clicking 'File', 'Export', 'Report', then choose 'Peptide Transition List'.
Please see attachment for the report output.
The peptide is LCVLHEK, its neutral precursor is 822.4 and I expect the 2+ ion is at 412.2.
However, in the report, the peptide is shown as 'LC[+57]VLHEK' and the 2+ precursor ion is at 449.74.
Do you know why? Did I do something wrong in the settings?



 BSA Peptide Transition List_1.csv  Transition Settings.docx 
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CE Optimization Observation
(3 responses) mburgess 2018-01-31
I am using version of Skyline on a Windows 7-run PC operating a Waters Xevo TQ-S. I have utilized the CE Opt tutorial in the past and generated a series of methods with +/- 5 steps around a calculated collision energy value. The output methods from Skyline produce the proper number of transitions in the MassLynx MRM method and the data is collected normally. However, when I input the data from the runs into Skyline, I am only observing +/- 3 steps currently. Not sure if anyone else is seeing this. Am I missing a setting somewhere?
Many thanks in advance,
Mike B
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Purple rectangle on graphs
(2 responses) ecanessa 2019-04-01

I am trying to use the graphs for skyline as images in a publication but a large purple rectangle appears when looking at a peptide as seen below. It does not appear for all peptides. Is there anyway to eliminate this?

Also is there anyway to keep the y-axis stable so one graph isn't showing an intensity of 0 - 250 10^3 while another has an intensity of 0- 80 10^3?

Emily Canessa

 Purple rectangle.png 
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MS3 spectrum reading by skyline
(1 response) maotian zhou 2019-04-01

Dear Skyline team,

I am trying to use skyline to read MS3 spectrum. Seems skyline can extract the MS2 spectrum through the Raw file easily. However seems there is no way to setup the MS3 level product ions, and extract the MS3 spectrum. Could you please let me know if there is a method to analysis the MS3 spectrum like what is doing on MS1 or MS2 level (can calculate area under curve).


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Export MRM method into Sciex 5500 QTrap
(4 responses) Richard Lam 2019-03-27

I generated the MRMs from peptides using Skyline and experienced an error when trying to export the MRM parameters into a method for Analyst (Sciex 5500 QTrap) - see attachment for the error message.


  1. I am using Analyst 1.6.3
  2. Skyline (64-bit)

Any solution to resolve the Export to Method issue?

Best regards,


 Export Method Error.docx 
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How to create a file with default settings and how to see the precises signal intensity?
(3 responses) t j f m voermans 2019-03-27


I was wondering if there is a way to create a new file with default settings, like when you created your first file after installing Skyline. I am asking because I get different signal intensities for a peptide between two files while the same results where imported. So I must have done something with the settings that changed the signal intensity. I tried to create a new file, but I still didn't get the same signal intensity as the first file I created.

Also, is there a way to see the exact signal intensity? Above the peak, I only see the retention time and the ppm difference. I tried various options provided by the right mouse button, but nothing seems to give me some numbers.



 Skyline signal intensity.JPG 
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Issue with AutoQC upload of wiff files
(1 response) katherine glenn 2019-03-21

I have run into a problem with the AutoQC upload. I am uploading a lot of data that has been acquired over a period of time, and I think it may not be working because I tried to upload in the wrong order of acquisition. Throughout the log file it says that the file it is trying to upload was acquired before the acquisition date of the last uploaded file and is skipping.
However I tried to create new folders on Panorama, which are now all empty, but it is still skipping files. I tried removing the “AcquisitionTimes.csv” and log files but this didn’t work either. Are you able to identify the problem here? Is there an easier way to upload these files together?

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Endogenous peptide overlaps with minor peak of heavy
jjotto 2019-03-26


I am using a heavy-labeled peptide (13C(6)15N(2) on Lysine). The neat heavy-labeled peptide shows multiple peaks all within a few minutes of each other with one major peak. I believe some of the peaks after the main peak are the result of deamidations. (the peptide is not ideal because of these modifications, but it has been selected for other reasons) There are also a few minor peaks before the major peak. When spiking the heavy-labeled peptide into real samples, the endogenous peptide seems to consistently overlap with a minor peak before the major peak of the heavy-labeled peptide. Other spiked heavy peptides and endogenous peptides within the same samples show very clear and complete overlap. I am trying to compare the endogenous peak area to the known amount of spiked heavy peptide, but this is causing problems. I'm attaching a few images that I think will help illustrate what I'm seeing. Any help would be appreciated.

Thank you

 Showing heavy and light with IDs.png  Showing heavy transitions with IDs.png  Showing light transitions with IDs.png 
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how to add new elements?
(1 response) lvyayao90 2019-03-20

Hi Skyline team
I tried to add Tb, Tm, and Ho as new elements in the unimod.xlm file in folder Skyline_4_2_0_19009 as following, but Skyline software still pops-up that Tb is not a valid chemical formula.
<umod:elem title="Tb" full_name="Terbium" avge_mass="158.9253" mono_mass="158.925343"/>
<umod:elem title="Tm" full_name="Thulium" avge_mass="168.9342" mono_mass="168.934211"/>
<umod:elem title="Ho" full_name="Holmium" avge_mass="164.9303" mono_mass="164.930319"/>

 <umod:brick title="Tb" full_name="Terbium" mono_mass="158.925343" avge_mass="158.9253">
     <umod:element symbol="Tb" number="1"/>
  <umod:brick title="Tm" full_name="Thulium" mono_mass="168.934211" avge_mass="168.9342">
     <umod:element symbol="Tm" number="1"/>
  <umod:brick title="Ho" full_name="Holmium" mono_mass="164.930319" avge_mass="164.9303">
     <umod:element symbol="Ho" number="1"/>

Is there anything else I could do to get Skyline to recognize the three elements? Thank you!


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Total Area MS1 differs slightly from sum of MS1 areas
(4 responses) Tobi 2019-03-25

Dear Skyline team,

sry to add to this topic but I sadly could not find this exact problem in the forum. It is more for understanding than an actual problem.

When calculating the total Area MS1 myelf in Excel and via Pivot (Sum of Areas) the number differs slightly from the Total Area MS1 from the results grid but the difference is more than what I would expect from rounding errors.

Quant is set to MS1 and DDA and Product Ions are non-quantitative (and I excluded product ion areas from the pivot sum of areas).

Is this behaviour expected or are some settings wrong? Do you have a recommendation on how to retrieve the area of a peptide as the sum of all charges and isotopes?

Thank you very much for the great support

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[M+4] and higher carbon isotopes
(2 responses) Tobi 2019-03-22

Dear Skyline Team,

in the target list, the number of natural carbon isotopes [M+X] for each charge of a peptide can be nicely set via Settings/Transition Settings/Full-Scan/Peaks.

While changing the value between 1 and 4 (Precursor to M+3) the isotopes in the target list follow exactly, but any input value higher than 4 will result at maximum in [M+3]. Am I doing something wrong or is it limited?

Just in case it is limited, could you think of increasing the limit since this feature is automatic and fast and easy to control?

Looking forward to get a short comment for your side, and thanks for the great software.


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importing failed due to RT predictor linear regression
(14 responses) brlawson 2018-03-15
Hi Skyline team,
I am currently working on using skyline to analyze DIA data. All of our samples had PepCal mix spiked in so, I created an iRT document that I have been importing before importing my raw files. I am working with a very large data set so I have been importing in batches. Out of the whole data set I had 3 files which I received the error "unable to finish importing chromatograms because RT predictor linear regression failed." I have looked at all the files in Sciex software and the RTs seem to be fairly consistent across all of the samples. Do you think there is an actual issue with these particular samples or is there some type of setting that I may need to change? Thanks for your help!
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skyline 4.2.1 error
(4 responses) hgoldsc1 2019-03-22
when i try to open a file i get this message:

Failure opening \\Mac\Home\Documents\Lab\POSTDOC!\Experiments (HLG_000)\M_000-APEX mass spec experiments\ms_013-test on-bead\skyline peptide list\
The document format version 4.21 is newer than the version 4.2 supported by Skyline (64-bit)
OK More Info
System.Reflection.TargetInvocationException: There is an error in XML document (2, 2). ---> System.InvalidOperationException: There is an error in XML document (2, 2). ---> pwiz.Skyline.Util.VersionNewerException: The document format version 4.21 is newer than the version 4.2 supported by Skyline (64-bit)
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadXml(XmlReader reader) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 530
   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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Large scale PRM data (msx count= 8)
(4 responses) Wael 2019-03-20

Dear Skyline team,
I have a question please regarding PRM analysis. In short, I have an inclusion list consist of about 300-500 peptides and I ran this in multiplex PRM mode (msx count = 8) without RT optimization, as a fast exploratory experiment.
I am getting the MS2 ions, but skyline is integrating the peaks for all peptides over 15 min (LC method 20 min), please find attached pic as an example.
My normal RT window for a pepetide is about 0.2 min. I have the resolving power 140,000 at 200m/z.

Is skyline able to process such a workflow and if yes how can I improve the peak integration, pelase ?

Please let me know if you would like me to share skyline file with you.

Many thanks

 Large scale PRM data.PNG 
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.NET framework
(1 response) Dan Itzhak 2019-03-21

Installation of Skyline 4.2 requested installation of .NET framework 4.7.2 on the instrument PC.

Following update of .NET to 4.7.2, there is no communication between the Lumos and the instrument PC.

The PC is windows 10 and the instrument is a fusion Lumos.

Is this a known problem? Is there any advice to resolve this issue.

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Peak area extraction from MS1 only data acquired in paralell with SRM data
(4 responses) wen ding 2019-03-12

Hi Brendan,
I can use Skyline to extract peak areas of ions from either MS only data or DDA only data without MS/MS being performed. But I can not do that from MS raw files with MS1 and SRM only data are acquired in parallel on LTQ or Quantiva. It appears that the results import process does take place since I can see the peaks being extracted very briefly , but eventually, no chromatograms or results are obtained. Is there a way to deal with that particular problem?

Thanks a lot in advance!


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Fixed integration/peak boundaries
seabiscuit 2019-03-21

Dear Skyline team,

I am doing regular SRM/MRM quantification of peptides on a Thermo TSQ Quantiva MS. We are testing a fast-and-dirty analysis process by eliminating the use of LC column for separation. So all the peptides we monitored with be introduced into the MS to form wide elution peaks that overlap with each other. The issue I have now is the algorithm Skyline uses to set the peak boundaries does not work well with this elution pattern, especially for peaks with lower intensities. So I am just wondering if you can add a feature letting the use set fixed peak boundaries for all peptides in all samples.
A typical elution pattern was enclosed for your reference.


Wenbo Zhi
Augusta University

 peak boundaries.emf 
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Share complete failure to open error
(3 responses) Brett Phinney 2017-11-01
When I share a complete skyline file and select current version and Try to open it again I get this error

Any ideas? I'm using the most uptodate version of skyline. I have repeated this several times

failure to open

System.Reflection.TargetInvocationException: There is an error in XML document (1, 1). ---> System.InvalidOperationException: There is an error in XML document (1, 1). ---> System.Xml.XmlException: Data at the root level is invalid. Line 1, position 1.
   at System.Xml.XmlTextReaderImpl.Throw(Exception e)
   at System.Xml.XmlTextReaderImpl.ParseRootLevelWhitespace()
   at System.Xml.XmlTextReaderImpl.ParseDocumentContent()
   at System.Xml.XmlReader.MoveToContent()
   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__DisplayClass128.<OpenFile>b__126(IProgressMonitor progressMonitor) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 257
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in c:\proj\skyline_3_7_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_3_7_x64\pwiz_tools\Skyline\Util\Util.cs:line 1940
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in c:\proj\skyline_3_7_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\skyline_3_7_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 131
   at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 261
 failure to open.jpg 
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fail to import results
(3 responses) chia-lin 2019-03-18


I failed to import results and I will paste the error test. Thanks for your help!
At 11:53:
Failed importing results file 'D:\sop\XX\PXXXXX\PXXXXXXX.raw'.
[ThermoRawFile] The system cannot find the file specified.

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