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

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

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

If your question is about an External Tool, please contact that tool's developers directly. Contact information can usually be found on Skyline's Tools | Tool Store... menu.  

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

  • A detailed description of your problem or question, including instructions for re-creating any problem that you are encountering. Screenshots are often helpful.
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Showing: limited to 100 requests
Get chromatograms for few peptides
(3 responses) shubham1637 2023-09-30 17:28

I have DIA files from small-amount samples. I did not spike-in iRT standards. Is there a way to get/see chromatograms for few candidate peptides? I am trying to load file in Skyline, however, since there are no iRT peptides, it is failing. I also tried CiRT but that also failed.


At 8:27 PM:
Failed importing results file 'F:\Users\rona\DIA-QE\DIA-QE\DIA\collinsb_X1803_171-A.mzML'.
The calculator test1-assay requires all of its standard peptides in order to determine a regression.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'F:\Users\rona\DIA-QE\DIA-QE\DIA\collinsb_X1803_171-A.mzML'.
The calculator test1-assay requires all of its standard peptides in order to determine a regression. ---> pwiz.Skyline.Model.Irt.IncompleteStandardException: The calculator test1-assay requires all of its standard peptides in order to determine a regression.
at pwiz.Skyline.Model.Irt.RCalcIrt.ChooseRegressionPeptides(IEnumerable`1 peptides, Int32& minCount) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Irt\RCalcIrt.cs:line 152
at pwiz.Skyline.Model.Results.ChromCacheBuilder.RetentionTimePredictor.CreateConversion() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 838
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 404
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252
--- End of inner exception stack trace ---

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Exporting Candidate peaks for ALL compounds
(2 responses) sstewart 2023-09-22 12:36

I'd like to export a report that contains the candidate peaks for EVERY compound. I'd like to do it from the command line. A report exists but (see screenshot) ONLY for one file at a time and with no command line access (unless I misread the docs).

Format would be the same as in the screenshot PLUS extra columns for identifying the compound: "File Name", "Compound Name", "Product Mz", and "Precursor Charge" (see below screenshot). Each row would be a candidate peak.

 extra_columns.png  existing.png 
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How to exclude lockspray channel
(1 response) lars debie 2023-09-29 04:18

In the data from a Waters Xevo G2-XS QTof with lockspray applied, the lockspray signal is stored as a separate channel. When the data is imported the data from the lockspray channel are included as datapoints. Since the compound of interested is not present at the point of lockspray, the peak or baseline is shown as zero when the lockspray was applied. This creates holes in the created chromatograms, see attachments. Is there a way to import only one channel and exclude the lockspray channel?

 Lockspray interference in peak.PNG  Lockspray interference in baseline.PNG 
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Pick children window closing instantly, 23.1 bug?
(1 response) warham 2023-09-29 10:45


I have been working on a file for a over a few days and today when I opened it and tried to open the pick children window, skyline stalls, uses 20% of my CPU for maybe 5 seconds and then the window opens and instantly closes so that I cannot use it. The file is other wise able to see the data, can access the data to generate the full scan data for example. The Pick children window was working on this file earlier, not sure how it might have become corrupted but closing and reopening both the file and skyline does not help. The modify peptide window is functioning properly as well. I'm not sure but the performance feels very much like a bug.


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Invalid methods for Agilent 6495 running MassHunter version 10.1 build 10.1.67
(1 response) michelle robinson 2023-09-28 08:01

Methods exported from Skyline-daily v for Agilent 6495 (both scheduled and standard MRM) are invalid in MassHunter version 10.1 build 10.1.67. The methods are valid on a different Agilent 6495 running an older version of MassHunter (version B.09.00, build 9.0.9037.0).

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Problem during EcyclopeDIA search
(1 response) aemmanue 2023-09-27 11:46

Dear Skyline Team,

I hope you are doing well. This morning I tried to process some DIA raw file with EncyclopeDIA search. There is an error message that I share with you. Thanks in advance for your help.

Best Regards.

 Skyline error_09282023.docx 
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Which formula to choose for combining peptides intensity into protein intensity ?
(4 responses) nicolas pierre 2022-05-05 04:43

Hello Skyline Team,

I have measured endogenous peptides with their heavy counterpart by SRM. I have also spike a protein in my sample for normalisation.
I have some questions for translated results at protein level.
When I divide the intensity of endogenous peptide by their heavy counterpart it become a ratio and in many cases this ratio totally change the ponderation of each peptide for their contribution to the protein intensity. Typically, peptides representing the highest intensity (the one which would contribute the more to protein signal with the addition of peptide intensities) for the protein can, when divided by its heavy counterpart, be the one with the lowest ratio among the other peptide of the protein. In this case this will totally change the ponderation. I am not very confident with this phenomena because usually signal with the lowest intensity are those with the lowest quality (near LOD, lowest level of precision, lowest quality of the peak...). It means that with a division by the heavy peptides I will give more importance (not systematically) to signal with the lowest quality.
In fact their is different solution to combine the signal of peptide into protein signal. For instance:

-Signal of protein A= (Signal of endogenous peptides 1/ signal of heavy peptide 1) + (Signal of endogenous peptides 2/ signal of heavy peptide 2)
-Signal of protein A = (Signal of endogenous peptides 1 + Signal of endogenous peptides 2)/(signal of heavy peptide 1 + signal of heavy peptide 2)

As explained, the first formula can change the ponderation of the peptides contributing to the protein signal, the second one will not. We could imagine other solutions...Also, I have spiked a protein for normalising my signal and I do not know where I can put it in the formula, where would be the best place..
Do you have some advises for this problem ? In there some Skyline solutions ?
Thank you in advance for your help.

Nicolas Pierre

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Problem exporting data files with the new skyline version 23.1
(2 responses) f bernuzzi 2023-09-27 08:47

Dear Skyline team,

I hope you are well. I am new to skyline so apologise if I may have missed something, but last week I did some analysis and exported the files without having any issues. This was using the old skyline. The other day I received an email that a new was released so I downloaded the newest version. Just now I finished analysing some samples as was planning on to export them but the software doesn't allow me to. I don't know whether with the new version something may have changed
The way I have been instructed to export the samples is through the following:
File > export > report. In the report then I would then select edit list, select samples in columns which would prompt me import a file. I would insert the samples columns, press import and when I first did the samples in columns became highlighted. This time however, it fails to recognise the file.
Any suggestion on how to deal with the issue and export the files. Worst case scenario I was thinking of uninstalling and reinstalling but don't know whether that may help. I have also attached several pictures, hopefully that will be more clear.
Many thanks for your help and support
Best wishes

Federico Bernuzzi, PhD
Cancer Research Scotland

 Screenshot (11).png  Screenshot (10).png  Screenshot (12).png  Screenshot (13).png  Screenshot (14).png  Screenshot (15).png  Screenshot (16).png 
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Failes on importing the data acquired with FAIMS and used CV value spectrum filter active
(4 responses) david 2023-09-27 03:16

Dear all,

I have some data acquired using Fusion Lumos with the FAIMS installed and two CV voltages set. When I import the file without any spectrum filter, the data are imported correctly and one can see zig-zag style of the MS1 level intensities due to the the intensities from the two CV values combined into a single graph. When I set the spectrum filter in a way to select only CV values of -50 and -70, it gives me the following error:

At 12:05 PM:
Failed importing results file 'U:\712006-Proteomics\Instruments\Orbitrap_Lumos\Data\2023\230926_IS270_Lumos1\IS270_Lumos1_74IS_FAIMS(-50-70)_HCD-OT_DDA_HeLa_50ng_01.raw'.
Times (4672) and intensities (2336) disagree in point count.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'U:\712006-Proteomics\Instruments\Orbitrap_Lumos\Data\2023\230926_IS270_Lumos1\IS270_Lumos1_74IS_FAIMS(-50-70)_HCD-OT_DDA_HeLa_50ng_01.raw'.
Times (4672) and intensities (2336) disagree in point count. ---> System.IO.InvalidDataException: Times (4672) and intensities (2336) disagree in point count.
at pwiz.Skyline.Model.Results.ChromCollector.ReleaseChromatogram(Byte[] bytesFromDisk, TimeIntensities& timeIntensities) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCollector.cs:line 120
at pwiz.Skyline.Model.Results.ChromGroups.ReleaseChromatogram(Int32 chromatogramIndex, Single retentionTime, ChromCollector collector, TimeIntensities& timeIntensities) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCollector.cs:line 633
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Collectors.ReleaseChromatogram(Int32 chromatogramIndex, ChromGroups chromGroups, TimeIntensities& timeIntensities) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1314
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.GetChromatogram(Int32 id, ChromatogramGroupId chromatogramGroupId, Color peptideColor, ChromExtra& extra, TimeIntensities& timeIntensities) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 619
at pwiz.Skyline.Model.Results.ChromData.Load(ChromDataProvider provider, ChromatogramGroupId chromatogramGroupId, Color peptideColor) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromData.cs:line 84
at pwiz.Skyline.Model.Results.ChromDataSet.Load(ChromDataProvider provider, ChromatogramGroupId chromatogramGroupId, Color peptideColor) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 285
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Load(ChromDataProvider provider) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 146
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 427
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 256
--- End of inner exception stack trace ---

I have tested this on the freshly opened Skyline document with just a single iRT peptide in the list of peptides to import. It does work with the positive CV values used and also with the negative CV values for which there are no data actually present in the raw file. It simply returns no traces at this case. But when I set any CV value that is in the data (-50 or -70), I get the abovementioned error.

I have used Skyline version.

Let me know if you would need any more data, I could share the raw file if necessary.

Thank you for double checking!


view request
Highlight fragment ion in peptide sequence
(1 response) Nadzeya 2022-06-19 07:21

Dear Skyline Team,

I do targeted mass spectrometry for phosphopeptides. Since the same peptide can be phosphorylated at different positions, it is crucial to have a specific fragment ion for site localization. Is there a function in Skyline to somehow highlight a selected fragment ion in a peptide sequence? For example, highlighting amino acids with another colour or a rectangle (see attachment). This would allow a quick visual assessment of the role of the fragment ion for PTM site localization. Now I'm counting with my eyes and moving a pencil on the monitor, both suffer :)

I really appreciate any help you can provide.

Kind regards,

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Problems with 23.1
(8 responses) djlj1 2023-09-25 04:57

Dear Skyline

We are having problems with exporting methods with the new version.

See the message in the attached .png file.

 exporting methods.png 
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Relative Quantification & MRM Transitions?
(1 response) megan kober 2023-09-26 18:27

Hi Folks!

I am utilizing Skyline Daily ( to analyze my QQQ data, where I aim quantify relative abundance of different intracellular metabolites. During sample processing, I have included an internal, isotopic standards for relative & surrogate normalization purposes. With Skyline, I have successfully imported a transitions list with the appropriate heavy/light formulas, set up a normalization method, and specified which isotopic standard should serve as a surrogate. However, I'm curious how Skyline is actually quantifying the peak area ratio to the defined surrogate.

In my transition list, the majority of my molecules (including my isotopic standards) have multiple product ions and I have not specified as qualitative vs. quantitative. But, when I examine a Document Grid displaying the normalized areas, there is only a single value despite multiple transitions. Does Skyline have a default way of setting a quantitative ion that I'm overlooking? Or, without distinguishing qualitative vs. quantitative product ions, does skyline sum all available product ions and use this "total peak area" for the calculation?

Attached are screenshots to help clarify what I'm describing above. Let me know your thoughts & recommendations!

Thanks in advance,

 SkylineQuantMethod.png  SkylineExampleTransitions.png  SkylineDocumentGrid.png 
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feature request - graphical layout manager enabling to switch between different graphs and or sample sets views
david 2023-09-26 07:38

Dear all,

not sure whether I am missing this feature being in Skyline already, but I would love to have an option to switch between different graphical layouts of the Skyline app, including e.g. different analyses grouping/shoving, different plots (like retention time comparison for replicates) displayed, positioned etc.

Or is this already possible please?

Might be related to this issue

Thank you in advance for your response/considering the feature request.


view request
Adding additional peptide after importing results
(1 response) s jager 2023-09-26 04:46

Hi skyline team!

I just imported my results (.raw files) after loading my fasta, but found out that my fasta was missing a pepitde... is there a way i can add this peptide now and integrate it for the result files? The loading of the files took about 4 hours, so i would prefer not having to start from scratch...

Best Shelley

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Feature request: include idotp when creating IMS library with Skyline
(2 responses) Juan C. Rojas E. 2023-09-19 05:39

Hi MS wizards,

I would like to request the addition of idotp as a metric that Skyline uses for deciding the correct center of the ion mobility window used for IMS-filtered EICs.

Attached is an example where the intensity maximization criteria (the only one used as far as I remember) leads to wrong selection of ion mobility values by co-eluting isobaric species. The wrong value was selected despite have identified the M+1 interference and marked it as non-quantitative.

Would it be possible to make Skyline to maximize both intensity and idotp (perhaps more weight on the idotp)?

As always, thank you for your time and help!
Juan C.

view request
wiff2 file MSMS transitions
(2 responses) Lisa Panzenboeck 2023-09-25 07:40

Hi Skyline-Team,
thanks to your amazing support regarding my last question ( I could import my .mzml files converted from .wiff and .wiff2 files into skyline.
However, for some reason I can only see my MS2 transitions of interest using .wiff files or .mzml files converted from .wiff files, but as soon as I try to import a .wiff2 file or .mzml file generated from the .wiff2, I don’t see any chromatogram information. The problem only seems to occur for my MS/MS transitions of interest, as the MS1 trace is visible irrespective of the file-type.

Do you have any idea how this could happen and how to circumvent this issue? Unfortunately, I need to use the .wiff2 file (or .mzml file generated from the .wiff2) for my data analysis. Using SeeMS I can clearly see that MS2 spectra are present.
I use Skyline 64 bit with Windows Enterprise and try to analyze data generated on a zenoTOF 7600 from SCIEX.

I would highly appreciate your input and look forward to hearing from you.

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Ratio to surrogate ...
(2 responses) a rocher 2023-09-06 02:25


I quantify small molecules on Skyline. I wanted to use metabolite B as an internal standard for metabolite A, so I defined marked metabolite B as a standard surrogate in the tree structure. Unfortunately, in the Normalization Method column of the Document Grid, the name of the metabolite is replaced by its InChIKey, which makes it very difficult to read (see attached file).

How can we make the "Ratio to surrogate..." option show the metabolite name (as defined in the tree structure) and not an identifier?


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Auto QC file type
(1 response) lkerton 2023-09-25 09:40

Very, very new to Skyline, so please be gentle:

We are working on our Quality Assurance plan, and I am looking into the possibility of importing endpoint Auto QC data into our existing quality control software and want to better understand Auto QC's output. Is there an output file other than the monthly zip file that could be saved externally? And if so, what is the format or file type? Any help would be appreciated!

Thank you,

Lindsay :)

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Mac os
(4 responses) 2965673435 2023-09-23 22:51

Sorry to bother u, my laptop is Mac m1 pro(ventura 13.5.2), i tried to run the download skyline exe on my parallel, but it was failed, can u help me?

 截屏2022-09-13 13.18.46.png 
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TurnoveR tool
(1 response) ghazaleh 2023-09-22 10:43

Good day,

I am trying to use TurnoveR tool in Skyline. I have a question regarding "Set Reference". I am not sure What can we use for Reference? is it like DDA of control sample (unlabeled sample)? can you please explain what Set Reference and why need to use?

Thank you,

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Import .d files
(1 response) Anastasia T 2023-09-19 02:16

Hi all,
newbie in skyline and I'm trying (unsuccessfully to import some data). I read that skyline looks in .d files for .tdf files. I do have .tdf files however skyline doesn't recognize it.

Any ideas/tips/tricks/help?

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MFragger EasyPQP library.tsv to populate Skyline target list
(3 responses) Tomas Vaisar 2023-09-18 06:26

Hi All,
Is there a way to populate Skyline target list with the library generated by MSFragger/EasyPQP (library.tsv)?


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Error uploading MSFragger DIA GPF results into Skyline
(1 response) Tomas Vaisar 2023-09-18 12:34

We are trying to upload MSFragger results from a search on DIA GPF, following the instruction ( using both DDA and DIA search results workflow in Skyline. But in both cases we get error that the spectra cannot be found. We have both interact-xx.pep.xml and the mzML files in the same directory, so I am not sure what is going on.
Apparently there has been previously similar issue with this when .mgf file were used.

Thanks for any advice.


 Error importing MSFragger Library to Skyline using GPF.pptx 
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Raw files moved
(4 responses) ingo.wohlgemuth 2019-09-28 01:12


thanks for the great software.
I analyzed a larger DDA dataset with MS1filtering. Unfortunately, the external disk with the RAW files crashed. I still have the skyline file on my computer and a backup of the maxquant search plus raw files of the corresponding analysis. However, Skyline expects the original location of the raw files on the broken external memory.

  1. Is it possible to reimport the data in the skyline files from a new folder?
    or 2. is there another way to reconnect skyline analysis and raw data (plus maxquant search)?

Thanks in advance!


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Right click to 'save template library item' to iRT database
(2 responses) sstewart 2023-08-30 12:43

Would like the ability to save item from Targets list to iRT database. This would allow user to work in a cycle of exploration / update targets library.

Maybe right click?

view request
Library details missing from spectra library explorer
(4 responses) Liyan Chen 2023-08-29 02:38

Hi Skyline developers,

I have been using Proteome Discoverer (Sequest node) with Skyline for building spectra libraries. This combination has worked fine for several projects this year till now. The PD result file, generated .blib and Skyline document seem to give a reasonable number of proteins and peptides, but library details are somehow not showing (screencap attached). Nothing in our workflow has changed between the previous projects and this current one except for the number of files in library run dataset: 80 files compared to 20-30 from before. I have also tried re-do of PD search and importing of library under different filename and get the same problem.

Thanks for your help.

 library details error.PNG 
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Error loading isotopic peaks from transition list
(3 responses) laura corveleyn 2023-09-11 06:27


I encountered an error when trying to import a transition list (which I exported from another Skyline project); it is not extracting the isotopic peaks. I tried looking into the transition settings, but I couldn't find any restrictions there for the isotopic peaks. I added the Skyline file and transition list in the attachment. Could you help me with this?

Thank you!


 230911_Skyline_full_template_snapshot_transition_list_refined.csv  230804 full skyline template snapshot  Error transition list.PNG 
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Peak Area or Intensity for both Heavy and Light Peptides
(7 responses) colin mcdowell 2023-09-11 10:51

Hello Skyline Team,

I have set up my skyline document to look for both heavy and light peptides. Is there a way, once i've imported my MS data, to have skyline show me the intensities and/or peak areas of the precursors and fragments for both the heavy and light peptides? Currently it only shows me the light peptides. Thanks!

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Transferring Uploaded Mass Spec Data Files Between Skyline Documents
(2 responses) colin mcdowell 2023-09-05 13:33

I no longer have access to the raw data files which were imported into a skyline document as results to establish retention times. Is there a way to transfer these imported results from one skyline document to a new one without having access to the original files?

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Prosit Spectral Library Error
(1 response) cbm11 2023-09-11 07:04

Good afternoon,
I have been trying to implement a Prosit Spectral Library in Skyline which I have done lots before with no trouble, but this morning a new error appeared. Any ideas what this could be caused by?

Best wishes,

view request
Making a Spectral Library from an .ssl file
(3 responses) david lewis2 2023-08-22 09:27

Hello Skyline Team,

I am looking to put together a spectral library using an .ssl file, and I was wondering if it is possible to create a spectral library using this file with MS2 scans that did not match from a database search? For example, instead of including the peptide sequence, when creating the spectral library, we can use different names, like this:

file scan charge sequence
demo.ms2 8 3 PrecursorID1
demo.ms2 1806 2 LAESITIEQGK
demo.ms2 2572 2 PrecursorID2
demo.ms2 3088 2 TTAGAVEATSEITEGK
demo.ms2 3266 2 DC[+57.0]EEVGADSNEGGEEEGEEC[+57.0]
demo.ms2 20919 3 VHINIVVIGHVDSGK
../elsewhere/spec.mzXML 00497 2 LKEPAQNTADNAK
../elsewhere/spec.mzXML 00680 2 ALEGPGPGEDAAHSENNPPR
../elsewhere/spec.mzXML 00965 2 FFSHEAEQK
../elsewhere/spec.mzXML 01114 2 C[+57.0]GPSQPL

In addition, can we only add the retention time column for this file and not add the score columns?

Thank you.


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Use one compound to calibrate different molecules in mixture
NewUser 2023-09-07 11:29

I am trying to generate a quantification method on skyline-daily where I have a set of calibration standards (of compound A) and then use this calibration curve to quantify two other compounds (A, B and C) in my actual samples. Is that possible using skyline? I tried using surrogate IS but that doesn't give me the curve correctly.

Note: Compounds B and C are not present in the calibration standards.

view request
TMT-labeled DIA
(1 response) beikih2 2023-09-07 10:19

Dear Skyline Support Team,

We are trying to use Skyline to process our TMT-18plex-7-tags DIA MS proteomics dataset. Could you please let us know if there is a good set up for this type of data? Also, how can I demultiplex this data?

Thanks in advance.


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DIA analysis on SKyline: some peptide does not appear in raw extracted chromatograms.
(1 response) bioc1213 2023-09-07 02:41

Hi, After successful creating and importing the library and analyzing the raw file (DIA data) on Skyline, I can see most of the extracted chromatograms of the peptides which are present in library. I noticed that for some peptides the raw chromatograms do not appear even they are present in the library. Please see the screenshot. Can you please guide me how to resolve this issue.

 Screenshot 2023-09-07 103132_SkylineSupport.png 
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"Apply Peak To All" to all molecules imported in Skyline
samartinez3 2023-09-05 08:28
Hello, I use Skyline for analysis of small molecules for metabolomics.

I have 100+ small molecules that I import via transition list, each with only one precursor I am integrating for quantification (MS1 peak area). When I inject my samples I use a separate external standard that consists of a neat solution containing all of the molecules being analyzed. When I import the results the external standard is imported along with the "unknown" samples and is used for confirmation of correct retention time integration for each molecule.

Currently I find myself going through the molecule list and using the "Apply Peak to All" function (using the standard result as the peak that is being applied to the rest of the unknown results). I'm wondering if there is a way to globally (across all molecules) apply the peak integration from one result file. Example: Apply the external standard peak integration values for all molecules to all the other results.

Thank you for your help!

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Pepsin cleavage sites
(4 responses) stefan ehling 2019-05-31 06:56

As far as I know, pepsin cleaves preferentially after phenylalanine, tyrosine, and tryptophan. Why are cleavage sites set to FL in Skyline? Are there any restrictions such as a proline preceding or following the cleavage site?

I am trying to detect peptide F.PGPIHNSL.P predicted by Skyline and I am failing at it.

Thank you.

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Skyline Problem
(5 responses) jean-christophe prost 2023-09-04 00:47

Dear all,

We have a problem with skyline . I tried different thinks, but with always the same result.
We had the newest version installed on both. Then the working station did not recognized the Soft anymore. I tried to install an older version. When we click on the skyline shortcut a dowload tries to be accomplished and then comes a pop-up window telling the download is not possible.
THe skyline files are not recognized anymore. THis started 3 weeks ago.
I desintalled and installed again the soft many times but nothing changed. I really do not understand what I can do more.
And this is only happening on two specific working stations

I put you everything the in the attachment bellow.^
Thank you for your support.

Kind regards, JC

view request
Skyline-daily asks reinstallation almost every 24 hours
(1 response) Yao Chen 2023-08-30 15:15

Dear Skyline Team,

My colleague and I have been facing a recurring issue with Skyline daily. It prompts us for reinstallation approximately every 24 hours. Furthermore, previously processed .sky files cannot be accessed unless we go through the reinstallation process.

Once reinstalled, the program operates as expected. Yet, it appears as if Skyline was never installed before — all our reporting methods, views, and setups disappear. While we can recover most settings by opening previously processed .sky files, the reporting methods are irretrievable.

Our IT department has confirmed that they don't have any mechanism in place that would auto-delete software.

Could you please advise on how we might resolve this issue? Your assistance would be greatly appreciated.

Best regards,


view request
Import Peak Integration Boundaries from command line
(3 responses) sstewart 2023-08-30 12:37

Do same as "Import > Peak Boundaries" from GUI.

view request
Absolute quantification with oxidized peptides
(6 responses) clemence balty 2023-08-25 04:39

I'm doing absolute quantification using the SureQuant method. I have a peptide with a methionine. I wonder if it would be possible to calculate the quantity of this peptide based on the sum of the reduced peptide and oxidized peptide. I mean set a calibration curve with the sum of both area in fonction of the quantity. Is there a way to do this in skyline or should I do it manually ?

view request
Issue with utilizing MSInspector
(2 responses) edoud 2023-08-25 12:28

As I develop targeted assays to share I would like to implement the CPTAC recommendations for assays using the MSinspector external tool - I've downloaded it but am getting an number of errors. I suspect many of the issues are related to it being coded in python 2.7 which is end of life. (Can't get pip installed, can't get pandas installed etc etc). All errors in the attached file. I've been messing around trying to install things in the cmd line and there are just too many issues getting versions for python 2.7.

Do you have any recs or could you give me the contact info for Yin Lu to see if they are planning on updating this tool at all? Thanks!

view request
Exporting data
(1 response) herath lakmini senavirathna 2023-08-28 11:14

Hi Skyline Team,

I have an issue with exporting the Excel file for peptide quantification from the Skyline file for a large number of data set (the number of results/raw files imported are close to 100). The generated Excel file cannot be opened or all the data are not exported as the number of rows that a Excel file can have passed the maximum limit. So, could please advise me on how to export all data? I am using iRT calibration as well.


view request
(1 response) ssedigh2 2023-08-26 10:37

Dear Skyline team ,

I am trying to search for the modifications I am interested in in my DIA data , and was wondering do you have a tutorial on what is the best way to peak the transitions and fragments ?


view request
How to group peptide sequences by peak quality icons
(9 responses) michel.petrovic 2012-09-12 05:48
Dear Brendan,

If I take for instance the tutorial 'Skyline Targeted Method Refinement' and go to the page 2, I can see the color explanation:

There are peak quality icons, and they indicate:
- Green – All transitions contribute a co‐eluting peak to the peak Skyline picked as the best.
- Yellow – At least half of the transitions contribute a co‐eluting peak.
- Red – Less than half of the transitions contribute a co‐eluting peak.

Our users would like to know if there is a way to view all information in a way that Skyline groups all peaks having a red icon together?
This will really simplify their manual control.

We will have much more protein sequences compared to your examples and this feature will be a must.

Best Regards,
view request
SILAC channels not showing up during DIA analysis
(2 responses) dghandour 2023-08-24 19:27

Hello! I'm relatively new to Skyline and this is my first time doing a DIA analysis, so I would appreciate some guidance. I'm running on Windows, Skyline daily.

I'm currently working on a proteomics DIA analysis. My process began with generating a few DDA runs on our Bruker timsTOF and running them through FragPipe for building a spectral library, and then generating a set of DIA runs (also on our Bruker timsTOF). The samples have a mix of light, medium, and heavy SILAC labeled proteins (R and K, R[6] and K[6], R[10] and K[8]).

After that, I followed the Skyline guide for spectral library generation and DIA analysis almost exactly (skipping the isolation window creation part).
Here's what I was following:

Although SILAC isn't explicitly discussed in the manual, I account for the SILAC channels during my analysis, in the Modifications section. When the data comes out, it shows two channels, a "light" and a "heavy", where the heavy channel probably accounts for both the medium and the heavy SILAC labels. (See "pic 1")

When I go to peptide settings and designate the correct labels to "medium" and "heavy" SILAC (see "pic 2"), then I go back to the chromatograms, nothing shows up for the medium or heavy SILAC channels. It says that there's no chromatogram available, for every peptide. (See "pic 3")

When I do all these SILAC settings for a DDA analysis, it usually works great. Is there something I'm doing wrong on Skyline, or do you think it's a problem with the original pep.xml files I used for making the spectral library?

 pic 1.png  pic 2.png  pic 3.png 
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Follow selected replicate/molecule in replicate/molecule comparison views
(4 responses) meowcat 2023-08-24 08:36

For batches with a large number of replicates and/or targets, the "peak areas" become less useful, as it becomes hard to visually evaluate the differences and the replicate / target labels are collapsed.

It is easy to zoom in on a subset of samples with ctrl-mouse wheel on the "replicate axis". It is also easy to quickly go through many samples with Ctrl-left and ctrl-right on the keyboard. However, when leaving the set of replicates onto which we are zoomed, the selection simply "leaves the window", i.e. we continue navigating through EICs but we don't see them in "peak areas". Also, selecting a replicate's EIC doesn't bring that replicate into the "zoom range" of the "replicate comparison" window.

It would be great if the range of visible samples would follow the selected replicate in a "moving window" kind of way. Also, if there was a scrollbar to move the window of selected replicates without having to zoom out and back in.

This is very hard to describe. Maybe I can make a screencapture to show what I mean, unless you already understand...

Note: the same applies to all of these replicate/molecule comparison views, like retention time, mass errors... 
view request
Include fragment ion annotation when "Copy Data" from Library Match tab
(2 responses) jonasbecker 2019-05-08 00:40

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
wiff to mzml import
(3 responses) Lisa Panzenboeck 2023-08-18 07:11

Dear Skyline-Team,
I have tried to convert the .wiff and .wiff2 files generated with the zenoTOF 7600 from SCIEX to mzml files using peak picking in ms convert. After the conversion it is possible to open the file in SeeMS but the import to skyline crashes with the following error message:

At 4:06 PM:
Failed importing results file 'herewasmyfilenameandpath'.
[pwiz::CLI::msdata::ReaderList::read] Invalid cvParam accession "1003293"

I'm using Skyline 64 bit 22.2.0527 with Windows 10 Enterprise.


view request
Thermo Altis method
(9 responses) avizrosenberg 2019-04-03 07:13

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?

view request
Request about Heavy/Light Peptides
(2 responses) philip remes 2023-08-22 17:47

We have a question similar to this one:

The situation is we have 800 peptides spiked into a sample. We wanted to find the RT's of the heavy/light peptide pairs and have a Skyline document with heavy/light pairs defined for all of the heavy peptides we could find.

Trial 1
In the initial peptide search, we included heavy K/R as a dynamic modification and imported this to Skyline. Using Accept Peptides, one could reduce to the list of peptides of interest, but not all of them would have both heavy and light in the document, because some peptides would just have an ID for the light, some just for the heavy.

Trial 2
We did a peptide search with static K/R modifications for just the heavies, and imported these to Skyline. Use Accept Peptides to make sure they are the ones we want. Now, how to add the lights? What we ended up doing was exporting the heavy transition list, appending the light transitions, and reimporting. We could make an external tool to enable this, but perhaps there is a better way already, or perhaps it would be a feature useful enough for the Skyline team to implement?

all_trans = []
for heavy in heavy_trans:
    light = deepcopy(heavy)
    seq = light['peptide_modified_sequence']
    light['precursor_mz'] = get_subtracted_mz(light['precursor_mz'], light['precursor_charge'], seq)
    if 'y' in light['fragment_ion']:
        light['product_mz'] = get_subtracted_mz(light['product_mz'], light['product_charge'], seq)


view request
Group Comparisons
(3 responses) ssedigh2 2023-08-22 05:25

Dear Skyline team ,

I am trying to do group comparison , and for some peptides even though it shows in the peak areas-replicate comparison that it is lower in my ICM condition than NF condition , when I do the group comparison , it shows that it has a positive fold change ( screen shot attached ) I was wondering can you help me figure out what I am missing here ?


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Retention time shift and integration
(7 responses) nbekhti 2023-08-15 09:31

Hello Skyline team,

I work on small molecules on Skyline and I have a retention time shift for some molecules. How can I "automatically" reintegrate these peaks with the observed RT instead of doing it manually, chromatogram by chromatogram as I'm having 200+ samples.

Can anyone help me with this please? thanks


view request
Unable to use MSFragger for spectral library
(5 responses) s jager 2023-07-31 06:52


I have been using MSFragger to analyze my timstof data, and i have been trying to upload the ‘interact.pep.xml’ files as spectral library. I put the '_uncalibrated.mzML' in the parent folder as had been described previously. However, i get the following error message:

"interact.pep.xml: To import an MSFragger search of timsTOF data (with ion_mobility attribute), the corresponding *_uncalibrated.mgf or *_calibrated.mgf file is required"

As of version 3.6, MSFragger does not output these files, and does the mxML counterparts instead (which I have). Can I somehow solve this and import the spectral library.

Or is there a workaround, for example a way i can import a list of peptide sequences with modifications, and that skyline processes this similarly to when I upload the FASTA?

thanks in advance!

view request
Skyline for small molecules, full scan-MS1, DDA-MS2
(5 responses) stepan koudelka 2023-08-11 01:05

Dear Skyline support team.
I am using high-resolution Orbitrap Tribrid to analyse small molecules. For an individual chemical standard, a precursor is detected by full scan MS1 as well as fragmented via DDA-MS2 using isotope label free acquisitions.
Precursors and the five most intense fragments of a corresponding precursor are summarized to create a Transition List feeding Skyline ( via Molecule Interface. For the graphs, I can monitor only precursor(s) Retention times, Peak Area, and Mass Errors after setting Full-Scan bookmark according to the used MS1/MS2 acquisition parameters. Are there any visualization settings and/or proper input format for the Transition List and the Transition Settings… to monitor these up to 5 fragments together with the corresponding precursor in a particular graph simultaneously (for the loaded results)?
Thank you.

view request
QuaSAR execution error
(5 responses) Alvaro Sanchez-Bernabeu 2023-08-16 07:55
Hey there,

I was trying to run QuaSar and I have got the following error messages:

At first, I got this message at the Immediate Window:

"C:\Users\Alvaro Sanchez\Documents\R\R-3.0.1\bin\R.exe" -f "C:\Users\Alvaro Sanchez\AppData\Local\Apps\2.0\4VDK9ATE.R6G\CXV3K6ZV.VLB\skyl..tion_2e441fc3bf6adc7f_0017.0000_f621a40fd78fad56\Tools\QuaSAR-1_32\QuaSAR-Skyline.R" --slave --no-save --args "C:\Users\Alvaro Sanchez\AppData\Local\Apps\2.0\4VDK9ATE.R6G\CXV3K6ZV.VLB\skyl..tion_2e441fc3bf6adc7f_0017.0000_f621a40fd78fad56\Tools\QuaSAR-1_32\QuaSAR.R" "C:\Users\Alvaro Sanchez\AppData\Local\Apps\2.0\4VDK9ATE.R6G\CXV3K6ZV.VLB\skyl..tion_2e441fc3bf6adc7f_0017.0000_f621a40fd78fad56\Tools\QuaSAR-1_32\common.R" "C:\Users\Alvaro Sanchez\AppData\Local\Temp\QuaSAR_QuaSAR_Input.csv" NULL Trial1 "light Area" 1 "heavy Area" fmol/ul 1 1 -1 1 0 0 0 150 150 1 0,2 0 NULL Trial1 0
El sistema no puede encontrar la ruta especificada.

Then, when copy and pasting the path to the file (C:\Users\Alvaro Sanchez\AppData\Local\Temp\QuaSAR_QuaSAR_Input.csv), it would open in Excel and then when I click again at the QuaSAR execution button while keeping it open, I get the following message:

Error exporting the report, tool execution canceled.
OK More Info
System.IO.IOException: Error exporting the report, tool execution canceled.
   en pwiz.Skyline.Model.Tools.ToolMacros.GetReportTempPath(ToolMacroInfo toolMacroInfo) en C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Tools\ToolMacros.cs:línea 266
   en pwiz.Skyline.Model.Tools.ToolMacros.ReplaceMacrosHelper(SrmDocument doc, IToolMacroProvider toolMacroProvider, ToolDescription tool, IProgressMonitor progressMonitor, String replacein, Macro[] macros) en C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Tools\ToolMacros.cs:línea 206
   en pwiz.Skyline.ToolsUI.ToolDescriptionRunUI.RunExecutableBackground(ToolDescription toolDesc, SrmDocument document, IToolMacroProvider toolMacroProvider, TextWriter textWriter, IProgressMonitor progressMonitor, Control parent) en C:\proj\skyline_23_1\pwiz_tools\Skyline\ToolsUI\ToolDescriptionRunUI.cs:línea 328
   en pwiz.Skyline.ToolsUI.ToolDescriptionRunUI.<>c__DisplayClass4_0.<RunExecutable>b__0() en C:\proj\skyline_23_1\pwiz_tools\Skyline\ToolsUI\ToolDescriptionRunUI.cs:línea 164

Excuse me for the spanish lines in the error messages... Any idea what might be going wrong?

Thanks a lot!

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SureQuant Chromatographic peak integration question?
(4 responses) 15702446287 2023-08-16 21:02

Hello skyline team,

We encountered two issues when using skyline to process surequant data.

Q1:The peak areas can be seen from the skyline,but no chromatographic peak?

Q2:The chromatographic peak of the heavy peptide cannot be triggered?

Is there a solution for this?



 surequant peak area question.pptx 
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Issues with Skyline Chromatography (Metabolomics)
(4 responses) victoria roberts 2023-08-16 10:03

I am having issues with peaks showing up on skyline, it shows that they are there in other softwares like qual browser and freestyle but whenever I try to look at it through skyline it looks like there wasn't really anything showing up although there was other random peaks (so it wasn't an upload issue). I think it may have to do with mass tolerance but I tried changing the transition settings to see if I the peaks would show up but they haven't, even when I went to a match tolerance of 0.5 m/z, although they definitely show up on other softwares within 0.005 m/z. I am not sure if there is something hard-coded that is preventing me from seeing the peaks or if I just don't know how to use the settings correctly, but I would appreciate any help I can get.

view request
System.IO.IOException: ERROR: No spectra were found for the new library.
(1 response) malikshagufa93 2023-08-15 23:15

I am trying to build spectral library using DDA mode using peptide atlas mouse plasma peptide atlas raw files.
3 data sets are giving the same error despite of using post translational modification information provided in the Article journal.
Below are the 3 data sets:-

I have tried to build library using each raw file individually and still the same error pops up everytime. Below is the error

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

Command-line: C:\Windows\System32\BlibBuild -s -A -H -o -c 0.95 -i spectral_library_PXD030951_REVIEW_UNREVIEW_1 -S "C:\Users\USER\AppData\Local\Temp\tmp7CAB.tmp" "D:\PXD030951\spectral_library_PXD030951(REVIEW+UNREVIEW)1.redundant.blib"
Working directory: D:\PXD030951
위치: pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) 파일 C:\proj\skyline_22_2\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:줄 161
위치: pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) 파일 C:\proj\skyline_22_2\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:줄 412
위치: pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) 파일 C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:줄 161

 화면 캡처 2023-08-16 150750.png 
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Group Comparisons
(5 responses) ssedigh2 2023-08-14 11:32

Hi ,

I am trying to do a group comparison for a peptides using my DIA data between a healthy and diseased group , for some reason the p-value for every peptide is showing 1, How can I trouble shoot that ?
also is there a way to export the peak areas ? the document grid doesn't have it .

Thank you

view request
surequant MS/MS peak extraction fails
(2 responses) 15702446287 2023-08-16 02:29

Hello skyline team,
We use the Exploris systems to acquire tPx data using the SureQuant method. We can see the target peptide (light and heavy) using xcalibur software, skyline also can extract the heavy peptide , but the light peptide detection fails. for more details please see the attachment.

Is this a known issue and is there a solution for this?

Many thanks!

view request
raw file import error : Expected sorted data
(3 responses) vbuser 2023-08-14 05:26

Hello guys,

we are pretty new in using Skyline. We have problems importing .raw files which are working completely fine in the Freestyle software from Thermo Fisher.
When importing the files into Skyline we got the error that the data could not be imported due to the error: "Expected sorted data".
The other .raw files from the same experiments are can be imported completely fine.
We added one of the files to this message.

Maybe you can help us, we would be really grateful.

Thank you


view request
Skyline Uninstalled Overnight
(5 responses) yadav 2023-08-11 11:51

Hi, not a major issue but a bit odd. I have been using Skyline on my computer for quite some time and when I went to open it the other morning I got a notification that I needed to install the software with the attached message. I could also not find the Skyline application on my computer. I had previously been using version 22.2. Another lab member had the same issue.


 image (5).png 
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raw file import error : Expected sorted data
vbuser 2023-08-14 05:16
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Peak Areas for replicate comparison
(3 responses) ssedigh2 2023-08-10 10:57

Hi , I was wondering is there a way to move the columns on the screen shot I send you , I want NF to come first followed by ICM and NICM ? also just to double check , a peak area for a specific peptide is an indicator of the amount of that peptide detected through out different samples right ?

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M+4 precursor selection on transition list
(1 response) Dzimow 2023-08-11 07:05

Thanks for your support.

I have a question related to the transition settings. I have a peptide, 9 residues, in which I would like to see the M+4 precursor.
This does not appear from the list when I expand the precursor list of this peptide, but it does for longer peptides.
Is there a way to select, also for shorter peptides, the precursor M+4?
It would be interesting because I need to check the incorporation of 13C into it and therefore I need this information.

Thank you

view request
parsing data to prm and dda mass filters
(6 responses) tara chouinard 2023-08-10 09:24

The prm/dda setting will not parse orbitrap data for separate peak integration for shared transitions despite the NCE being unique (22% v. 30%). Is there a way to parse data in skyline similar to mass filter parsed data for methods with both DDA and targeted experiments?

 prm mode in skyline 493.emf 
view request
Display area of isotope peaks in document grid
(4 responses) karin preindl 2023-08-09 05:58

Dear Skyline support team,
Is there a possibility to display the isotope peak areas (number defined in the transition settings) in the document grid or do i have to create a seperate molecule for each isotope peak?
Thank you and best regards,

view request
Peaks integration with different boundaries between light and heavy
(5 responses) Anne Incamps 2013-11-06 07:09
Hi Brendan

I don't manage to integrate manually light and corresponding heavy peptides with different boundaries.
If I clic on the light peptide and integrate the right peak, it automatically shift the same boundaries for the heavy one. I cannot set different boundaries for light and heavy. How can I sort this out ?

Thanks for your answer,

view request
Importing MSAmanda mzid.gz file "Error getting score type for this file"
(3 responses) nicole pontarin 2023-07-17 18:55

Good morning Skyline team,

And thanks in advance for considering this support request.

I am using MS Amanda standalone version to expand my search to 5 amino acid-long peptides. I think that Skyline uses the default MS Amanda settings, searching for 6-30 aa-long peptides. I would like to receive your support on:

  • either how to modify the min and max peptide length in the Skyline "Import peptide search" from .raw data;
  • or how to resolve the "Error getting score type for this file" when I try to import files generated by MS Amanda. I tried both Skyline and Skyline daily in their up to date versions.

With thanks and regards,

view request
Allow Custom Report to Export Chromatogram Time in Units of Indexed Migration Time
(6 responses) sstewart 2023-07-21 10:09

Right now, when building a custom report, I can only select 'Raw Time' under Chromotagrams. Instead, I'd like the option to select 'Indexed Migration Time'.

view request
Updating PanoramaWed to Support Version 22.2?
(2 responses) raweed 2023-08-03 11:24


I upgraded my Skyline Daily software to 22.2 and following that, my AutoQC loader started erroring out and gave me this error [03 Aug 2023 10:55:13,669 WARN : The version of this Skyline document is 22.2. This is newer than the highest supported version 22.13] whenever I tried to upload data to Panorama Web. Is there any possibility to update Panorama Web to be able to support newer versions of Skyline daily? If not, where can I find older versions that I could install and are supported by Panorama Web?

Thank you!

view request
Problem with importing msfragger output into skyline
(2 responses) jdemeter 2023-08-03 10:31


I am trying to import peptide search results from msfragger into skyline (timstof pro, dda). This process used to work with the pep.xml, mgf and .d files, but now it stopped working and I get an error message (see attachment) of not finding the appropriate source files. When I use mzML with the pep.xml files, the library is generated fine, but the peaks are not imported correctly (see second attachment). I tried this using the latest skyline and skyline-daily with the same result.

Any help would be appreciated.

Thank you,

 error_mgf.PNG  peaksPNG.PNG 
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MRM windows are smaller than in the measurements
(4 responses) schweitzer theresa 2023-07-28 04:39

we are currently working with a 7500 QTrap and are measuring scheduled MRM data of cell lysates. We also use spiked in SIS peptides of our peptides of interest and saw that heavy and light peptides show some retention time shifts. Everything fine so far, we increased our MRM window to 60s instead of 30s and can see the data being collected in Sciex OS (attached file: Troubleshooting Skyline.jpg) at 9.1 to 11.1 min, however when importing the data to Skyline we only see the signal from 9.6 min to 10.6 min which would fit our old 30s windows (Troubleshooting Skyline_RT.jpg).

We already checked every skyline setting, but nothing seemed to fix the problem and now we are running out of ideas, so if you have some idea how we could get all the collected data shown in skyline that would be awesome.

Thanks in advance.

Kind regards,

 GTPasen Skyline_60s  Troubleshooting Skyline.JPG  Troubleshooting Skyline_RT.JPG 
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Problems with relative quantification
(1 response) sarita 2023-08-02 05:42

Hi to everyone, I have to calculate the relative quantification of three proteins in five patients before and after a treatment. I have the peptide modified sequence and the PRM raw data.
I don't have any internal standard heavy peptides. I know that each peptide was added at the same amount in each sample. I have to perform the relative quantification, for each protein of interest, comparing the relative amount between the 2 conditions (pre- and post- treatment). How can I calculate the light/heavy ratios for each peptide in each sample? and how to obtain the adjusted p-value and foldchange of each comparison for the three proteins?

view request
Offset windows in DIA analysis
(3 responses) sweaver4 2023-07-31 07:44


I'm looking through some DIA bottom-up proteomics data acquired on a timsTOF where the isolation windows were 24 m/z, but offset (i.e. 400, 424, 448... 412, 436...). How should I setup the transition settings to reflect this? I can point the isolation scheme to the .d file which successfully pulls in the m/z values used for the windows. Should I set the Deconvolution to overlap in this window?

My goal is to look at the chromatograms and have the intensity traces for each fragment ion reflect only MS2 signal that was present in spectra where the precursor isolation windows were appropriate. (So if an example precursor ion was 430 m/z, fragments would show up in isolation windows 424-448 and 412-436, but not 400-424). Is this how the chromatogram extraction is automatically designed, or do I have to perform additional deconvolution before uploading my .d files?

Thanks very much for any help, and let me know if I can clarify anything! I looked through the tutorials and documentation and couldn't find any specific examples for this, but apologies if it is somewhere and I missed it.

Simon Weaver.

view request
Pressure output?
(11 responses) alejandro.cohen 2020-02-20 09:43

Hi Skyline people,

I work with an Agilent 1290 coupled to a Qtrap5500 running MRM metabolomics which I analyze using Skyline (.dam methods, .wiff datafiles). Analyst is recording the column pressure output signals, however viewing these using Analyst is cumbersome and time intensive when working with big sample batches. We have encountered erratic behaviour of our chromatography, accompanied by unusual pressure profiles for samples, which tend to correct themselves after a few runs.

As part of our QC, it would be great if skyline would be able to add (overlap) the pressure traces on each chromatogram. Is this possible? I know each vendor 'hides' the pressure traces in different output and channels... but the information is there :)

I know this request has been done in the past... here another kind 'reminder'



Alejandro Cohen, Ph.D.

Scientific Director
Biological Mass Spectrometry Core Facility. Room N-105
Life Sciences Research Institute

view request
Spectrum IDs
(5 responses) paul.derbyshire 2023-02-01 05:16

I would like to include the number of spectrum IDs per peptide when exporting a document report. For example, if I have 8 samples and each chromatogram has 1 ID then I would be looking for a value of 8.

Hope you can help.



view request
Spectral library corruption
(8 responses) jfoe 2023-07-05 06:03

Dear Skyline Team,

I have recently updated to (841287d47) and have just noticed that I have by now corrupted several of my spectral libraries which is a critical problem for me now.
I am doing a workflow where I create my spectral libraries manually in skyline. I typically have several files loaded and select the best quality peak only, such that it gets included in my spectral library.

When I create spectral libraries like this (Export -> Spectral Library) and then inspect it in skyline, I get "Failure loading spectrum. Library may be corrupted.".
What I found by now is that this seems to occur specifically when the peak for a precursor is not selected in the last results file.

I am attaching an example skyline file and the spectral library that I exported from it. Loading the spectral library with skyline then gives the error text as illustrated in the .png that I am also attaching.

I would be very grateful for your help with this.

 230705 skyline speclib  230705 skyline speclib issue.png 
view request
Skyline after upgrade 22_2 and update about AutoQC possibility to importe single injections from wiff file
(5 responses) jean-christophe prost 2023-07-17 03:27

Dar Skyline team,

I am sorry for that but we have a small problem. We observed big discrepancy between XEVO and Sciex measurements using skyline. And comparing the raw data processed with the their own software, the area obtain between TargetLynx and Sciex OS are really close.

In the attachement for exemple, I put in Skyline QTRAP and XEVO measurements of ERNDIM 1 and 2 controls used for our Acylcarnitine assay.
In skyline you have the feeling, we lost all sentibility with the sciex instrument, and when you compare the data in their process soft, you see, you are very close to each other.

I have the feeling, since we updated the new release of we have such problems. We are only able to install the unpluged version due to our firewall restrictions. When I go the mail from Brendan or Brian to install the new 22.2 release, I desinstall all the the skyline versions and install the new versions, and did not the "recovering from a broken Installation" workflow.

I really do not understand how it is possible to observe such area differences in skyline, which are not present at all in the respective processing softwares.

In advance thank you for your help.

Sorry I am coming back to you regarding questions I had in February abour AUtoQC (2023-02-22 01:41:18), since I never had any feedback from you. And I am no more able to sign in and ask again to these Email flow (QC trend report)
Vagisha told me it is not possible to implement in AutoQC single injections from wiff file. COuld you fix this issue? Since we have only Sciex instruments it would be really nice to be able to implement AutoQC without import always all data, and then exclude replicates you do not want.

In advance thank you for your help.
Kind regards,

 Skyline_After_Update_22_2.docx  029_Acylcarnitines_Template_Final.skyd 
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importing peptide search
(3 responses) ssedigh2 2023-07-25 09:20

Hi ,
when importing peptide search I keep having this error pop up , how do I fix it ?
"importing the FASTA did not create target proteins"
thank you,

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Dark Mode
(1 response) andy bennett 2023-07-24 18:50

This seems to have been brought up before, but I'm unsure if it was implemented.
It would be really nice to have a dark mode for Skyline.

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Why the iRT peptides not showing
(1 response) guo xue 2023-07-24 03:13

I have searched my Hela +iRT data from timsTOF using PEAKS. then I am able to build library with iRT conversion. however the iRT peptides were not showing. could you please help me to get the iRT list? Thank you.

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modification of precursor m/z order in targets and results panels
(6 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,

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Crash when importing raw files
(24 responses) erin weisenhorn 2022-05-25 14:18

I am running Skyline Daily with version on Windows 10 and am trying to import MRM raw files from a Xevo G2-XS. When I import the files Skyline crashes and gives no error. I have replicated this on multiple computers. Under transition settings I have the mass analyzer set to 'TOF' and not centroided as another user reported causes error. I would really appreciate help with this and would be happy to provide the raw files.

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Import Peak Integration Boundaries in time units of indexed migration time instead of minutes
sstewart 2023-07-21 10:22

Two ideas:

  1. Checkbox on import dialog from "Import > Peak Integrations"
  2. Or, more generally, two report new variables "Start Time (in indexed migration time)" and "End Time (in indexed migration time)" then import code uses minutes / indexed time depending on which columns present.
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peptide selection for PRM
(6 responses) ssedigh2 2023-07-21 06:33

on my first try to chose peptides for a PRM study, a couple of peptides came up that they are not coming up on my other tries. I am sure I am using the same peptide setting and filters . all the paptides that are not showing were in the first 50 AA of the protein sequence , can you help me with trouble shooting this ?

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Problems with transition list
(16 responses) fdalessa 2023-07-12 08:54

we are measuring porphyrins via HPLC-MS in our lab. We've been using an eatablished protocol for a few months now. However, we had to adapt the HPLC-MS method in order to measure different kinds of porphyrins within one run. Scince that adaption we can't evaluate our Chromatograms with our established transition list as it falsly detects our MS°2 measurements as MS^3. Although we can clearly see our added target molecules and their fragments in the chromatogramms, skyline doesn't detect them at all.

Do you have any idea to solve our problem?

Thank you in advance!!

 Problem 1.pdf  Problem 2.pdf  skyline transition list porphy_01.csv  chromatogramms.pdf 
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Quantification of multiple small molecules
(1 response) matthew smith 2023-07-19 01:17


Skyline has been a great tool for my lab to generate peak areas normalised to IS or surrogate standard, however I was excited by the ability to generate multiple calibration curves and thus quantification using the 'Concentration Multiplier' as outlined at

I am a little confused as to how to complete the workflow, since the Concentration Multiplier only seems visible in peptide mode (not molecules).

Perhaps I should load my transition lists as peptides, however this seems tricky due to the requiements of specific information which does not exist for small molecules.

I am using skyline version

Any information would be appreciated.

Best wishes,

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.pdresult fail to build skyline library
(1 response) guo xue 2023-07-19 02:31

I ran Hela cell lysate with iRT spiked in and searched the data using Proteome Discoverer Sequenst which has 47k proteins. Then I used the .pdresult file to build library but it gave the error: no new peptide will be added to the database; one run was not converted due to low correlation. could you please help to understand what is the reason? thank you very much.

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Instrument index not available for requested device Parameter name: instrumentIndex
(4 responses) malikshagufa93 2023-07-16 17:20


I am trying to build spectral library using peptide atlas raw files dataset but i am facing issues multiple issues with 4 datasets particularly, I have attached the screenshots of the file.
I would like to send the raw files dataset for you to evaluate. Please help me to fix this issue.
Below are the 4 dataset:- PXD003772, PXD030951, PXD010664, PXD008969.

 PXD003772 EROOR.png  PXD030951.png  PXD010664 ERROR.png  PXD008969 ERROR.png 
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Importing libraries in Skyline with no Q-value
(2 responses) lars kristensen 2017-07-04 05:03
Is there a workaround to import libraries in Skyline from a Proteome Discoverer 2.1 Sequest HT search without q-values from Percolator (e.g. because the number of hits were too few for the Percolator node to calculate a q-value due to the use of a small database)?
I tried to set the cut off score to 0 but it still required the q-values.
I’m using the latest version of Skyline (64-bit)

My aim is to quantify PRTC peptides in a HeLa background. Performing a PD 2.1 serch using parameters to identify both HeLa and PRTC peptides will result i q-values allowing me to import the identified PRTC peptides/fragments. However, only a few of the PRTC peptides are identified this way.

I have tried to make two separate searches, one optimized for HeLa and one optimized for PRTC and then combine them by creating a multiconsensus report. I checked that the PRTC peptides were indeed in the multiconsensus report. When I used this PD result file to create a library in Skyline I could create a library in Skyline without an error presumably because there were q-values in the PD result file. However only one PRTC peptide matched the spectral library (presumably because the PRTC peptides were not associated with a q-value and the single match was a random event.

Thank you very much for any assistance
Kind regards
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Skyline for small molecules
(10 responses) zhong li 2023-07-13 12:05

Have a question for the small molecule quantitation (MRM data from Triple Q instrument) : I need to quantify Compound A (with Internal Standard B), but there is no standard Compound A. I want to use the standard/calibration curve from Compound C’s signals (with Internal Standard B) to quantify Compound A. Is it possible in Skyline? Thanks.



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MPP Report doesn't work after upgrade to 22.2
(6 responses) denina simmons 2023-07-08 17:55

Hi there,

I am processing my first set of data since I upgraded to 22.2.

I use the MPPreport external tool to export my DDA results, and the tool no longer works. I have attached a screenshot of the error string from the "immediate window".

Can this be repaired? I am trying to find a different way to export my results, and I tried to export report from the file menu, and it sort of works, only my protein groups are not rolled up together, and I am not sure if there is a way to fix that? Or just to fix the MPP report external tool?



 MPPReport tool error.png 
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Issue running encyclopedia
(1 response) joel steele28028 2023-07-14 10:07
OK More Info
System.IO.IOException: Error occurred running process.
[03:03:40] Missing .DIA file:
[03:03:40] Encountered Fatal Error!
[03:03:40] Can't read file C:\Users\jste0061\AppData\Local\Apps\2.0\A27R8HNY.TN2\HJZCM6HZ.ERX\skyl..tion_2e441fc3bf6adc7f_0017.0000_f621a40fd78fad56

Command-line: C:\Users\jste0061\AppData\Local\Apps\2.0\A27R8HNY.TN2\HJZCM6HZ.ERX\skyl..tion_2e441fc3bf6adc7f_0017.0000_f621a40fd78fad56\Tools\openlogic-openjdk-jre-8u342-b07-windows-64\bin\java.exe -Xmx12288M -jar "C:\Users\jste0061\AppData\Local\Apps\2.0\A27R8HNY.TN2\HJZCM6HZ.ERX\skyl..tion_2e441fc3bf6adc7f_0017.0000_f621a40fd78fad56\Tools\encyclopedia-2.12.30-executable.jar" -Duser.language=en-US -Duser.region=US\Windows\Temp -v2scoring "True" -i "" -libexport -o "C:\Data_Analyses\RS Human SwissProt Jun2020-z3_nce33.elib" -f "C:\Data_Analyses\RS Human SwissProt Jun2020.fasta" -l "C:\Data_Analyses\RS Human SwissProt Jun2020-z3_nce33-prosit.dlib"
Working directory:
   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 180
   at pwiz.Skyline.Model.Lib.EncyclopeDiaHelpers.GenerateLibrary(String encyclopeDiaLibInputFilepath, String encyclopeDiaElibOutputFilepath, String fastaFilepath, IEnumerable`1 diaDataFiles, IProgressMonitor progressMonitor, IProgressStatus& status, EncyclopeDiaConfig config, Boolean quantLibrary) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Lib\EncyclopeDiaHelpers.cs:line 629
   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 633
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Feature Request - Remove Replicate/Result from Chromatogram or Replicate Right-click menu
(2 responses) Will Thompson 2023-07-13 06:56

Hi Brendan and Team,

I wanted to ask for a simple feature to make managing files which are missed injections a little easier/faster. Please consider adding the ability to remove a replicate/result file completely from the document (similar to edit/manage results) in the right-click menu. The workflow would be if you observe a run to have no data of interest, you can right-click and then instead of only "remove peak", you could "remove replicate" (or "remove result").




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Skyline dropout with Astral data when using wrong window scheme in DIA
(4 responses) telgierari 2023-07-13 10:41

when extracting spiked in Pepcal peptides we noticed dropout in the signal on some of the peptides, this was effecting primarily methods with small DIA widows, the Skyline document was wrongly set to " SWATH(100VW)", once the transition setting is changed to "all ions" peaks looked excellent.

 After correcting transition setting.PNG  Before correcting transition setting.PNG 
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Skyline minimized while changing from 2 screens to mirrored does not restore
Mark Belanger 2023-07-13 10:56

Hi y'all! Brian Searle noted this bug while we were in between presentations for the UW Skyline course. Steps to repro:

  1. Start with a mirrored (to a projector, for instance) Windows session
  2. Open a PPTX, project with Slide Show settings of Automatic, Use Presenter View
  3. Open Skyline, use it, minimize it
  4. Close Powerpoint
  5. Attempt to restore Skyline.
    Effect: Nothing happens, Skyline does not restore.

Thanks for your consideration!

  • Mark
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Surrogate External Calibration
(2 responses) jrenders 2023-07-12 10:44

Hi there,
I have a unique challenge I was hoping Skyline would be able to help me solve. I have a compound I would like to quantitate but standards do not exist this compound. I have an isotopologue standard of the compound and have a calibration curve using that isotopologue. However, I am unable to find a way to quantitate a compound against a calibration curve if they do not share the same precursor mass. Is this possible? For context: I have standards for the light variant of the compound (no heavy atoms) and I have a standard for the triply deuterated version, however, in my unknown samples I will be observing a version of the compound that has 6 heavy carbons despite not having a standard for this 13C6 variant. Thanks for any insight.

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Error in EncyclopeDIA search
(1 response) rj8 2023-07-12 15:20
For some reason, I bombed in the prosit library bit. Here's the info:

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 608
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IC-HRMS data for metabolomics
(5 responses) samartinez3 2023-07-11 13:11

Hello, I recently heard about Skyline after ASMS 23 (everyone was using it!) and I have a few questions! I'm fairly new to the mass spec community and currently use compound discoverer/tracefinder for processing small molecule data for the metabolomics core I work in. These softwares are a bit more new-user friendly (AKA faster for me to be trained on/put to work on my own) but now that I know a little more about mass spec, I'm trying to find more efficient ways to process my data as we have over 250 targeted metabolites we screen per sample and have 100+ samples weekly. I'm wondering if I'm using Skyline correctly, as I find myself still having to manually go through each molecule and ensure the correct RT is chosen for all my samples (replicates).

Here is the current workflow I have been using:

  1. Import small molecule transition lists (list of 250+ molecules) with RT, m/z values, adducts, and formulas
    Transition Settings are on Full scan; Count/Orbitrap/70,000ResolvingPower at 400m/z /PRM/Orbitrap/70,000ResolvingPower at 400m/z/Include all matching scans
    Molecule Settings are all on default, I do have a question about libraries I will ask below, and as for labels I only have heavy checked (I'm still not very clear on if I should have both or neither checked? I think heavy checked was the default for small molecules.
  2. Import Results (samples)
  3. Expand all precursors and ensure the correct peak is being integrated for each molecule (each molecule only has one precursor) while using the Synchronized Integration for all replicates.
    RT shift does not usually occur from replicate to replicate but does happen from batch to batch (ex: data processed from samples injected on Monday will likely have a small RT shift ~0.2 min compared to samples injected on Friday after 100+ samples have been injected). Is there any way to adjust this globally? Such as, shift all molecules RT +/- 0.2min? As of now I manually check the peaks for a few compounds, note the RT shift, and adjust the RT values on excel then reimport the transition list with the "new" shifted RT.
  4. Export the molecule name, replicate name, and area (MS1 total area)
    These are the only features I will be needing from all the data I process as we have an internal pipeline we analyze it with. I have a standard that is injected separately as a sample that I use to ensure the correct peaks are being integrated. Is there any way to align all the molecules RT and integration to the standard if it is set as a sample on its own (a replicate)?

This workflow is repeated each time I make a new batch, is this the correct way to be working (importing the Transition List each time)? Is there a way to save this workflow/method/save the manual changes I have made to the molecules? Ex: I change the integration range but have no way of keeping that range when importing the transition list at the start of a new batch. I have been trying to get around this is by importing peak boundaries from previous batches, but still, as mentioned before, my RT shifts between batches so this isn't always useful to do.

Regarding libraries, I have made a spectral library using mzVault but am unsure how to import that into Skyline/if that is possible or if I need to use another software for creating the library/changing the format of the mzVault library file.

Sorry for all the questions! Please let me know any tips/help you have, I appreciate all of it and am excited to learn more about Skyline! :)


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