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Chromatogram information unavailable
(8 responses) liuh1 2024-02-26 13:35

I use skyline-daily to process Agilent 6495C QQQ data. With several metabolites I always have problem to integrate the peak area. It shows the message: "Chromatogram information unavailable" I have followed the answer about the same problem, but it can't solve my problem.
Last week I worked for several times to get one file with less "Chromatogram information unavailable", but more metabolites show "Chromatogram information unavailable" after I added a few data file to the project.

I have checked the data file on Masshunter Qual, the metabolite shows great peak and right retention time.

What can I do next?


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error importing fragpipe output into skyline
(4 responses) jdemeter 2024-02-27 16:34


I have a similar problem to #63338: I am trying to import pep.xml files from fragpipe output for a group of DDA runs. I get the same error about not finding the scans. After doing the recommended modification of the pep.xml files, a new error occurs: please see the attached screenshot.

I am using Skyline-daily (64-bit) (a978938a7) - the previous version did not have this problem. Skyline (64-bit) is also able to read in the files without any problem.

can you please look into this?

Thank you,

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Spectral library
(4 responses) andreas kalagasidis 2024-02-24 03:38

I am trying to create Spectral library for use with OpenMS analysis. I created Spectral library using DDA files. When I export this as a Report, it contains no fragment charge in the Skyline transition list due to the use of MS1 filtering flow.

Could this have a major impact on subsequent steps in the Open Swath workflow (I haven't been able to complete the workflow)?

Any suggestions?

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Failure opening Skyline-Daily file
(1 response) samartinez3 2024-02-28 09:50

Hello, I have been receiving an error when trying to open a file that has been previously analyzed. It gives me the options to click OK or More Info, More info gives the information below:

System.IO.IOException: Failure attempting to load the window layout file W:\FilePath
Rename or delete this file to restore the default layout.
Skyline may also need to be restarted. ---> System.ArgumentOutOfRangeException: Index was out of range. Must be non-negative and less than the size of the collection.
Parameter name: index
at System.ThrowHelper.ThrowArgumentOutOfRangeException(ExceptionArgument argument, ExceptionResource resource)
at System.Collections.Generic.List`1.get_Item(Int32 index)
at DigitalRune.Windows.Docking.DockPanelPersistor.LoadFromXml(DockPanel dockPanel, Stream stream, DeserializeDockableForm deserializeContent)
at pwiz.Skyline.SkylineWindow.LoadLayoutLocked(Stream layoutStream) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\SkylineGraphs.cs:line 526
at pwiz.Skyline.SkylineWindow.LoadLayout(Stream layoutStream) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\SkylineGraphs.cs:line 481
at pwiz.Skyline.SkylineWindow.UpdateGraphUI(SrmSettings settingsOld, Boolean docIdChanged) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\SkylineGraphs.cs:line 308
--- End of inner exception stack trace ---
at pwiz.Skyline.SkylineWindow.UpdateGraphUI(SrmSettings settingsOld, Boolean docIdChanged) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\SkylineGraphs.cs:line 317
at pwiz.Skyline.SkylineWindow.OnDocumentUIChanged(SrmDocument documentPrevious) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\Skyline.cs:line 588
at pwiz.Skyline.SkylineWindow.SetDocument(SrmDocument docNew, SrmDocument docOriginal) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\Skyline.cs:line 706
at pwiz.Skyline.SkylineWindow.RestoreDocument(SrmDocument docUndo) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\Skyline.cs:line 903
at pwiz.Skyline.SkylineWindow.SwitchDocument(SrmDocument document, String pathOnDisk) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\Skyline.cs:line 861
at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\SkylineFiles.cs:line 397

I'm using a Skyline-daily with a special "apply to all" feature (from Nick). Please let me know what I need to do to properly open the files.

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Issues importing Small molecule transition list
(13 responses) m3g4n 2023-12-04 14:07

Hello! I'm having an issue where I'm trying to copy-paste a list of 30 compounds into skyline and it's breaking apart some of the entries (quant and qual are separate). I don't know how to add them together, or insert one into the other, etc. If anyone knows what I should be looking in depth into in terms of excel syntax that would be super helpful.

Thank you!

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(1 response) herath lakmini senavirathna 2024-02-27 12:01


I would like to know the difference between the total ion current area and the TIC chromatogram.
The total ion current area of each replicate can be obtained through skyline file---> export--> report--> peptide quantification.
TIC chromatogram can be obtained through Skyline file--> export-->chromatogram.
These two give different values and would like to know the difference.


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DIANN out in Skyline
(1 response) tania karasiewicz 2024-02-26 05:17


I have just started to use DIANN for some SWATH-DIA data. I am trying to visualise the data and do the stats in Skyline. I have managed to import the library and the peak boundaries of DIANN in skyline for visualisation of the different acquisitions.
I am having problem to do the stats, since I didn't run mprophet, is it possible to import the obtain q value from DIANN some how ?

Or in anycase if you have any tips for DIANN in Slyline, that would be great.

Have a nice day,


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Chromatogram information unavailable
(10 responses) Pa Nic 14 2024-02-23 13:02

I am working with a Q exactive in PRM mode. I created a transition list with my internal standard and thiol precursors. All compounds work well except my internal standard. I get a message saying Chromatogram information unavailable. I am sure about my retention time and fragmentation (I got them with a full ms d2). I don't understand that with the same parameters, I get a signal on Chromeleon for my internal standard. It is too bad cause I need my internal standard signal to continue working with Skyline, which is way more user-friendly...
Is there anything I could try

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Error Exporting to MassLynx Only with Scheduled Method
(7 responses) cbm11 2024-02-22 01:28
Good morning,
I have quite a large document with ~1000 peptides which I want to develop a method for, so I split them into a few 10 min runs with RepLiCal RT Predictor and am now using that to predict RT and schedule them all in one much longer run. The issue is that I am getting this error (which I assume might be a MassLynx problem?) but it only occurs when I try to export the method as "Scheduled", I am able to export the method with no issues if I just export it as standard unscheduled. I am also able to export other scheduled documents as expected, this one in particular using RT prediction just throws up the error. I was wondering if you could help me out with this problem please? Thank you!

(I am sharing the Skyline doc on fileshare as "RT Prediction Export Issues" too)


An error occurred attempting to export.
Error generating Quanpedia databank

Command-line: Method\Waters\BuildWatersMethod -w 1.5 -m "D:\Gez.PRO\ACQUDB\BCR 2 SRM Plus IStd.exp" "D:\ColleenM.PRO\ACQUDB\rgi0ks4h.fwn\transitions.txt"
Working directory: D:\ColleenM.PRO\ACQUDB
OK More Info
System.IO.IOException: Error generating Quanpedia databank

Command-line: Method\Waters\BuildWatersMethod -w 1.5 -m "D:\Gez.PRO\ACQUDB\BCR 2 SRM Plus IStd.exp" "D:\ColleenM.PRO\ACQUDB\rgi0ks4h.fwn\transitions.txt"
Working directory: D:\ColleenM.PRO\ACQUDB ---> System.IO.IOException: Error generating Quanpedia databank

Command-line: Method\Waters\BuildWatersMethod -w 1.5 -m "D:\Gez.PRO\ACQUDB\BCR 2 SRM Plus IStd.exp" "D:\ColleenM.PRO\ACQUDB\rgi0ks4h.fwn\transitions.txt"
Working directory: D:\ColleenM.PRO\ACQUDB
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass, Boolean forceTempfilesCleanup, Func`3 outputAndExitCodeAreGoodFunc, Boolean updateProgressPercentage) in C:\proj\skyline_23_1\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 181
   at pwiz.Skyline.Util.Extensions.UtilProcess.RunProcess(ProcessStartInfo psi, String stdin, String messagePrefix, IProgressMonitor progress, IProgressStatus& status) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Util\Extensions\UtilProcess.cs:line 45
   at pwiz.Skyline.Model.MethodExporter.ExportMethod(String exeName, List`1 argv, String fileName, String templateName, Dictionary`2 dictTranLists, IProgressMonitor progressMonitor) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Export.cs:line 4731
   at pwiz.Skyline.Model.WatersMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Export.cs:line 4523
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Util\Util.cs:line 1922
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 204
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\skyline_23_1\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2313
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Exporting scheduled MRM method to *.exp Masslynx
(8 responses) per larsson 2024-02-01 09:05

Dear Skyline support,
I run lipidomics using waters xevo tqxs. Managing the many MRMs in the Masslynx software is to time consuming (adjusting RTs manually) so I am trying to learn skyline. My main objective is to be able to write all MRM experiments into a *.csv file that can be used as a compound database. I can then use skyline to build the *.exp method for each application and update the RTs in excel.

I manage to insert transition list with the parameters needed in a way that seems correct into Skyline but I have problems to get all needed settings exported to the *.exp file.


  1. The problem is that when I export method I am not able to get the right polarity set for each MRM in skyline into the *.exp file, they will al be ES+ even if listed as ES- in my Skyline file.
  2. I have the retention time and retention time windows in Skyline but am unable to get export this to the *.exp file the start and stop time will be at the same value.

If I can find a solution to these problems I would be able to write and modify all my methods from templates in excel and export them very quickly as needed to Masslynx. Changing RT times and windows would be a very simple task in excel and making selections for MRMs for a new application would be very simple as well. To make this work I need to find the solution to export the scheduled RTs and the polarity for all MRMs from skyline to masslynx.

I am using Masslynx 4.2 SCN 1045 and Skyline and run this on the workstation used for controlling the instruments.

In case any one can help me I attach my csv file that I use to import into skyline, pasting into "insert transition list"
I attach my skyline file
I attach my .exp template for the export
I attach a raw file with results from the lipids in the template

Would be extremely nice to find a solution and I am think there should be lots of other users that would have use of this solution.
Excel->Skyline-> Masslynx for method setup would be great


 Skyline problem data.7z 
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Error when Upload MSFragPipe output file to skyline
(1 response) wchu3 2024-02-23 10:44

Good afternoon Skyline team

Recently, I tried to use Skyline to analyze the output files from FragPipe. I found a tutorial (Untargeted analysis of DIA datasets) about how to use Skyline to analyze the MSFragger data and it worked for the tutorial data but for my AAV data, it's always displaying the attaches error. Do you know what happened? Looking forward to hearing from you, thank you.

Best wishes,


 IMG_0092.jpg  IMG_0091.jpg  Tutorial-5-DIA-Fragpipe.pdf 
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Distinguishing between heavy carbon and heavy hydrogen
(7 responses) molly hopper 2024-02-22 11:24

I'm working on a small molecule tracing experiment where we've added a duterated molecutle (D5 Glutamate) as in internal standard in our study, and have a heavy labeled carbon tracer. We're trying to trace the heavy carbons, and in an unlabeled control, we've noticed a slight signal for [M5C13-H]. I am interpreting this as an issue with the masses being so close that skyline believes them to be the same peak. We have the resolution to see the difference between [M5H2-H] and [M5C13-H] on the instrument (Thermo Exploris 240). I'm hoping for some help with how we can resolve the peaks in skyline, as we're soon to start a study that uses C13N15 tracers.

Current Transition Settings:
MS1 Filtering, precursor mass analyzer: Orbitrap, resolving power: 240,000 at 200 m/z
Instrument: Method matcho tolerance 0.055 m/z.

I've tried changing both of these settings with no change to the integrations.

Windows 10, Skyline 64-bit


 Screenshot 2024-02-22 142143.png 
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error using "DIA raw" option when performing Import Peptide Search
(4 responses) wphipps5 2024-02-21 14:53

Hi there-- I am trying out this "Start from: DIA Raw" option during Import Peptide Search of our DIA results. It runs for several hours but then gets an error message. Here is how it ends:

"[2024/02/21 12:08:14] INFO: Issued command:
[2024/02/21 12:08:14] INFO: percolator --results-peptides D:\dia_mzml\231101_dia_mzml_rename\crux-output/ --decoy-results-peptides D:\dia_mzml\231101_dia_mzml_rename\crux-output/percolator.decoy.peptides.txt --results-psms D:\dia_mzml\231101_dia_mzml_rename\crux-output/ --decoy-results-psms D:\dia_mzml\231101_dia_mzml_rename\crux-output/percolator.decoy.psms.txt --verbose 2 --protein-decoy-pattern DECOY_ --seed 1 --subset-max-train 0 --trainFDR 0.01 --testFDR 0.05 --maxiter 10 --search-input auto --no-schema-validation --protein-enzyme trypsin --post-processing-tdc D:\dia_mzml\231117_dia_mzml_rename\
[2024/02/21 12:08:14] INFO: Started Wed Feb 21 12:08:14 2024
[2024/02/21 12:08:14] INFO: Hyperparameters: selectionFdr=0.01, Cpos=0, Cneg=0, maxNiter=10
[2024/02/21 12:08:14] INFO: Reading tab-delimited input from datafile D:\dia_mzml\231117_dia_mzml_rename\
[2024/02/21 12:08:14] INFO: Features:
[2024/02/21 12:08:14] INFO: retentiontime rank abs_ppm isotope_errors log10_evalue hyperscore delta_hyperscore matched_ion_num complementary_ions ion_series weighted_average_abs_fragment_ppm peptide_length ntt nmc Charge1 Charge2 Charge3 Charge4 Charge5 Charge6 Charge7
[2024/02/21 12:08:14] INFO: Found 28 PSMs
[2024/02/21 12:08:14] INFO: Concatenated search input detected and --post-processing-tdc flag set. Applying target-decoy competition on Percolator scores.
[2024/02/21 12:08:14] INFO: Train/test set contains 28 positives and 0 negatives, size ratio=inf and pi0=1
[2024/02/21 12:08:14] FATAL: An exception occurred: Error: no decoy PSMs were provided.
[2024/02/21 12:08:14]
[2024/02/21 14:20:21] Search canceled."

The full output is attached as well as a pdf of my settings. I can provide the mzML files and the fasta.


 settings.pdf  output.txt 
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Cannot Import Chromatograms from MassLynx
(5 responses) cbm11 2024-02-19 06:40

Good afternoon,
We are trying to import chromatograms from MassLynx to Skyline 23.1 and only just today have started receiving the below error:

"At 2:46 PM:
Could not load file or assembly 'pwiz_data_cli.dll' or one of its dependencies. The specified module could not be found.
System.IO.FileNotFoundException: Could not load file or assembly 'pwiz_data_cli.dll' or one of its dependencies. The specified module could not be found.
File name: 'pwiz_data_cli.dll'
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache()
at pwiz.Skyline.Model.Results.ChromatogramCache.Build(SrmDocument document, String documentFilePath, ChromatogramCache cacheRecalc, String cachePath, MsDataFileUri msDataFileUri, IProgressStatus status, ILoadMonitor loader, Action`2 complete) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Results\ChromatogramCache.cs:line 732:"

We have tried old skyline documents/raw data files that used to work and also tried uninstalling/reinstalling 23.1. Any idea what else could be happening? I've attached an example .raw file (zipped) and a skyline doc where this is happening

 DME trial patient 
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Explicit Retention Time from msp library for small molecules
(10 responses) meowcat 2024-02-13 02:56


we are using MSP libraries from in-house small molecule standards with specified retention time.
This is correctly loaded into the MSP (NIST) library. However, the retention times from the library aren't used as explicit retention times when adding the compound.

I made a github issue and coded a provisional "solution":

Aside from the fact that it would need to be improved, maybe this isn't needed at all:

  • Is there an existing way to add an explicit retention time (e.g. from a CSV file) in batch to molecules imported from a Spectral Library?
  • Is there an existing way to associate spectra from spectral libraries to existing molecules in a document (rather than to insert new molecules from spectra)?
view request
skyline batch installation
(4 responses) chi nguyen 2024-02-21 16:45
dear Skyline team,
I am trying to install the skyline batch most recent version ( but I was not successful so far. Below are the error details.
many thanks

2024-02-21 16:02:57,329 [INFO] SkylineBatch: Saved configurations were found in: C:\Users\NguyenD14\AppData\Local\Apps\2.0\Data\X823W123.JCK\94EG05GC.7Y7\skyl..tion_e4141a2a22107248_0015.0002_29e5eb00463e294d\Data\\user.config
2024-02-21 16:02:57,943 [ERROR] SkylineBatch: An unexpected error occured during initialization.
System.NullReferenceException: Object reference not set to an instance of an object.
   at SkylineBatch.MainForm.ListViewSizeChanged()
   at SkylineBatch.MainForm.MainForm_Resize(Object sender, EventArgs e)
   at System.Windows.Forms.Control.OnResize(EventArgs e)
   at System.Windows.Forms.Form.OnResize(EventArgs e)
   at System.Windows.Forms.Control.OnSizeChanged(EventArgs e)
   at System.Windows.Forms.Control.UpdateBounds(Int32 x, Int32 y, Int32 width, Int32 height, Int32 clientWidth, Int32 clientHeight)
   at System.Windows.Forms.Control.UpdateBounds(Int32 x, Int32 y, Int32 width, Int32 height)
   at System.Windows.Forms.Control.SetBoundsCore(Int32 x, Int32 y, Int32 width, Int32 height, BoundsSpecified specified)
   at System.Windows.Forms.Form.SetBoundsCore(Int32 x, Int32 y, Int32 width, Int32 height, BoundsSpecified specified)
   at System.Windows.Forms.Control.ScaleControl(SizeF factor, BoundsSpecified specified)
   at System.Windows.Forms.ScrollableControl.ScaleControl(SizeF factor, BoundsSpecified specified)
   at System.Windows.Forms.Form.ScaleControl(SizeF factor, BoundsSpecified specified)
   at System.Windows.Forms.Control.ScaleControl(SizeF includedFactor, SizeF excludedFactor, Control requestingControl)
   at System.Windows.Forms.ContainerControl.Scale(SizeF includedFactor, SizeF excludedFactor, Control requestingControl)
   at System.Windows.Forms.ContainerControl.PerformAutoScale(Boolean includedBounds, Boolean excludedBounds)
   at System.Windows.Forms.ContainerControl.PerformNeededAutoScaleOnLayout()
   at System.Windows.Forms.ContainerControl.OnLayoutResuming(Boolean performLayout)
   at System.Windows.Forms.Control.ResumeLayout(Boolean performLayout)
   at SkylineBatch.MainForm.InitializeComponent()
   at SkylineBatch.MainForm..ctor(String openFile)
   at SkylineBatch.Program.Main(String[] args)
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Skyline Error on MS data file import
(1 response) db432 2024-02-21 04:43

Dear Skyline Support,

I am using Skyline to import MS data collected on waters Xevo TQXS and Skyline fails to open the file with the error below.

"At 12:28 PM:
Could not load file or assembly 'pwiz_data_cli.dll' or one of its dependencies. The specified module could not be found".

I have uninstalled Skyline from the PC, deleted all command files, restarted the PC and reinstalled the most up to date version (Skyline 64-Bit) (cf25ad847) but still the error appears.

I also note that my Skyline files on the PC do not have the Skyline logo but just plain files (see attached PDF file).

Can you please advice on what to do?

 Skyline Errror.pdf 
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Regression fit
(2 responses) danielacgranato 2024-02-16 11:53

We are analyzing our calibration curves in Skyline for peptide absolute quantification. We have observed that Skyline now has different regression fits besides linear. Would like to know, if we should consider any criteria when applying one type instead of the other. Do you have any suggestions?

Thank you very much in advance. Best, Daniela

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Analysis of Sciex MRM3 data from 7500 QTRAP
(3 responses) h l elfrink 2024-02-08 08:01

Dear Skyline team,

For our publication we are considering uploading the targeted MRM3 data (Sciex 7500 QTRAP) that we generated to the Panorama repository. If I understand correctly, then compatibility of the data with Skyline is a prerequisite, but this is not true for our data. When trying to open a data file Skyline cannot find any chromatogram. This issue was raised previously by a colleague of mine a couple of years ago, the proposed solution, using the the MS3 fragment instead of the MS2 fragment did not work for us.

Could you provide another round of feed back on this issue, please? Should we look for an alternative repository to share our data for now?


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Error in generating .mzid file
(2 responses) elvistc 2024-02-15 22:09

Has anyone experienced this error before? I saw another post regarding this and someone mentioned to update the visual C++ version. The current version installed on my computer is correct and I still see this error.

Thank you in advance.

 mzid error.jpg 
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Importing fractionation sample to Skyline
(1 response) GH 2024-02-15 13:23

I have DIA sample which is fractionated and I have total of 12 .raw fractions data files.
My question is how can import raw files to Skyline? is there anyway to import as combine all fractions? or we need to combine before importing?

Thank you, Ghazaleh

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Filter peptide contains specific peptide in library
(2 responses) GH 2024-02-14 11:43

I generated my library from other software and want to import to skyline (import library assay). I want to filter a peptide containing only L (leucine). I tried in peptide setting -->Filter-->Exclude peptides containing. I created the one to be excluded (attached file here). I did refine and advanced, however it does not filter peptides and still see all peptides.
my question is that is it possible to filter peptide for library? or Do you have any suggestion to solve this problem?

Thank you, Gazale

view request
Comparing MS1 data with and without FAIMS
(2 responses) paul.derbyshire 2024-02-15 04:07

Hi Skyline team,

I've been running some samples on two different Thermo mass specs - Orbitrap Fusion and Orbitrap Eclipse with FAIMS. When I build the data in Skyline I see distinct saw-tooth chromatograms from Eclipse/FAIMS samples but not from Fusion samples (see attached screenshots). Applying smoothing masks the saw-tooth appearance. Is this a effect of the FAIMS ion separation or is there something in transition settings that needs to be applied before data import?

Thanks in advance.

Paul Derbyshire

 Skyline_MS1_no_smoothing.png  Skyline_MS1_with_smoothing.png 
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a-ions from fully SIL labelled amino acids
(4 responses) jfoe 2024-02-14 04:35

Dear Skyline-Team,

I have a workflow where I create spectral libraries from Skyline. I recently noticed that I am missing some a-ions from my SIL-labelled peptides, if the fragmentation is next to the SIL label.

The reason is, that while the common b- and y- ions fragment neatly at the peptide bonds, the a-ion loses a C-atom that belongs to one of the adjacent amino acids.
Skyline always subtracts a 12C. When the amino acid is fully SIL, this should be a 13C. I suspect this is tough to implement in a general sense but it is very common that SIL are fully labelled so maybe skyline could implement an exception where a 13C is removed if there is no 12C at all in the amino acid.

I am including a screenshot that hopefully shows you why this causes me to lose signal. In that specific case there is actually some peak at the wrong a6+, too, but I am missing the main, correct a6+ peak. In effect, this is quite bad because the spectral library will end up with a bad dotp when detecting the natural non-labelled peptide because it expects only a very weak a6+ ion.
Also, I am including the skyline file.
Skyline version is x64 on Windows.

Let me know if anything is unclear.

Best wishes,

view request
How to get Skyline to produce different molecule calibration curves in a single instance?
(2 responses) manesh chand 2024-02-14 15:46

I have 115 molecules that I want to calculate concentrations for using Skyline. We have ran a calibration curve of all 115 molecules pooled together at varying concentrations and performed a serial dilution that we ran on our SCIEX 6500 QTrap, files are labelled inslicht 753884 cal 0-9. I followed the tutorial steps for the small molecule quantification and I am able to input the analyte concentration values for one of the 115 molecules on the document grid view and get a calibration curve for. However, this does not allow me to add the other 114 molecules that have different concentrations inside those cal 0-9 files that I want to calculate concentrations for using a singular Skyline instance. Is there a way I can format the document grid view in Skyline to where I can input the other 114 molecule known concentrations into the analyte concentration column without having Skyline auto filling those values down. I have attached pictures of the molecule setting and document grid view, skyline files, and raw data. Thank you for the help!

 OxyEndo Skyline  OxyEndo Skyline  OxyEndo Skyline Template.skyd  OxyEndo Skyline Template.skyl 
view request
Spectrum annotation inconsitencies
(2 responses) jfoe 2024-02-14 05:10

Dear Skyline-Team,

I often try to identify peptides via skyline. In this effort, the "Show peak annotations" feature of the spectrum view can be really handy. But unfortunately, I really struggle to understand how it works, i.e. what leads it to match some peaks and not match others.

I am uploading two screenshots, one (spectrum_annotation_issue_control.png) shows how the transitions are matched from the canonical way of having transitions active in the targets panel. Note e.g. y7 and especially y8 are matched nicely between 900 and 1100 m/z.
Switching to the peak annotation view (spectrum_annotation_issue.png), I lose these matches (red box). I struggle to find any reason why this is so inconsistent.
The view is really helpful in many situations and e.g. in the example reveals the b3 match, which I didn't have enabled and I think it's a great feature!

Skyline version is x64 on Windows.

Let me know if anything is unclear.

Best wishes,

 spectrum_annotation_issue_control.png  spectrum_annotation_issue.png 
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Spectral Library and Data processing
(6 responses) damlaaygun09 2024-02-12 04:46


I'm trying to use Skyline version for my thermo.raw proteomics datas. I have 2 diseased and 1 healthy data, and each one has 3 fragmentation. I want to get group comparision result LFQ and also need shared and different proteins between subjects. I watched tutorials from YouTube (MQSS 2023 - Dmitry Alexeev). I solved many problem but some points are still unknown for spectral peptide library. I have downloaded from Peptide Atlas and NIST but software is not accept these file types. I'm not sure about am I wrong when I download the library? The other problem is when I can get result for group comparision I see the adjust p-value as 1.00 so I don't know what is the actually correct steps for my aim. Can you help me?Thank you!


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Building a spectral library using msp
(11 responses) laura corveleyn 2021-02-02 03:24


I am trying to build a spectral library in Skyline using an msp file based on predicted spectra. Since we work with histones, we derivatize our peptides using propionylation, so all unmodified lysines and N-terms are blocked with a propionyl group. Therefore, Propionyl (K) and Propionyl (N-term) are fixed modifications, unless another variable modification is present. However, Skyline does not seem to recognize the modification "Propionyl" from the msp, although I also defined it as a modification in the peptide settings. All other modifications (e.g. Acetyl, Dimethyl,..) that I defined, are used correctly in the library. After some research, we figured out that Skyline recalculates the precursor m/z for each peptide (based on its sequence) and doesn't use the one that is stated in the msp. So for a peptide that carries a propionyl group, the m/z is recalculated as if the peptide would be unmodified because Skyline does not recognize the propionyl.

In the attachment you can find a short example of a predicted msp and the Skyline document with the corresponding library I built with this msp. In the msp you can see that the Propionyl group in the header is stated in the exact same way other modifications are, so I don't understand where the problem is.

Thank you in advance for helping me out!



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Chromatogram information unvailable
(4 responses) gazelezi 2024-02-13 12:42


I am a new Skyline user and trying to get some chromatographic information bout several target small molecules. Following the instructions from the site, I was able to insert a transition list and import raw file results from one of my test runs. Initially, I was able to see the chromatogram pieces of information for all the target molecules except for one. After several trials, I wasn't able to fix the issues.
So, I decided to reload the transition list and the raw files. After that, I could not see any chromatogram information for all target molecules. I would appreciate any help.

Attached is the file I am working on.

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building a MS2 transition list
(6 responses) p de blaauw 2024-02-09 01:27

Hello skyline,
I have been using a transition list for small molecules in Skyline for high-resolution data (MS1 scan) for years now. We now have another machine, namely an orbitrap where we generate MS2 spectra in besides the MS1 full scans. What is the best way to create a transition list to analyze the (known) MS2 fragments in Skyline?"

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uploading pdresult files without raw files
(1 response) ssedigh2 2024-02-12 12:18

Hi ,

I was wondering if there is a way to open a PD file in skyline if I don't have access to the raw files?
Thank you for the ongoing support.

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DIA to SRM assay gradient
(3 responses) akhilabrai 2024-01-22 21:37

Hi Skyline team,

I was looking at the tutorial on developing methods for SRM/MRM assay from DIA chromatogram libraries. I had a list of precursors of my interest. It was suggested to run iRT in the qqq instrument. I planned to run Pierce iRT in the Sciex QTRAP in triplicates.
My questions are

  1. What concentration of iRT to be taken?
  2. How to choose the gradient and the run time while running in the QTRAP?

Please help me out.

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When importing Agilent .D files is there a way to read the Sample Group information?
(2 responses) cian monnin 2024-02-05 13:17

Hello Skyline Team!

When making a worklist on Agilent's Data Acquisition software there is a Sample Group column. Is there a way to import or populate a column with this information in Skyline? It's stored in the the sample_info.xml as

    <DisplayName>Sample Group</DisplayName>

Specifically the <Value>20x_MEF_NPRL2</Value> is of interest for us.


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autoQC explicit retention time window
(3 responses) halwaseem 2024-02-06 10:26

I am running serum samples using an MRM method on an Agilent triple quad. Only product ion data is collected (at specified RT windows). There is no full MS data. I would like to use autoQC to monitor the signals of the deuterated internal standards spiked into a quality control sample. I have tried including and excluding the explicit retention times but Skyline isn't correctly integrating the ISTD peaks due to inter-batch retention time shifts(~0.2-0.8 min). Even with a window that captures the shift, Skyline is going in an peak picking noise at the explicit retention time instead of the nicer peaks.

I have attached 3 examples of 3 different compounds in the autoQC transition list that are being properly/improperly integrated.

Since this is for SST, I would prefer for this to be automated and not have to go in and manually adjust the peaks so that they show up correctly in Panorama. Is there something in the settings that can be adjusted to address this issue? Is there a way to set a minimum peak intensity for autoQC integration? There are many examples where there is an obvious peak in the RT window, but Skyline integrates noise instead.

If a iRT calculator was added to molecule settings, would Skyline be able to apply that as raw files came in via autoQC?  autoQC issue.docx 
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PDA Data in Thermo Raw File Prevents Skyline Import
(6 responses) ddrruss 2024-01-31 08:44

I am using the most recent version of skyline, on a windows 10 OS.

I have a PDA detector inline and am collecting UV as well as MS1 and MS2 (DDA using a thermo ascend).
I am using a byonic mzid file to build my library.
When I try to upload my raw file I get an error that says something along the lines of PDA chromatogram not recognized. It looks like Skyline is also trying to import the PDA data from my raw file in addition to the mass spec data. I can send the raw data file for inspection.

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Unable to import Spectra from Bruker DDA analysis.tdf or analysis.tdf_bin using BlibBuild
(42 responses) lubwen 2024-01-08 10:31

I ran into this error of 'No spectra were found for the new library' when importing DDA data from Bruker's raw analysis.tdf (or analysis.tdf_bin) data.

I am using a tab-delimited .ssl result file (954.ssl, see attached), with the following example lines:

file scan charge sequence
HT_20221220_Vividion_sampleB12_1000ng_Slot1-19_1_954.d/analysis.tdf_bin 177793 3 [+42.0]AAAAAAVGPGAGGAGSAVPGGAGPC[+324.2]ATVSVFPGAR
HT_20221220_Vividion_sampleB12_1000ng_Slot1-19_1_954.d/analysis.tdf_bin 176745 3 [+42.0]AAAAAAVGPGAGGAGSAVPGGAGPC[+324.2]ATVSVFPGAR
HT_20221220_Vividion_sampleB12_1000ng_Slot1-19_1_954.d/analysis.tdf_bin 176808 3 [+42.0]AAAAAAVGPGAGGAGSAVPGGAGPC[+324.2]ATVSVFPGAR
I am attaching the error screenshot to this thread. The original tdf and tdf_bin files are too large (~760MB) for this forum.

Could someone help me track down the issue?

Thanks a lot!
Bingwen Lu
 Error.png  954.ssl 
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Different calibration curve concentrations and fittings for different molecules
(1 response) cian monnin 2024-02-05 13:07

Hello Skyline team!

We are doing relative concentration (mix of approx 260 authentic standards in a 9 point curve). Some standards are at different levels. Is there away to have different values for different molecules other than using the Concentration Multiplier?

Is there a way to select different Regression fit for different molecules?


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DIA with overlapping windows, MSFragger output files, import peptide search creates Warning: Did not find spectrum
(3 responses) jennifer schwarz 2024-01-31 04:35

Dear all,

I am using overlapping windows in DIA and demultiplex the spectra with MSConvert.
The created mzML files I use for the search with FragPipe(v21.1)/MSFragger (4.0).
The created interact-*.pep.xml I use for the "import peptide search" (Skyline .
This created the error shown in the screenshot. "could not find scan number" and "did not find spectrum".
Different score values don't make a difference.
This issue was not there in cases when I did not have overlapping windows and did not need to use MSConvert first.

All relevant files are in one folder.
I have the following files:
interact-naming_rank1.pep.xml files
naming_rank1.pepXML files
the mzML files from MSConvert used for the search
and the Thermo raw files.

The difference to the runs without prior usage of MSCovert is that those files are having the naming:

 Screenshot 2024-01-31 131943.png 
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Importing Thermo Biopharma Finder Search Results to Skyline
(3 responses) Yao Chen 2024-02-02 00:25

Dear Skyline Team

Would it be possible to create a targeted search list in Skyline from Thermo's BioPharma Finder search results?


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Peak inversion over y-axis with new skyline version
(1 response) dhaupt 2024-02-02 08:11

Hi Skyline Team,

I recently downloaded the latest update ( and now all of my peaks appear split with the earlier half of the peak being above the y-axis and the later half falling below the y-axis. The peak areas themselves don't appear to be affected, however, I can't seem to figure out how to fix this. I sent multiple files to a colleague using an older version of skyline and they observed typical peak shapes. Wondering if there is a way to disable this or if there is a way to revert to an older version of skyline. Will attach a screenshot of an example chromatogram along with my skyline project so you can check my settings. Thanks for your help.

Dan Haupt

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Building a spectral library from Proteome Discoverer Output
(7 responses) r g beniston 2013-07-24 09:00
To whom it may concern (so to speak),

I'm trying to use the Mascot search results from within Proteome Discoverer 1.3/1.4 on OrbiTrap Elite generated .raw files (Nodes used are: Spectrum Files, Spectrum Selector, Mascot, Annotation and peptide validator) to build a spectral library for future use. I've exported the PSM results to a .pep.XML file and tried to build a library in Skyline v1.4 but I get the error message that the file is not from one of the recognized sources and that no spectra were found for the new library. Help, new to Skyline, and use of spectral libraries generally. Is there a get around?
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Refine/Results/Max transition peak rank setting
(6 responses) Zac 2024-01-31 10:59

Refine/Results/Max transition peak rank...i have being setting this value to 3 to try to filter results to only show the top 3 ranked transitions. I have noticed that for most peptides i get rank 1, rank 2 and rank 3, but for some peptides there may be a different rank included. Is this a bug or is another transition rank included if there is no signal for one of the top 3 ranks? In some cases all three top transitions have signal but they are still not chosen.

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(4 responses) vanyabangera 2024-01-28 06:01

Hello Team,

I have generated tryptic peptides using skyline.

Could you please confirm whether the method I followed is correct or not?

a) I have edited peptide settings with and without (none) background proteome
b) Edit>import>FASTA>and paste by peptide sequence from the FASTA file

I would require clarifications on background proteome and edit>import>FASTA options. At first, I had the whole FASTA file of an organism as background proteome and pasted the whole FASTA file of an organism in edit>import>FASTA options. It gave me some tryptic peptides.
Secondly, I gave none as background proteome and imported a peptide sequence of interest on edit options. It gave me some tryptic peptides

But different results. why is that so? What exactly do background proteome and edit>import>FASTA options do in this case?

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Data processing on Skyline
(2 responses) nbekhti 2024-01-22 07:31

Hi Skyline team,

I have a bench of questions on the data processing using skyline, below:
(1) Is it possible to do baseline correction on Skyline?
(2) Is it possible to have noise canceling on Skyline (Smoothing)? I can see on: View>transform>Savitzki-Golay smoothing, is this the right tool to use for this purpose?
(3) Before doing the integration, does skyline apply resolving? meaning identify shoulder peaks and split into individual features?
(4) Does skyline have alignment tool, meaning aligns detected peaks in different samples?

Thanks a lot for your help,

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isotopes/labeled forms not reported in mProphet command line export
(3 responses) Will Thompson 2024-01-30 11:44

We have been looking at utilizing the mProphet command line export to help fix incorrect candidate peak assignments, and we notice that none of the isotope labeled, iRT, or surrogate standards are in the report. Is this on purpose or a bug? This removes the ability to fix misassignments of some of the isotope labeled standards in our workflows, particularly for small molecules where some isotope labeled standards share a mass. Skyline document attached; specifically the mProphet entries are missing for molecules in the 'iMT' and "IS" molecule lists.



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Chromatogram information unavailable JUST for the Heavy Label
(1 response) cvarcini 2024-01-30 11:41


I'm encountering an issue with my internal standard, a heavy label of one of my molecules, in Skyline. While I can see all the chromatograms, the one for the heavy label isn't processing properly. I've attempted this with both Skyline and Skyline Daily, experiencing the same issue on both platforms. I've uploaded two different transition lists, one with the heavy label having the same name as the molecule, and another with a different name. Unfortunately, neither has worked. I've verified the raw data, and I'm confident the peak is present.

How can I resolve this problem and successfully use Skyline to quantify samples using my internal standard?  VitD.skyd  VitD.skyl  TransitionList.csv  TransitionList2.csv  Std12.raw  HeavyLabelPeak.png 
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How to analyze Shimadzu .lcd files?
(7 responses) AWY 2024-01-19 21:37

I outsourced LCMS running and received .lcd files for targeted metabolites.

Files contain (samples with replicates, blanks , standard curv / dilution series ).

I don’t have access to shimadzu software to process targeted LCQQQ .lcd files. This was not possible with MSDIAL, I heaerd the best open source software for processing targeted LCQQQ data.

How can I analyze .lcd files from targeted metabolics?

I would be grateful if you could share any tutorial please.

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Merging calibration curve and samples measurements skyline files
(1 response) ddkale chemistry 2024-01-29 01:52

I have a calibration curve skyline file of few analytes, and another skyline file related to measurement of samples. I do not have calibration curve for all analytes, In this case I would like to merge two skyline files and use data from calibration curves to predict concentration of analytes in samples,
Please let me know if there is any way around to use calibration curves from another skyline file with or without merging both files.
Many thanks

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How to change the language from Chinese to English?
(2 responses) xx huang 2024-01-28 04:26

Hi, thanks for your work in skyline! I am a student in Shanghai Jiao Tong University, Shanghai, China.
I installed the skyline and opened it to find that the default language is Chinese. As I get more familiar with skyline, I want to use it in Englisgh. I wonder how I can change the language into English or where I can download the English version? Thanks in advance!

Best wishes

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"Failed importing results...the calculator requires all targets list in order to determine a regression."
(4 responses) lh48280 2024-01-25 13:24

Hi Skyline Team,
I have been trying to develop a PRM method for a targeted DIA experiment in skyline. I have eight targets of interest which have been built into a transition list, but I do not expect all my samples to have all 8 of these targets. I have been successful in building the calibration curves from spectra for my targets and setting up the iRT calculator, however, when attempting to upload results for sample quantification, I keep receiving this error:

"Failed importing results file.
The calculator requires all 8 of its standard peptides to be in the targets list in order to determine a regression."

I have followed the PRM and iRT tutorials and looked through other support requests, but unfortunately have not been able to fix this. The tutorial data works without any trouble, but once applied to my own spectra throws an error. I have attempted to re-import spectra, and rebuild the calculators, but the same error still occurs. Any advice/help would be greatly appreciated!


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Import of data into Skyline
(1 response) kuechler 2024-01-26 01:23

I am trying to do host cell proteome analysis and imported the FASTA of a whole genome consisting of 45000 proteins.
When I now import my DIA measurements, the needed amount of storage space is enormous (>100 GB per sample) and currently exceeds the storage space of my computer. So, I am wondering, if there is any opportunity for reducing the storage space needed for the import of my samples.

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unstable results importing, chromatograms only partially visible, again
(1 response) warham 2024-01-25 15:07

Hi Nick (probably)

As per your instructions earlier today I uninstalled skyline. Restarted, reinstalled, restarted and imported results, and that worked. For multiple imports over multiple projects. However, just now, on another import, the chromatograms are disappearing. Here, there not completely gone, but they only exist for a minute or so of the whole trace. I will uninstall and reinstall again, but this instability seems new with 23.1? I've attached the unfortunate file.


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Interference peaks and peak tailing
(3 responses) thetrung126829412 2024-01-25 07:26

I ran DDA samples to check a few synthetic peptide pairs (light and heavy, run separately) for purity before spiking the heavy peptides in my biological samples. Based on the extracted chromatograms shown in the skyline, some of the peptides are not so pure (95% purity from the manufacturer). These strong interference peaks (both MS1 and MS2 levels) are however still distinguishable thanks to the intended synthetic peaks of the heavy or light synthetic peptides.

However, when I spiked the heavy peptides in my biological samples (PRM experiments), due to the low signal intensities of the endogenous peptides, the peaks were quite difficult for me to distinguish.
For example, in peptide 1, the peaks y7 and y6 of the endogenous peptide have some really strong interference peaks, I could of course remove these peaks and still use the rest of the peaks for quantification. However, I am afraid the other three peaks might not be enough to confirm detection of the peptide 1.
For peptide 2, there are only three product peaks for both the endogenous and the heavy spiked-in peptide. It looks quite similar to the DDA run of the peptide 2. The interference peaks are very strong and eluted later than the intended peaks. So, it could be safe to conclude that the endogenous peptide was not detected in this sample.
For peptide 3, there are 4 product peaks for both the endogenous and the heavy spiked-in peptide. It looks quite similar to the DDA run of the peptide 3. However, in this case, the intended peaks have much higher intensity values than the interference peaks. The interference peaks eluted later than the intended peaks and they almost looked like tails. So, I think I could conclude that the endogenous peptide was detected in this sample but I am not 100% sure.
Could you please help me take a look at these pictures and let me know what you think? I appreciate your help.

 Peptide 1- DDA.PNG  Peptide 2- DDA.PNG  Peptide 3- DDA.PNG  Peptide 1- PRM.PNG  Peptide 2- PRM.PNG  Peptide 3- PRM.PNG 
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Transition with precursor m/z in MS2
(4 responses) dhabaker 2024-01-23 06:44

Hello Skyline Support,

For some of the precursors in my PRM data, the MS2 spectra have the precursor m/z as the base peak. While mentioning the same m/z for product m/z in the transition list, Skyline does not give a separate product ion transition peak for this precursor (shown in the attached snapshot). Is there a way to have a separate product ion transition peak when the precursor and product m/z are similar?
I am using Skyline 23.1.
Please let me know if additional details are required to convey the issue if its unclear.
Thank you.


 Similar mz for product transition peak.PNG 
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View chromatogram not available for all runs
(2 responses) bart van puyvelde 2024-01-24 04:54

Dear Skyline team,

Probably I am not the first with this issue, but after browsing through the support page I could not immediately find a solution to my problem.
I was wondering if there is a limit on the number of runs that can be viewed as chromatogram?
In total, 256 MRM (Waters) runs have been imported but for some reason I cannot see them all (only 70%). Through the document grid, I can select the runs which are not shown directly, but than other runs are removed from Chromatogram view.
Is there an option to have all runs visualized at the same time?

Ghent University 
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Ion mobility settings using timTOF Bruker DDA data
(2 responses) Luisa Nieto Ramirez 2024-01-23 10:48

I am currently developing a PRM method for a timsTOF Flex Bruker instrument. I have some questions that I hope you can help me with:

  1. In the SOP provided by Bruker, using Skyline to export the PRM method, they have under the peptide settings--> Prediction tab, the option for ion mobility predictor as shown in the first picture attached. However, in the current version of Skyline, we are not able to see this ion mobility predictor option. Do you know if that is no longer possible in the skyline versions after 2020? And what is used instead?

  2. We have been experiencing difficulties when trying to adjust the ion mobility isolation window to the actual values. We have seen the actual ion mobility values are shifted from the window set it by Skyline (see screenshot2). We tried to improve that, by manually integrating the peaks and then re-importing the data, however, it is still shifted (not working). That made us manually change the ion mobility window in the timsTOF software by looking at the heatmap observed in skyline. Do you have any suggestions on how to adjust the ion mobility window using skyline less prone to human errors?

  3. In the same line, under these ion mobility issues what would be the advantages or disadvantages of selecting "use spectral library ion mobility values when present" under the transition settings-->ion mobility.

This is a similar concern reported previously, but unfortunately, I was not able to generate a report with the "Explicit Ion Mobility Value" since the column for that variable was empty.

Thanks in advance for your help and time.

Luisa Maria

 Screenshot1.png  Screenshot2.png 
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Issues with import peak boundaries
(1 response) Juan C. Rojas E. 2024-01-23 05:21

Hi all,

Has there been a change as how the Import -> Peak Boundaries function work?

I have noticed that after importing a peak boundaries file (.csv export generated by Skyline) the boundaries are not implemented for all targets and I end up with something as the attached image.

I recall that in the past the same boundaries were enforced in all precursor and fragment ion targets when I used this approach.

Juan C.

 Peak_boundaries_issue.png  Peak_boundaries_issue_zoom.png 
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Spectral library with .ssl format for wiff2
(1 response) Thierry Schmidlin 2024-01-22 06:58

Hi all

We are trying to generate spectral libraries for skyline based on standard compounds measured in IDA mode on a Sciex ZenoTOF 7600 (generating .wiff and .wiff2 files). In this process we use Sciex OS to help annotating spectra. We would now like to implement these semi-manually annotated spectra into a library within Skyline.
In a similar approach based on Orbitrap instruments we previously successfully used the .ssl format and a conversion of the .raw file to .mzML in order to build spectral libraries in Skyline (shown in screenshot "raw ssl.png"). A similar approach using a .ssl file with ZenoTOF .wiff2 files converted to .mzML files (shown in screenshot "wiff ssl.png") resulted in an error message regarding the scan number ("Error sciex.png"). There seems to be an issue with the "native id format" used in .wiff/.wiff2 - however, the use of .ssl files seems to completely rely on the scan column being present and populated. Is there any advice known, on how to successfully link specific ZenoTOF MS/MS spectra within a run to particular compounds when builing spectral libraries in Skyline?

Thanks a lot for any help on this issue and kind regards


 wiff ssl.png  Error sciex.png  raw ssl.png 
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nbekhti 2024-01-22 07:22

Hello Skyline team,

I started using the AutoQC and Panorama lately to monitor system suitability runs and find it impressively helpful, but on the Skyline template generated I've noticed that not always the right peak is integrated and due to this we can notice a shift in RT or difference in the peak areas on Panorama. I tried to manually correct the integration on this Skyline template but noticed that it hasn't been updated on Panorama, can anyone help me with this please?


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Reporter Ions - Just a thank you!
Juan C. Rojas E. 2024-01-20 06:33

Hi Skyline team,

I just wanted to thank you for adding the support of visualizing reporter ions in the spectral library match.

I have been re-analyzing some data that requires checking some reporter ions and whoever coded this into Skyline has made my life significantly easier!

Juan C.

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Collision Energy (CE) Optimization for PRM
(3 responses) minsu907 2024-01-19 10:28

Dear Skyline team,

We are trying to optimize CE for our PRM.
We could find a tutorial for CE optimization, but it only covers MRM using QQQ.

Could you let us know if CE optimization for PRM is feasible on Skyline program?

Minsoo Son

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jm007 2024-01-19 15:41

Brendan, I am trying to reach you to discuss a dev project. Please email me @

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mass error (ppm) for peptides
(1 response) kmsd 2024-01-19 12:42

I am trying to find the mass error for BSA QC sample to compare the replicates from day to day run. I know mass error for small molecules are available in skyline but when I tried to do it for peptides using TSQ Altis plus it was not showing up in skyline. I am using

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export intensity
(1 response) 1638393444 2024-01-19 08:41

I am trying to export peak intensity of my MS data of PRM on QE , but I could not find it, how can I do that?

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Import Failing
(4 responses) michele tinti 2024-01-18 04:49
Hi, I'm trying to import a raw file from a DIA run using Skyline-daily (64-bit) (f33716d12).
The file fails with this output:

At 12:30:
Failed importing results file 'E:\DH_anna\new_database\280_2023_DUN_DH-AT-LKO1+EGTA.raw'.

pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'E:\DH_anna\new_database\280_2023_DUN_DH-AT-LKO1+EGTA.raw'.
 ---> pwiz.Skyline.Util.AssumptionException
   at pwiz.Skyline.Util.Assume.Fail(String error) in C:\proj\pwiz\pwiz_tools\Skyline\Util\Util.cs:line 2041
   at pwiz.Skyline.Model.Results.TimeIntensities.GetInterpolatedIntensity(Single time, Int32& index) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\TimeIntensities.cs:line 438
   at pwiz.Skyline.Model.Results.TimeIntensities.<GetInterpolatedIntensities>d__34.MoveNext() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\TimeIntensities.cs:line 428
   at System.Linq.Enumerable.WhereSelectEnumerableIterator`2.MoveNext()
   at System.Collections.Generic.List`1..ctor(IEnumerable`1 collection)
   at System.Linq.Enumerable.ToList[TSource](IEnumerable`1 source)
   at pwiz.Skyline.Model.Results.MedianPeakShape.GetCorrelation(TimeIntensities chromatogram) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\MedianPeakShape.cs:line 89
   at pwiz.Skyline.Model.Results.ChromPeak..ctor(IPeakFinder finder, IFoundPeak peak, FlagValues flags, TimeIntensities timeIntensities, IList`1 rawTimes, MedianPeakShape medianPeakShape) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromHeaderInfo.cs:line 1154
   at pwiz.Skyline.Model.Results.PeakIntegrator.IntegrateFoundPeak(IFoundPeak peakMax, FlagValues flags) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\PeakIntegrator.cs:line 97
   at pwiz.Skyline.Model.Results.ChromData.CalcChromPeak(PeakGroupIntegrator peakGroupIntegrator, IFoundPeak peakMax, FlagValues flags, IFoundPeak& peak) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromData.cs:line 332
   at pwiz.Skyline.Model.Results.ChromDataSet.GeneratePeakData(TimeIntervals intersectedTimeIntervals) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 761
   at pwiz.Skyline.Model.Results.PeptideChromDataSets.PickChromatogramPeaks(ExplicitPeakBoundsFunc explicitPeakBoundsFunc) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 224
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.ScoreWriteChromDataSets(PeptideChromDataSets chromDataSets, Int32 threadIndex) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 107
   at pwiz.Common.SystemUtil.ProducerConsumerWorker`2.Consume(Object threadIndex) in C:\proj\pwiz\pwiz_tools\Shared\CommonUtil\SystemUtil\ProducerConsumerWorker.cs:line 186
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.PostChromDataSet(PeptideChromDataSets chromDataSet) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1288
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 433
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 256

DIA-NN uses the file, and it opens with Xcalibur without a problem.
Do you please have any suggestions?

Thanks for your attention,
view request
How to add glycostlation for o-and n-glycosylation on hte peptide sequence?
Jenny Albanese 2024-01-18 14:06
view request
New version (V23) can't properly work on the data processing for CE optimization
(1 response) hsiaoyc 2024-01-17 04:02

Chromatogram doesn't show multiple transition in "single" transition view, but in "all" transition view.
Adjusting peak boundaries will lose ramping data of peak area.

The former version (V22) was workable, and where should I get the install for the V22. I urgently need to process the CE optimization data.

view request
About Skyline
(1 response) saito-yurie 2024-01-16 04:31

Hello Skyline Teams

How many people useing Skyline(or How many download In recent years)

I'm looking forword your reply.

view request
Integration Help
(1 response) pmj4 2024-01-16 07:52

I have a question about integration settings. The current method file integrates multiple peaks at the same retention time window. How do I change some of the peaks to manual integration without affecting all the other ones?

view request
Missing transition peak areas
(4 responses) dhabaker 2024-01-08 08:56

Hello Skyline Support,

I am currently encountering issues observing transition peak areas of an analyte in a skyline project,

As indicated in the screenshots, the precursor peak area is clearly visible but when trying moved to individual transitions of the same precursor, nothing can be visualised (not even the yellow zone of the explicit retention time window).

However, when the raw data are inspected separately in bruker's data analysis, the presence of the same transition ions can be seen from that precusor ion confirming the presence of both the listed transitions in the skyline's transition list.

I am currently using Skyline 23.1 for prm-PASEF based data, please let me know if any further details are required regarding this.

Thank you.

 1_Precursor peak area.PNG  2_Corresponding transition peak area.PNG  3_In Raw data.PNG 
view request
MRMHR on ZenoTOF for small molecules
(4 responses) viragsagikiss 2024-01-10 15:45

Hi all,

Apologies if this has been discussed before I normally work on low resolution targeted small molecule workflows.
I ran the MRMHR mode for small molecules a SCIEX ZenoTOF 7600 system, could you help me if there is a tutorial for that?
I used the small molecule tutorial combined with the PRM tutorial for high res instrument. I could create the transition list and read in the data but only MS1(Precursor ) data shows up, no fragments. I added 'f' in the transition list settings.

Thanks you for pointing me in the right direction,

view request
Import DDA Peptide Search
(3 responses) s84063 2024-01-06 01:25


I wanted to make IMPORT DDA PEPTIDE SEARCH. I made everything correctly with tutorial (files .mz5 and FASTA downloaded from SkyLine) and I created results. So the SkyLine works correclty. But when I wanted to process my own data from Preotome discoverer and I stocked. Every time I got an error information with description below.

It is definitely some problems with my own .raw files. I changed files from raw. to mz5- no success (exclude error in .raw or .mz5). I changed .fasta file for a new one from uniprot- no success (exclude error in FASTA). I have no idea what is wrong and what I should do next.

Best regards,
Piotr Szatkowski

 Import DDA.pdf 
view request
Toggle off protein overlay information
(1 response) Juan C. Rojas E. 2024-01-10 05:40

Hi Skyline wizards,

I am facing serious performance issues when I accidentally hover the protein node of Skyline documents. This is more significant the bigger the document is and specially if it has PTMs on it. I am not sure what is calculated when this is done, but it freezes the software completely. If I am lucky after some minutes of not clicking anywhere it will finish processing and let me continue. But I have had several instances I had to force shut the software and loose all unsaved progress.

I suspect these issues was introduced when the protein sequence coverage visuals were included some time ago (maybe 1-2 years). This might have a good appeal for many, but in my case I find it more troublesome than helpful so I was wondering if there is a way of disabling that feature or focus some rework on it to avoid these "freezes"?

Thank you for your time and help.
Juan C.

view request
Error in negative mode mzML file import
(2 responses) chang_he 2024-01-09 14:44

Hi, skyline support team,

I exported Waters Unifi analysis files to mzML and tried to import the data into Skyline 23.1. The following error was reported:

"At 2:42 PM:
Failed importing results file 'C:\Users\che\Desktop\Test\2_3_mix_6pt25uM [1A,5] [3].mzml'.
This document contains only negative ion mode transitions, and the imported file contains only positive ion mode data so nothing can be loaded."

It seemed like Skyline could not recognize the mzML file as negative mode. Is there a good way to fix this issue?

Thank you.

view request
Peak normalization with iRT
(2 responses) c frampas 2024-01-09 07:52


I was wondering if it was possible to use iRT for peak normalization? If so, could you explain how to go about it?
Due to a large number of samples, I had to divide them into 2 batches. Unfortunately, the mass spec had an increase of sensitivity and therefore I can't compare samples. Your help would be appreciated.

Many thanks,

view request
Exporting protein name from proteome FASTA file.
(3 responses) jpbelair 2024-01-02 12:06

I have a list of 328 gluten / gliadin peptides found in the wheat proteome of 1,750 wheat proteins. I have a candidate list of 28 peptides with very good signal and fragmentation. The sample matrix is Human urine and I am capturing the gluten / gliadin peptides by immunoprecipitation using a series of mAbs A1, G12, and two custom-made mAbs. I have imported the FASTA file of the wheat proteins. I am putting together a list of candidate peptides that serve as a biomarkers for Celiac Disease. The peptides are found in multiple wheat species. I can see the peptides associated with multiple proteins when I go to Edit --> Unique Peptides... I've tried playing around with the report template without success. Does anyone know how to export the protein name(s) (and possibly more data) from the FASTA file? Copying the data from the window does not capture the column header information showing the protein name.

sp|A7XUQ5|AVLB5_WHEAT Avenin-like b5 OS=Triticum aestivum OX=4565 PE=2 SV=1

All I need is the protein name "A7XUQ5|AVLB5_WHEAT Avenin-like b5".

The data was acquired on a Q Exactive Plus with DDA acquisition.

Skyline version (28f9b9301), Windows 10 Enterprise.

Thank you, Jeff

view request
modification of precursor m/z order in targets and results panels
(12 responses) heuillet 2023-06-16 04:51

I use Skyline to process my MS data of 13C-fluxomics approaches. I study the 13C-incorporation inside my metabolites of interest.
In my transition list I associate different m/z to one compound. For example, for glutamate analysis I need to integrate:precursor,[M+1], [M+2], [M+3], [M+4], [M+5]. The number of m/z depends on the total number of carbon inside the molecule.
After loading my transition list, the order in the targets panel is not kept and the [M+1] is always the last m/z of the list for all metabolites.
How can I change that and put the [M+1] juste before the [M+2]?
thanks a lot,

view request
Exporting no retention time to transition list
(1 response) Jeff Whiteaker 2024-01-05 10:57

Recently updated to the latest daily version of Skyline (
I tried exporting a scheduled transition list from the attached document using SCIEX settings.
The values for retention time were not exported (i.e., zero in all rows).
It works if the document is shared using a previous version (23.1).

Maybe I missed something? Many thanks for your help tracking this down.
view request
Importing peak bounderies
(2 responses) evy timmerman 2024-01-05 05:18

I always get the following error:
'index_outside_the bounds of the array_import_peak_bounderies'

Could you please check out for me how i can fix this? Or can this be simplified? The goal is to get the peak picking from DIA-NN instead of skyline and perform mProphet on the data set....

A 'stronger' computer also failed.
On the other hand only importing a small subset works.
This is a dataset with 18 raw files and >200,000 precursors.

Kind regards,

skyline file could not be saved to share, also there the daily version gave an error.

 index_outside_the bounds of the array_import_peak_bounderies.JPG 
view request
Zooming not working properly
(3 responses) Tomas Vaisar 2014-02-19 08:32
Hi Brendan,
 have noticed that the zooming using mouse stops working after awhile especially with larger data (big DIA files or many SRM files) loaded in the Skyline. Initially after opening the Skyline project it works fine, but at some point Skyline stops responding to the mouse zooming. Going to the menus and applying zoom through menu options still works. This is not related to the current release as I have been noticing it for some time. I guess I got to the point I should mention it. Perhaps the problem is with my computer/mouse.

view request
Keyboard shortcuts for scrolling through targets
(2 responses) matthew murphy 2024-01-05 08:17
I appreciate the keyboard shortcuts that have been provided for many of the functions in Skyline. One I would hope to see is a keyboard shortcut to scroll through the targets after interacting with them (e.g. setting boundaries).

It would be great to have a key combo that will allow you to jump to the next or previous molecule after you have interacted with the chromatograms. You can scroll through the compounds with the "up" and "down" keys, but once you set boundaries in the chromatogram, you have to select the next compound with the mouse.
You can scroll through the separate chromatograms for each sample using the "ctl + up" or "ctl + down", so if for a particular compound you need to set different boundaries for different samples, you can move through them more quickly this way than having to select the tab or use the dropdown menu using the mouse.

Thank you!
Matthew Murphy
view request
log2 scale for group comparison
(3 responses) hwang2 2024-01-04 07:30

I'm using Skyline and 23.09. For group comparison and p-value calculation, current version has three normalization methods: none, equalize to medians and ratio to heavy. Could you add log2 option for p-value calculation?



view request
Fold change
(6 responses) ilaria palmisano 2024-01-04 07:22

Dear all,
I am a beginner and I have a question about how skyline calculates the Fold Change between groups. I understood that, asking a Clustered report, one can get the protein abundance values used to calculate the FC. But, using those values, I am not able to reproduce the FC calculated by Skyline.
Could you please help me? Thanks

view request
Skyline not compatible with Shimadzu LCMS-8060
(14 responses) William 2021-04-19 21:10

I am porting the method to our new purchased Shimadzu LCMS-8060. I exported transition list from Skyline (choose Shimadzu QQQ) to txt file. However, when importing compound list to Shimadzu, it is completely chaos. I then check the title of each rows and find they don't match. Here I attach the compound export file from Shimadzu QQQ. Pls update Skyline so that exported file can be recognised by Shimadzu.

view request
Development MRM method from a high resolution MS data
(1 response) oyku su yildirim 2024-01-04 03:02

I am a master's student who is a new Skyline user. I am wondering if a MRM method can be developed from a high resolution MS data. If available, which webinar or tutorial can help me?

view request
Why can't the predicted retention time be displayed?
(4 responses) caixue 2023-12-25 20:48


I created a CiRT predictor with an r value of 0.99, but the predicted RT can not display as shown in attachments.

I don't know if there is a problem with the parameters or settings.

Looking forward to response

Xue Cai

 1703565973741.png  1703565931622.png  1703565907792.png 
view request
Removing Redundant Transitions
(5 responses) eajarett 2022-11-28 14:07


I am trying to exclude all redundant transitions in a DIA assay library from quantitation. I was able to export all transitions and manually determine which ones were identical with others based on their molecule list name, precursor m/z, precursor charge, product m/z, and fragment ion.

I tried manually deleting all transitions in my DIA assay library and reimporting the "mixed transition list" containing only the non-redundant transitions, but this didn't work. Is there a better way to exclude the redundant transitions from quantitation?

Thank you so much!

view request
support forum posting policy question
(2 responses) bobxiong 2023-12-24 10:35

Hi Skyline Team,

I would like to know if it is permitted to have a url link to a commercial website in a response. I checked the Skyline support page but did not find a specific language on it.

Thanks and Happy Holidays!

Bob Xiong

Founder, ProteinQuantSRM-Dx

view request
.d files do not seem to be recognized by Skyline batch
(2 responses) Shawn Rice 2023-12-15 07:06

I am trying to run TIMS-tof .d files using Skyline batch. When I try to save the configuration file, I get the error attached. It says the data folder is empty, but my .d files are in the folder. It seems that skyline batch is not recognizing the .d files as raw data. Note Skyline Batch is v.


view request
predicted retention times not working for random subset of library
(1 response) Will Thompson 2023-12-15 05:30

Dear Skyline Team,

I'm having some trouble in recent version(s) with retention time/iRT not producing predicted retention times for a seemingly random subset of compounds which are in the library. Can you take a look at the attached document and see if you can help me figure out what is going on? Every compound in the document should be in the retention time library. Every run has all iRT indexing compounds identified...and yet a predicted retention time (and appropriately narrow RT window for extraction) is not produced for all the compounds. I just can't seem to figure out why some of these compounds would work and others not. Moreover, it doesn't seem to happen all the time, and routinely all the iRT molecules which it needs to produce predictions for the other compounds, are always found.

Help! I appreciate it.



view request
predicted retention times not working for random subset of library
(1 response) Will Thompson 2023-12-14 19:31

Dear Skyline Team,

I'm having some trouble in recent version(s) with retention time/iRT not producing predicted retention times for a seemingly random subset of compounds which are in the library. Can you take a look at the attached document and see if you can help me figure out what is going on? Every compound in the document should be in the retention time library. Every run has all iRT indexing compounds identified...and yet a predicted retention time (and appropriately narrow RT window for extraction) is not produced for all the compounds. I just can't seem to figure out why some of these compounds would work and others not. Moreover, it doesn't seem to happen all the time, and routinely all the iRT molecules which it needs to produce predictions for the other compounds, are always found.

Help! I appreciate it.



view request
Extract method file from Thermo Raw file
(11 responses) Josh Baeza 2023-10-25 08:51
Hello Skyline Team,

Would it be possible to extract the method file path and name (C:\Path\To\method.meth) from a Thermo .raw file and export this field in the report?

view request
Cannot read Bruker .d with MS1 only data
(4 responses) ho-tak lau 2023-12-11 10:34

Hello Skyline team,

I have some .d files containing .tsf that I want to analyze using Skyline. These are MS1 only files. I am using Skyline-daily (64-bit) (6a818db84). And as soon as I import the data, Skyline will close.

I can share a file if that helps.


view request
Issue running EncyclopeDIA search
(2 responses) lisa walrond 2023-12-13 08:04
I'm trying to import an EncyclopeDIA search. It is failing for me in both Skyline and Skyline daily. I have been able to run these searches successfully before. I've included error messages from both failures below.

Thank you, Lisa

OK More Info
pwiz.Skyline.Model.Prosit.PrositNotConfiguredException: Intensity model '' does not exist
   at pwiz.Skyline.Model.Prosit.Models.PrositIntensityModel..ctor(String model) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Prosit\Models\PrositIntensityModel.cs:line 52
   at pwiz.Skyline.Model.Prosit.Models.PrositIntensityModel.GetInstance(String model) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Prosit\Models\PrositIntensityModel.cs:line 65
   at pwiz.Skyline.Model.Prosit.Models.PrositHelpers.PredictBatchesFromPrositCsv(String prositCsvFilePath, IProgressMonitor progressMonitor, IProgressStatus& progressStatus, CancellationToken token) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 408
   at pwiz.Skyline.FileUI.PeptideSearch.EncyclopeDiaSearchControl.Search(EncyclopeDiaSettings settings, CancellationTokenSource token, IProgressStatus status) in C:\proj\skyline_23_1\pwiz_tools\Skyline\FileUI\PeptideSearch\EncyclopeDiaSearchDlg.cs:line 632

OK More Info
Skyline-daily (64-bit) (6a818db84)

pwiz.Skyline.Model.Prosit.PrositNotConfiguredException: Intensity model '' does not exist
   at pwiz.Skyline.Model.Prosit.Models.PrositIntensityModel..ctor(String model) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Prosit\Models\PrositIntensityModel.cs:line 52
   at pwiz.Skyline.Model.Prosit.Models.PrositIntensityModel.GetInstance(String model) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Prosit\Models\PrositIntensityModel.cs:line 65
   at pwiz.Skyline.Model.Prosit.Models.PrositHelpers.PredictBatchesFromPrositCsv(String prositCsvFilePath, IProgressMonitor progressMonitor, IProgressStatus& progressStatus, CancellationToken token) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 408
   at pwiz.Skyline.FileUI.PeptideSearch.EncyclopeDiaSearchControl.Search(EncyclopeDiaSettings settings, CancellationTokenSource token, IProgressStatus status) in C:\proj\pwiz\pwiz_tools\Skyline\FileUI\PeptideSearch\EncyclopeDiaSearchDlg.cs:line 693
view request
Error when importing .raw files on Mac Virtual Machine
(2 responses) filippa qvist 2023-12-12 05:05

I recently had an issue where I got an error message (see attached image and full error text below) when I tried to import .raw files into Skyline.
For context, I was running from a MacBook Pro with a virtual machine called Parallels Desktop, and I subsequently spoke to another Mac VM user who had experienced the same issue. It happened when importing with both Skyline 23.1 and Skyline Daily.

I was able to import .mzXML and .d (TimsTOF file folders) into Skyline as normal, but when importing .raw files I encountered the error listed below. This issue was only present when the .raw files were stored in a Mac directory (\Mac\Home\Documents), but can be resolved by moving the files to either the C: drive or any network drive. In those cases, the .raw files open as normal.

I have now just changed where I store the files I want to import, but was asked to still report the error.
Thank you!

Full error:
At 1:59 PM:
Failed importing results file '\Mac\Home\Documents\GroupedStudies1\HeartFailure\raw\D_102_REP1.raw'.
[RawFileImpl::ctor()] Corrupt RAW file \Mac\Home\Documents\GroupedStudies1\HeartFailure\raw\D_102_REP1.raw
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file '\Mac\Home\Documents\GroupedStudies1\HeartFailure\raw\D_102_REP1.raw'.
[RawFileImpl::ctor()] Corrupt RAW file \Mac\Home\Documents\GroupedStudies1\HeartFailure\raw\D_102_REP1.raw ---> System.Exception: [RawFileImpl::ctor()] Corrupt RAW file \Mac\Home\Documents\GroupedStudies1\HeartFailure\raw\D_102_REP1.raw
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, Boolean combineIonMobilitySpectra, Boolean trimNativeId) in C:\proj\skyline_23_1\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 201
at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 295
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 191
--- End of inner exception stack trace ---

view request
signal to noise
(1 response) jelliott11 2023-12-12 14:56


I wanted to ask about how to determine signal to noise of a specific feature/ion on my spectra. Is there a way to calculate it on the spectra level?

Justin E :)

view request
Error occurred while importing the assay library
(1 response) caixue 2023-12-11 04:35
When I tried to import the assay library in TSV format, I encountered the following error message:

Failed reading the file D:\lab\project\DIA_PRM\TPDlibV2_FAIMS_FP_iRT_nonIsoform20422Fasta_20230621.tsv. Unexpected SQLite failure reading D:\lab\project\MFT\FAIMS_TPD_lib-assay.blib.
OK More Info
System.IO.IOException: Unexpected SQLite failure reading D:\lab\project\MFT\FAIMS_TPD_lib-assay.blib. ---> System.Data.SQLite.SQLiteException: SQL logic error or missing database
no such table: RefSpectraPeaks
   at System.Data.SQLite.SQLite3.Prepare(SQLiteConnection cnn, String strSql, SQLiteStatement previous, UInt32 timeoutMS, String& strRemain)
   at System.Data.SQLite.SQLiteCommand.BuildNextCommand()
   at System.Data.SQLite.SQLiteDataReader.NextResult()
   at System.Data.SQLite.SQLiteDataReader..ctor(SQLiteCommand cmd, CommandBehavior behave)
   at System.Data.SQLite.SQLiteCommand.ExecuteReader(CommandBehavior behavior)
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteLibrary.<>c__DisplayClass72_0.<ReadSpectrum>b__0(SQLiteConnection connection) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Lib\BiblioSpecLite.cs:line 1319
   at pwiz.Skyline.Util.PooledSqliteConnection.ExecuteWithConnection[T](Func`2 function) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Util\PooledSqliteConnection.cs:line 95
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.PooledSqliteConnection.ExecuteWithConnection[T](Func`2 function) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Util\PooledSqliteConnection.cs:line 106
   at pwiz.Skyline.Model.Lib.CachedLibrary`1.LoadSpectrum(Object spectrumKey) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Lib\Library.cs:line 1118
   at pwiz.Skyline.Model.Lib.SpectrumInfoLibrary.get_SpectrumPeaksInfo() in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Lib\Library.cs:line 2526
   at pwiz.Skyline.Model.Lib.SpectrumMzInfo.GetInfoFromLibrary(Library library) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Lib\Library.cs:line 2425
   at pwiz.Skyline.SkylineWindow.ImportMassListIntensities(SrmDocument& doc, List`1 librarySpectra, Boolean assayLibrary, BiblioSpecLiteSpec& docLibrarySpec, BiblioSpecLiteLibrary& docLibrary, Int32& indexOldLibrary) in C:\proj\skyline_23_1\pwiz_tools\Skyline\SkylineFiles.cs:line 2400
   at pwiz.Skyline.SkylineWindow.ImportMassList(MassListInputs inputs, String description, Boolean assayLibrary, DOCUMENT_TYPE inputType, Boolean forceDlg) in C:\proj\skyline_23_1\pwiz_tools\Skyline\SkylineFiles.cs:line 2106
   at pwiz.Skyline.SkylineWindow.ImportAssayLibrary(MassListInputs inputs, String description) in C:\proj\skyline_23_1\pwiz_tools\Skyline\SkylineFiles.cs:line 1874
   at pwiz.Skyline.SkylineWindow.ImportAssayLibrary(String fileName) in C:\proj\skyline_23_1\pwiz_tools\Skyline\SkylineFiles.cs:line 1864
May I ask how to solve this?
view request
Quantify with the calibration curve of an other compound
(6 responses) heuillet 2023-11-21 00:26

I want to quantify a compound with the calibration curve of an other (with structural similarities). I didn't find the way to do it even with the tutorials I found.
Is it possible? If yes, could you help me?
thanks a lot,
kind regards,

view request
Can Skyline export to Agilent QqQ method? Can you do segments?
(2 responses) wangqingok 2023-12-07 10:00

Hi Skyline Team,

Can skyline export to Agilent method in a segmented manner? I could only export to Agilent method with a static SRM method but I wish to know if I want to have multiple segments in the method, such as for time 1~10 minute, do MS2 scan, and for time 10~20 minute, do a transition table, and how do do this from skyline and export? I have a lot of transitions and methods to export in this way. Many Thanks!

view request
Isotope prediction issue upon data import
(1 response) dbc007 2023-12-07 13:10

Hello --

When a new document is started (i.e. - set molecule/trans settings, transition list imported via copy/paste from spreadsheet, raw data imported as Agilent .D files or Thermo .RAW files) the isotope prediction algorithm does not include the isotopologue of highest abundance (what we might think of as the “main” monoisotopic signal for each imported compound). To be clear, these are high-res MS1 data. Therefore the idotp does not get calculated correctly. I can fix this by simply saving, closing, and re-opening the Skyline file, which isn’t such a big deal, but this happens every time I start a new skyline file. Happens to others in my lab also across different computers, and it's been happening across version releases. I think it occurred in version 22, continues in the latest v23 release, and also in the most recent Skyline Daily release (attempted and replicated issue on 7 Dec 2023).

I have attached a PPT file with screenshots and some more description of what we see. As mentioned, if I close the file and re-open it, the issue is gone forever (in that file, but comes back when we make a new one). There is some (rather ugly) data shown in the file, but it illustrates my specific problem.

Has anyone else seen this issue? If it was a one-off bug I would have just thought nothing of it, but it's been happening routinely for at least a few months.


view request