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.  

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

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

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

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

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

If you are including a Skyline document, please use Skyline's File | Share menu item (choose "Complete" if asked), which prepares a single zip file with your document and all the needed supporting files in it. Then upload that file to the Uploads page. If the actual raw data files are needed to illustrate a problem, those will need to be zipped up and uploaded separately.
[File Upload]

[Terms of Use] [Privacy Statement]

Showing: limited to 100 requests
Error in Turtorial DdaSearchMS1Filtering
(2 responses) Irene_G 2021-03-03

I want to learn how to analyse our DDA files from Waters SynologyG2Si and have therefore run tutorial Skyline DDA Search for MS1 Filtering. When run the tutorial I got error; "Unable to open file QE_140221_02_UPS1_300fmolspiked.mzid (file does not exist)". I use Skyline-daily (64-bit) (32d27b598). The computer 64-bit, 64.0 GB RAM with Windows 10 Pro. I got the same error when I try with my own files.

Please, can someone help me to solve this?


 Spectral Library Import Peptide Search.PNG 
view request
Older Skyline versions
(1 response) n-ochiai 2021-03-03

Hello, Skyline support,

I would like to install older versions of Skyline, but I cannot find any download links. Is it still possible to get them?

When I tried install Skyline 20.2, an error occurred installing .NET Framework 4.72 (attached error massage).

My company laptop runs an older version of Windows 10 (build 10240) which I am unable to upgrade.

Since this version does not support .NET Framework 4.72, I would like to install an older version of Skyline on my computer.

Is there a link for installation of older version of Skyline versions in your web site?


 Skyline setup error.png 
view request
Prosit unavalaible
(4 responses) laure bastide 2021-02-26
Hello Skyline team !

Thank you very much for your wonderful work !
When I am trying to do a Prosit Library this message appears and the server is unavailable when I am going to Tools > Options > Prosit.
Is there anything that you can do ? (It seems like a repetitive issue...)
Maybe it is not a problem with skyline and I create an issue too in the Github of Prosit but no answer so far.

Thanks a lot for your help and I wish you a good week-end !

Status(StatusCode=Unavailable, Detail="failed to connect to all addresses")
OK More Info
pwiz.Skyline.Model.Prosit.PrositException: Status(StatusCode=Unavailable, Detail="failed to connect to all addresses") ---> Grpc.Core.RpcException: Status(StatusCode=Unavailable, Detail="failed to connect to all addresses")
   à System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
   à System.Runtime.CompilerServices.TaskAwaiter.HandleNonSuccessAndDebuggerNotification(Task task)
   à Grpc.Core.Internal.AsyncCall`2.UnaryCall(TRequest msg)
   à Grpc.Core.DefaultCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   à Grpc.Core.Interceptors.InterceptingCallInvoker.<BlockingUnaryCall>b__3_0[TRequest,TResponse](TRequest req, ClientInterceptorContext`2 ctx)
   à Grpc.Core.ClientBase.ClientBaseConfiguration.ClientBaseConfigurationInterceptor.BlockingUnaryCall[TRequest,TResponse](TRequest request, ClientInterceptorContext`2 context, BlockingUnaryCallContinuation`2 continuation)
   à Grpc.Core.Interceptors.InterceptingCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   à Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, CallOptions options) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:ligne 96
   à Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, Metadata headers, Nullable`1 deadline, CancellationToken cancellationToken) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:ligne 86
   à pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:ligne 230
   --- Fin de la trace de la pile d'exception interne ---
   à pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:ligne 234
   à pwiz.Skyline.Model.Prosit.Models.PrositModel`6.PredictBatches(PredictionServiceClient predictionClient, IProgressMonitor progressMonitor, IProgressStatus& progressStatus, SrmSettings settings, IList`1 inputs, CancellationToken token) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:ligne 314
   à pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibraryOrThrow(IProgressMonitor progress, IProgressStatus& progressStatus) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:ligne 112
   à pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibrary(IProgressMonitor progress) dans C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:ligne 68
view request
Calibration curve - including information from replicates
(1 response) Kuba 2021-03-02

Hi Skyline Support,

I have a question related to how the calibration curve is presented.

I am trying to create a calibration curve for my heavy peptides at different injected concentrations. The injections were done in triplicates for each concentration of the peptide.

At the moment I can see calibration curves with concentration data points individually for each replicate. What I would like to see instead is a single point as the average of the three runs and the standard deviation. Is it possible in skyline? or do I need to export peak intensities for each peptide concentration individually and plot that externally?

Many thanks


view request
Lipodomics: How to apply single calibration curve (done with one representative molecule) to all members of a lipide class??
(4 responses) StefanW 2021-02-15

Because of the diversity of lipids they are normally separated in group, which share a common structural motive, like head groups etc. Typically these groups shares a common MS2 fragment.

The LipidCreator tool is very helpful in creating the corresponding transition lists.

For quantification it is usually to generate a calibration curve with one of the group member (i.e. PS (18:1, 16:0) ). Subsequently these calibration curve is used to quantify all group members. For this purpose the "slope" of PS(18:1,16:0) has to be assigned to all PS species (i.e. PS(20:0,16:0) or PS(16:1, 18:1) etc etc).
To my knowledge, such a assignment is unfortunately not possible in Skyline. Or ???

Inclusion this feature will procreate skyline to a unique and outstanding lipid my mind....

Thanks a lot for a comment on this support case.


view request
Strange looking chromatograms
(2 responses) Kuba 2021-02-28

Dear Skyline Support,

During the analysis of my PRM runs, I observed that for some of the peptides the chromatograms are strongly distorted - shattered.
Have you seen such data in the past? What could be a reason for it.
I attached screenshots of few examples.
If needed I can provide more technical info about the experiment.

Many thanks for your input


 1.png  2_heavyvslight.png 
view request
Parent-ion scanning
(11 responses) r bagshaw 2015-07-22
Will Skyline be able to interpret a RAW file from a parent-ion scanning experiment? In the experiment I specifically want to see parents of a product that is 85. Setting a transition consisting of a parent mass of something I expect to see in the sample with a fragment ion of 85 does not produce a chromatogram in skyline. (this is small molecule work)
Can you comment on this?
view request
Protein level FDR in Skyline?
Chinmaya k 2021-02-28


Does Skyline calculates protein level FDR?


view request
How to create library (BiblioSpec spectral library), background Proteome (Background Proteome file:- human, yeast, BSA, and others).
(9 responses) shelkarmicrobio23591 2021-02-25

I need all primary files to prepare the library (BiblioSpec spectral library), background Proteome (Background Proteome file:- human, yeast, BSA, and others).

So in your first tutorial that you have mentioned the interact-prob.pep.xml file which is not getting.

Please help to design the SRM experiment.


view request
Collision Energy Optimization Thermo Fusion Lumos
(3 responses) anne sanner 2021-02-25

Dear Skyline team,

I am currently setting up a PRM assay on the Thermo Fusion Lumos and would like to use Skyline's collision energy optimization function. In the default settings "Transition Settings -> Prediction -> Collision energy", there are only linear equations given for triple quads. Can you tell me what the regresison parameters for the Fusion Lumos are? Thank you!


view request
No chromatograms for transition ions
(1 response) michael-e-wright 2021-02-26

I wonder why transition ion chromatograms are not available for some peptides? This peptide was quantifiable using TMT across eight different samples. I'm not familiar with MaxQuant pipeline for scoring and quantifying TMT-labeled peptides. But this must be a lousy ID, correct? A lot of sketchy MS/MS spectra filter through .90 to 1 probability cut-off scores using MaxQuant search results. Do MaxQuant peptide scoring filters correlate with PeptideProphet scores? Am I looking at this the wrong way?

view request
Signal/Noise für die Nachweisgrenze ermitteln
(2 responses) alexandria balac 2021-02-26

Hallo , ich brauche bitte eine Unterstützung um das Signal/Noise zu ermitteln bei der Peptidmessungen . Wollte meine Nachweisgrenze darüber ermitteln.

Wo finde ich den Wert bzw. die Anzeige?

Vielen Dank für die Unterstützung Alexandria Balac

view request
Enable Peptide settings / Digestion / Enzyme == None for top-down
v delcourt 2021-02-26

Hi Brendan,

I think this is rather an improvement than an issue since it's still possible to get the result by another trick. However, it'd be convenient to have the protease choice to be set to "none" for MS1/MS2 quantification of intact proteins / peptides that were not subjected to proteolysis.

Let me know what you think.
Thanks once again for the magnificent tool you bring to the community.


view request
settings for importing timsTOF files in Skyline
kirti pandey 2021-02-25


I wanted to know if there are any specific settings say for e.g. in peptide transition whilst importing timsTOF files onto Skyline to visualise MS1 data.

Thanks in advance

view request
" don't know how to read "when uploading dia data in raw format
(3 responses) 1010419931 2021-02-24

Dear skyline development team, when using skyline's Data independent acquisition function, I encountered the " don't know how to read " prompt as shown in the figure when uploading dia data in raw format .I used Thermo Scientific's Q exictive HF to get the above data in raw format.I updated the skyline software, but it didn't solve the problem effectively.
What is the reason for this? How can I solve it?I hope I'm the first one to ask this question. I'm looking forward to your reply. Thank you.

view request
Can Skyline generate iRT assay library file used in Mobi-DIK script?
(1 response) Z. Jiang 2021-02-24

Dear Skyline team,

I am recently trying to use Mobi-DIK to analyze diaPASEF data. I was wondering if there is any way using Skyline to create the iRT assay library file that is used in the script "python --pasefdata data.d --mqout path/to/maxquant_data --irt irt_file.tsv"?


view request
How to use CIRT to predict retention time?
(5 responses) caixue 2020-05-20

I am trying to use the peptides selected by myself as CiRT to predict the retention time. The irt value of each CiRT peptide has been calculated and saved as a new Retention time predictor. Please see the attachment. However, when I chose the predictor, the document could not import the cirt peptides I selected, so I could not import the result. What should I do?
Looking forward to a reply. Thanks!

 Skyline cirt problem.pdf 
view request
Errors creating a spectral library and MS1 filtering
(2 responses) michael-e-wright 2021-02-23

I keep getting errors that RAW files are missing when I build a spectral library or carry out MS1 filtering. I'm processing MS data from an external source and I want to make sure the missing MS files are truly the source of the processing errors. Thanks.

view request
Generation of proteotypic peptides
(1 response) hogas aurel 2021-02-24

I have a suggestion regarding the Skyline analysis software. I am working with it for 1-2 years now, and I am having difficulties establishing the best peptides to analyze because I have to keep searching for softwares that help me generate the list of proteotypic peptides for my proteins of interest. Do you think you could integrate some form of software (in Skyline) which can help in generating the protetotypic peptides for the proteins of interest?

Thank you for your patience in reading my suggestion,
Kind regards!

view request
Group Comparison with multiple parameter
(1 response) david hoi 2021-02-24

Hello everybody,

I am trying to set up a group comparison were I want to compare a treatment vs a control at several time points in triplicates which I annotated . However, the group comparison only allows for annotation of 2 parameters such as treatment vs control and replicate, leaving me no option to also select the time points. Is there a way how I could overcome this and combine the analysis or will I have to do this separately?

Many thanks in advance,

view request
Import all peptides option using SkylineCmd
(1 response) jpaezpae 2021-02-23

Hello everyone,

I am trying to import a spectral library and add to the document all peptides in such library.

I am aware that using the GUI, this task can be accomplished by going to View > Spectral Libraries > Library > "<Add...>" > {add the library} > {click on the Add All... button} and it "just works" but I trying to use the command line interface for skyline and don't know exactly how to do this and cant seem to find any CLI flag for it.

As a workaround I think I could generate a transition list from my .sptxt by parsing all peptide names, duplicating precursor mass as a column and filtering the peaks that match y and b ions ... But I really feel like there should be a easier version.

Highly appreciating all of your help
Kindest wishes,

view request
Missing peaks MS1 quant centroid data
(14 responses) mstokes 2021-02-17

Hello everyone! We routinely use Skyline for MS1 quant with profile mode data and it works well. I have some centroid data that I'm trying to push through and I keep seeing missing peaks in certain files. When I do an XIC I can see the peak is there, the RT is good, the MError is good, but nothing in Skyline. I have tried running quant with both "Orbitrap" at a variety of resolutions and RT windows as well as "Centroid" with various ppm ranges and RT ranges, but to no avail. Many times it even looks as though there was a good MS/MS in the run that shows up as blank.

I have attached a screenshot of one example, peak is missing in Skyline in 2nd file but present in the XIC.

Has anyone else seen this issue or has ideas for a fix? Thanks!

 478.9123_Skyline_10ppm1min.png  478.9123_XIC.png 
view request
SYFT Data support
(4 responses) Christina lucas 2021-02-22

Hi there,

I was wondering whether anyone has every tried to implement data of a SIFT MS of syft technologies for small molecules (mainly VOC's). Datafiles are *.csv and *.xml, unfortunatly no mzML or mzXML available.

Data is quite simple you select a reaction ion (First Q), Reaction, product ion (Second Q), supported are SIM and Fullscan in the Second Q.

I attached some Datafiles in csv /XML, there no proprietary data format.

It would be great to get it into Skyline :)

Best, Christina

 Terpene_AromaticCitrus-BO 4.0 1h-20210122-131019.csv  Terpene_AromaticCitrus-BO 4.0 1h-20210122-131019.xml  Full scan 200 AMU pos only-Fullscan 5.0-20210122-134542.csv  Full scan 200 AMU pos only-Fullscan 5.0-20210122-134542.xml 
view request
Problems saving mprophet features
(5 responses) rmagni 2021-02-19
Hello Skyline Team,

I am having troubles saving the mprophet features in csv. When I attempt to do so I see this message:

Failed attempting to save mProphet features to C:\Users\rmagni\Desktop\GMU_projects\Tuberculosis_DIA\TB_DIA_dilution_curve\try.csv.
Access to the path is denied.
OK More Info
System.Reflection.TargetInvocationException: Access to the path is denied. ---> System.UnauthorizedAccessException: Access to the path is denied.
   at System.IO.__Error.WinIOError(Int32 errorCode, String maybeFullPath)
   at System.IO.File.InternalMove(String sourceFileName, String destFileName, Boolean checkHost)
   at pwiz.Skyline.Util.FileStreamManager.<>c__DisplayClass24_0.<Commit>b__1() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\UtilIO.cs:line 713
   at pwiz.Skyline.Util.Helpers.TryTwice(Action action, Int32 loopCount, Int32 milliseconds) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1892
   at pwiz.Skyline.Util.FileStreamManager.Commit(String pathTemp, String pathDestination) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\UtilIO.cs:line 715
   at pwiz.Skyline.Util.FileSaver.Commit(IPooledStream streamDest) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\UtilIO.cs:line 1217
   at pwiz.Skyline.FileUI.MProphetFeaturesDlg.WriteFeatures(String filePath, MProphetResultsHandler resultsHandler, IList`1 calcs, CultureInfo cultureInfo, Boolean bestOnly, Boolean includeDecoys, IProgressMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\MProphetFeaturesDlg.cs:line 146
   at pwiz.Skyline.FileUI.MProphetFeaturesDlg.<>c__DisplayClass13_1.<OkDialog>b__1(IProgressMonitor b) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\MProphetFeaturesDlg.cs:line 111
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_20_2_x64\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_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1944
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 202
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.FileUI.MProphetFeaturesDlg.OkDialog() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\MProphetFeaturesDlg.cs:line 118

My goal is to have a final list of peptides and a q-value associated with them so that I don't have to refine manually. Am I doing the right thing trying to export the mprophet features or should I look for some other report?

Thank you very much and keep up the awesome work!

view request
Duplicate transitions
(1 response) hober 2021-02-22

I regularly perform screening of peptides where I look for all potential +1 and +2 product ions from the different precursors I have selected. However, I often end up with cases where there are multiple identical precursor ion-product ion pairs, e.g. a b3 ion with the same m/z as a y3 ion and so forth.
This is unfortunately not limited to identical m/z, but also includes product ions with small differences in m/z that after rounding by the MS control software end up with the same m/z.

Would it be possible to implement something similar to the "Remove Repeated Peptides", but for transitions in Skyline?

view request
All-ions fragmentation data workflow
(8 responses) dmesasan 2021-02-18


I'm having some issues with importing results and exporting chromatograms of Agilent data that has been acquired using an all ions fragmentations mode. Maybe I need to adjust my settings somehow to accommodate this type of acquisition? I've attached an example data file. Thanks for the help!

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

Hi Skyline Team,

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

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


view request
Can I run without .NET 3.5?
(1 response) DWheatcraft 2021-02-11

I'm unable to install Win 10 Enterprise new workstations that are being setup, but it keeps trying to install .net 3.5 which is quite outdated. I can't get further and my IT doesn't want it on the workstation since it's so old. Is there a version of Skyline that can avoid this install?

Thanks, Dorothy

view request
Lipid Peak Integration Boundaries
(2 responses) dpeake 2021-02-17

Hi everyone,

I’m investigating using Skyline for integrating lipid annotation and QA/QC.
I have been very impressed with Skyline’s ease-of-use for quantitation.

I have several questions regarding adjusting integration parameters, in particular, the integration window that is displayed. When integrating a batch of lipids, some windows for the same species are displayed in a wider window than others. This window appears to change when the program sets different peak boundaries. This results in missed areas or integration of the another isomer, the largest peak in the window [see attached].

I would prefer to set a single integration window and peak boundary for all samples for a given lipid, thereby reducing the need for manual intervention.
After looking through this forum, I can't say that I have found any method that works. Adjusting the peak boundaries and "Apply to All" does not work.
Can anyone suggest a workaround other than manually changing peak boundaries one file at a time?


 Skyline 20.2 Lipid Integration.pdf 
view request
Q-TOF .raw data search stops after certain retention time
(10 responses) Yao Chen 2019-08-13

Dear Skyline Team,

I am importing Waters .raw data generated by Q-TOF instruments into Skyline. The whole file collects data for 120 min (pic 1), however, the searches in Skyline seems stopped at around 60-70 min for almost all the transitions I was monitoring (pic 2 shows four examples).
I could see a very clear signal of a spiked-in standard peptide in the raw file, which eluted at 77 min (pic 3), however, its retention time was not reached in the search of Skyline (pic 2, upper left). How should I change my settings so I can do a full chromatographic search, please?

I have my partial skyline processing file attached.



 pic 1 _ base-peak intensity chromatogram.PNG  pic 2.png  pic 3_raw chromatogram of standard peptide.png 
view request
Problem with error model from mProphet
(2 responses) Sangram 2021-02-16

Hello Team Skyline,

Thank you for this great application and wish you the best in the year ahead.

I am new to proteomics and trying it with toddler's step.

So basically I have SWATH data from sciex triple TOF of cell line knock out model. I am trying to quantitate differentially expressed proteins between wild type and knock-out cells. The steps followed were mostly from the following webinar and parameter suggestions from the following paper.

So my mProphet model looks like the fig attached. I think there is something wrong as the target and decoys they have huge overlap. What might have gone wrong ?
Secondly the Pval looks absurd (can be seen in the volcano plot attached) ??
Thirdly there were some proteins like ABL1 and MDM2 which was there in the search result (generated through PeptideShaker at 1% fdr) but was missing from spectral library ?? is there any default filtering that leads into this observation ??

Thanks and regards

 mProphet_model_report.png  p53_comparison_volcano.png 
view request
Peak boundaries for small molecule shotgun lipidomics
(7 responses) bharat 2017-07-19

I am interested in using skyline instead of Analyst for processing data from shotgun lipidomics. These runs are generated without a column, so all we're interested is an area under the curve from a start time to an end time. After these raw areas are generated, all other work on deconvolution, quantitation, etc are done externally. So, all we would need is for the integration to be done somewhat robotically from start time t1 to end time t2. I've been very successful, thanks to the good documentation, in getting all the transitions in (>140) and the integrations done. But, I can' get to the final step, which is this automatic peak boundary picking. So, you can see from the attachment that sometimes we get the good, and sometimes the needs adjustment. I've tried the "import peak boundaries" option, but that doesn't seem to work for me for small molecules. Anything I'm missing here?


 GoodIntegration.PNG  NeedsAdjustmentIntegration.PNG 
view request
Feature Request - Cycle Time
(6 responses) Tobi 2021-02-15

Hi Skyline Team,

could you please think about adding a calculated average cycle time to the report features? It is a really important feature to check in PRM and DIA runs repeatedly. While it can be easily calculated from RT start, RT end and Points Across the Peak, having it in Skyline directly would be a big plus.

With best wishes,

view request
Unknown error when opening LC-IMS-MS chromatograms
(9 responses) rozas 2021-01-28

Hello SkyLine Team,

I would like to ask you about a problem that I have using the software ( Skyline version 20.2). The data that I want to treat was was generated with a Synapt G2-Si.

I can open the MS files when IMS was turn off but it is not possible to open the data when Ion mobility was working. The following error appears:

At 12:17 PM:
Failed importing results file 'G:\My Drive\30-REC-20-ERGO.PRO\Data\20201204-004.raw'.
[pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Unknown Generic Error

if I click in more info:

At 12:17 PM:
Failed importing results file 'G:\My Drive\30-REC-20-ERGO.PRO\Data\20201204-004.raw'.
[pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Unknown Generic Error
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'G:\My Drive\30-REC-20-ERGO.PRO\Data\20201204-004.raw'.
[pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Unknown Generic Error ---> System.Exception: [pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Unknown Generic Error
at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, Boolean getBinaryData)
at pwiz.ProteowizardWrapper.MsDataFileImpl.HasSrmSpectraInList(SpectrumList spectrumList) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1064
at pwiz.ProteowizardWrapper.MsDataFileImpl.get_SpectrumList() in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 538
at pwiz.ProteowizardWrapper.MsDataFileImpl.get_HasCombinedIonMobilitySpectra() in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 599
at pwiz.Skyline.Model.Results.FileBuildInfo..ctor(MsDataFileUri msDataFileUri, MsDataFileImpl file) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1430
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 216
--- End of inner exception stack trace ---

I would appreciate if you can help me.

Thank you,

have a nice day.


view request
QuaSAR and SProCop and AVG-DIA Problem
(2 responses) hatice akkulak 2021-02-15

Dear Skyline team,

I am trying to analyse my data through QuaSAR and SProCop tools; however, I am getting an error message all the time. According to the immediate window, there is a problem about some of the packages needed to use that tool. I tried to install these packages through R studio but I still couldn't get to use these tools. I tried to use these tools through other computers but I got the same error message. I don't know what to do. I'll be glad if you could help me. Thank you.

Best regards.

view request
Setting Transitions to Non-quantitative is not synchronized across light/heavy precursors
Tobi 2021-02-15

Hi Skyline Team,

hope you are doing well.

When working with isotope-labeled peptides I noticed that setting a specific fragment to non-quantitative is not synchronized between the light and heavy form, even if synchro was explicitly checked and when deletions of fragments were synchronized. Observed in Skyline and daily.
Please find an attached file.

With best wishes,
view request
Import Peptide Library from PEAKS Online from TIMS TOF data
(13 responses) ed3 2020-10-09


I have been trying to import a peptide search from PEAKS online into Skyline but I keep getting an error and I was hoping somebody could please help me understand what is going on. The spectrum was acquired with a Bruker TIMS TOF, and when I try to import the pep.XML file in the "Peptide Search" option, I get an error saying "The .pep.xml file is not from one of the recognized sources." I have the IMS-TOF spectrum in .mzXML format in the same folder as the pep.xml file, and I am not sure why I get this error.

Thank you very much for your help.


view request
parse rules MS Amanda
(5 responses) kguehrs 2021-01-05

Hi Skyline team,

First of all: Happy New year with good health and ongoing success in all things of your personal and professional life.

I have tried to generate a spectral libraries from DDA runs with MS Amanda integrated in the newest Skyline version. In my first approach, I have used a limited but refined (adapted to UniProt headers) database. MS Amanda performed well and created a library as expected. In my second approach, I used the same DDA files but a larger but not refined in-house generated database. MS Amanda did all the searches and created the mzID files but failed to create the library due the failure to parse the database entries. The database was generated in our institute and has a header format that is obviously not recognized by MS Amanda in its default state of integration into Skyline. Unfortunately, the standalone version of MS Amanda is not very handy for generating results of multiple searches and I actually have no idea how to change parse rules in MS Amanda.

I there any documentation about the parse rules MS Amanda applies and which type of database headers are supported. This would be helpful to adapt the databases if necessary. The other way would be to have a possibility to adapt/change the parse rules when using MS Amanda in Skyline to generate libraries.

Best Karl-Heinz

view request
Error when importing .speclib spectral library from DIA-NN
(3 responses) levasseurmaxence 2021-01-11

Hi Skyline Team,

I am trying to import a DIA-NN spectral library (.speclib) but get the following error message:

ERROR: Unable to read peaks for redundant library spectrum 1, sequence , charge 2.

Command-line: C:\Users\maxen\AppData\Local\Apps\2.0\WXXH5JG8.6M9\H2BPMA06.WGC\skyl..tion_e4141a2a22107248_0014.0002_5cab26a4bb7d0a9e\BlibFilter -b true "C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test\test.redundant.blib" "C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test\~SK41BE.tmp"
Working directory: C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test
OK More Info
System.IO.IOException: ERROR: Unable to read peaks for redundant library spectrum 1, sequence , charge 2.

Command-line: C:\Users\maxen\AppData\Local\Apps\2.0\WXXH5JG8.6M9\H2BPMA06.WGC\skyl..tion_e4141a2a22107248_0014.0002_5cab26a4bb7d0a9e\BlibFilter -b true "C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test\test.redundant.blib" "C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test\~SK41BE.tmp"
Working directory: C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibFilter.Filter(String sourceFile, String destinationFile, IProgressMonitor progressMonitor, IProgressStatus& status) in C:\proj\pwiz_x64\pwiz_tools\Shared\BiblioSpec\BlibFilter.cs:line 52
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 211

After reading that post I am pretty sure Skyline supports .speclib files but I nonetheless tried to convert the .speclib library to a .blib library using BiblioSpec. However, I get the same error message when I tried to import that .blib file instead (I assume that is how Skyline imports .speclib files in the background?).

Am I doing something wrong here? I am using the latest version of Skyline (

Thanks for your help!


 20210105_bnpagemsms_O7_speclib.tsv.speclib  20210105_bnpagemsms_O7_speclib.tsv 
view request
Triple Dimethyl Modification not imported from MaxQuantSearch
(8 responses) ida suppanz 2021-02-02

as I have now been struggeling for three days on this problem, I hope that someone can advise me. I am using Skyline regularly for MS1 filtering of ICAT data which works very fine. Now I have a dataset with triple dimethyl labelling, that means light +28, medium +32 and heavy +36, respectively on each N-terminus and Lysine residue . I do manage to set the modifications in the peptide settings in way that the correct m/z values are present in my test fasta (see screenshot). However, every time I try to use the "import pepide search" function with the msms.txt generated by MaxQuant, these settings vanish. If I try to do the "import peptide search" first (without adding the fasta first, only with the settings), Skyline tells me that there are no transitions, although I clearly have the m/z values corresponding to the Light and Heavy label in my msms.txt (see screenshot). If I try to import the whole msms.txt (not just the peptide I am interested in) with settings that worked for my ICAT data, Skyline correctly informs me that I do have Carbamidomethyl [+57] on C, and and Dimethyl [+28] on K, but that the following modifications cannot be interpreted: K [+32] and K[+36]. When I try to add, eg C'2H'6 - C2H6 as a heavy modification on K, Skyline tells me that this modification already exists and I cannot add it.
How can I get the chromatograms in Skyline?

 Settings.png  msmsScreenshot.png 
view request
small molecule integration filtering
(1 response) sufyan pandor 2021-02-11

For a large scale screening experiment (ion mobility 6560) i want to be able to have skyline firstly not integrate a peak if the mass accuracy is outside a specified range and secondly not integrate a peak if an M+1 M+2 is missing and not co-eluting or better yet not at the correct ratio from the theoretical fit

Is any of this possible to get through hundreds of data files/compounds?


view request
Routine Absolute Quant Method
(3 responses) alaine garrett 2021-02-11


I would like to set up a routine absolute quant method for peptides using PRM. Is there a way to save the set up in Skyline that would allow me to save all the setting and add data files as needed without going through the set-up as outlined in the Absolute Quant Tutorial?

Thank you

view request
Number format bug
(3 responses) jrenders 2021-02-10

Hi all,
Apologies if this is the wrong place to report a bug - I looked and was not authorized to add a new "issue" to the /home/issues page.

The issue is regarding number formats in the document grid. Previously, these number format designations would remain active until you changed the selected report. However, recently skyline began to reset all number formats every time the current report was filtered in any way.

As an example, I often change the "Calculated Concentration" from scientific to "#,###.##" to show commas at the thousands place and two digits after the decimal. Staying on this same report, if I search for a molecule by typing into the "Find:" text box above the table, the number formats all reset.

This is a new reaction and (I'm pretty sure) was not an intentional change since it is pretty inconvenient for the user. I have attached my .zip so you can see. I am running the most recent version of skyline daily.

Let me know if you need more info or if I should log this somewhere else. Thanks! 
view request
A fixed and variable modification on the same amino acid
(17 responses) laura declerck 2016-07-13

I study modified histone peptides. Because of the derivatisations every N-terminal AA contains a propionylation on the N-terminus. This modification was put fixed in Mascot. Sometimes the N-terminal AA is a Lysine which almost always contains a PTM.
I know skyline cannot recognize 2 different modifications on the same AA, so i have build new modifications for all the PTMs with the mass of propionyl added. However, Skyline cannot recognize any of these self-made modifications.

I think this is because Mascot reports the fixed and the variable modifications as separate delta masses and not as one.

Do you know how I can ensure that these peptides still get into my library?

Kind regards,
view request
Detection issues
(3 responses) luc camoin 2021-02-05

Dear Skyline Team,

I think it's probably a trivial question but I can't find the solution. I want to follow the signal of a peptide using Skyline. I know this peptide is present in my RAW file (see attached file). When I load the raw file in Skyline, I can see the beginning of the signals of the peptide (M, M-1, M-2 and ions fragments) but the signal is suddenly cut off (see the attached file). Apparently, Skyline did not load the rest of the signal from the raw file. Can you help me to solve this problem?


view request
Detection Q Value shows #N/A for solid peptides
(1 response) Z. Jiang 2021-02-05

Dear Skyline Team,

I am currently working on analyzing DIA data on Skyline. One thing I noticed is that quite a lot peptides with very good XICs and dotp scores have "Detection Q Value" shown as #N/A. For example, attached figure (Detection Q Value NA.png) shows the XICs of one of the iRT peptides. It is definitely in my sample and has very well-aligned XICs, intensities and dotp scores. However, the detection Q value is #N/A. Actually, all my iRT's detection Q values are #N/A. Does #N/A mean that the peptide is not significant?

I performed "Refine - Reintegrate" and trained "mPhrophet" model with target-decoy strategy (see figure "Skyline_Reintegrate.png"). After that, I selected "Only integrate significant q values" with a cutoff of 0.01. Did I do anything wrong?

Thank you,

 Detection Q Value NA.png  Skyline_Reintegrate.png 
view request
Display of protein abundances in PRM experiment
(3 responses) bpfister 2021-02-05

Dear Skyline team,

I was very excited about the recent implementation to show the actual protein abundances of each replicate within the Group Comparison grid, but am having problems with displaying them correctly. As my experiment included spiked-in heavy labelled peptides, I used "ratio to heavy" as the normalization method for the group comparison (see screenshot). I understood that I should also apply this normalization method in Peptide Settings --> Quantification (screenshot), but when I do that, it will only display me N/A for all replicates and proteins in the Group Comparison grid (why?).
When using "Equalize medians" in Peptide Settings --> Quantification, the protein abundances are displayed but they don't give me values that would lead to the fold changes calculated by Skyline (also not if I change the normalization to "Equalize medians" for the group comparison). Which settings do I need to use to get exactly the data that underlies the fold change calculated by Skyline?

I am using Skyline version

Thanks a lot for your help!

view request
How to use explicit retention time in proteomics interface?
(3 responses) yoneyamat 2021-02-03

Dear skyline team,

I am trying to measure peptides in SRM mode, and would like to use an explicit retention time on the chromatogram view. The small molecule interface has a column to paste the explicit retention time. Edit>Inset>Transition List. However, there is no column in proteomics interface to paste the explicit retention time for each peptides.
I would like to know how to use explicit retention time in proteomics interface.

Thank you in advance for your help.

view request
How to develop a MRM Method from scratch for known proteins (protocol/tutorial)
(2 responses) AWY 2021-02-03

I am interested in developing MRM biomarkers for specific plant proteins (Arabidopsis and Maize)

May I know if anyone could share any practical tutorial on how to develop a QqQ method from interested proteins.

Doesn't have to bein plant example protocol from any organism is OK to understand the workflow.

From theoretical digestion, how to identify unique peptides, to develop QqQ method.

Thank you.

view request
proteotypic peptides
io 2021-02-03

Dear all,
In order to choose proteotypic peptides, do I have to go first to peptide Atlas to select peptides or Skyline have this in consideration?
I have tried to install the proteotypic peptide viewer tool but my PC have problems in the installation.
Best regards

view request
Simple precursor ratios
(3 responses) chiva cristina 2021-02-02

Dear Skyline team,
I hope you are all doing well!
I have a question regarding the last version of Skyline daily. I have noticed there is a new checkbox (or maybe it is not so new but I have just discover it) in the Quantification tab under Peptide Settings called "Simple precursor ratios". Could you tell me what does it does and when can be helpful?
Thanks a lot

view request
Building a spectral library using msp
(3 responses) laura corveleyn 2021-02-02


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!



view request
Exporting Skyline document as report to include modified amino acid position on protein
(4 responses) paul.derbyshire 2021-01-29

Hi Skyline Team,

I have a question regarding document export as a report, i.e., File > Export > Report. Is it possible to extract the modified position of an amino acid within a protein sequence? In the jpg example attached, this protein is phosphorylated at position S134. Can such information (S134) be included in the report and if so which of the various option(s) should be selected?

Thanks in advance.


view request
copy/paste error from excel
(4 responses) Will Thompson 2021-01-29

Hi Skyline Team
On Windows 10 machines it seems when i copy/paste from excel into the document grid (for instance when pasting a small set of values for calibration standards), the 'receiving' column in Skyline document grid skips a row between all the values. So it pastes all the values, but it will leave an empty row between every value, so only the first one is in the right place and the rest are in the incorrect location. Its really annoying to not be able to copy and paste a list of values. :) Verified on two different machines. Can you verify?

Thank you


view request
MS1 filtering
(2 responses) paul.derbyshire 2021-01-28

Hi Skyline team,

I am having a go at MS1 filtering in Skyline and seems to be working quite well. If I click on the chromatogram peaks a new window opens up showing the precursor and it's isotopes (M, M+1, M+2 and so on). If I zoom in to say, the precursor (see attaches image file) I see that this peak is made up of other peaks. I am assuming these are subtle isotopic variants around this m/z and reflect their natural occurrence. Is this interpretation correct? Look forward to your reply.



 MS1 precursor zoom.jpg 
view request
Response of CE optimization (attached files)
(7 responses) hjl 2021-01-27

Dear Skyline team,

As you were asking a sample files of Skyline on my question of CE optimization, I prepared the files.

My workflow of CE optimization is described as below.

1.Specified 1 transition per compound

2.Based on specified transition, set methods for 1st CE optimization( step size 6, step count 3) = 1st analysis
Before) I used 4 step size and 6 count, I thought many step counts may draw some confusion to measure the best transition, I shorted it as 3 instead increasing step size( 4 to 6)

3.Analyzed and clarified the best CE for each transition

4.On the basis of 1st result, set methods for 2nd CE optimization( step size 2, step count 3) = 2nd analysis

5.Analyzed and acquired the final optimized CE for every transition each

6.Compared two methods( one = original method ; the other = obtained by CE optimization)

  • in the process of optimization, I specified each compound's RT as dragging the peak on the chromatogram, but in the sample file I attached, some of compounds might not be assigned for its peak. Please consider that flaws of those files.

My question is on the 1st optimization, I could clearly see that the CE optimization is needed for every compounds
then, after setting the optimized method( on the comparison step) Original data showed far better results than the other.
If the original data is better than modified one, what made the optimization result (especially that of 1st optimization) different ?

Thank you for your help as always.

 routine_1transition_1st(sample).sky  routine_1transition_2nd(sample).sky  routine_1transition_3rd_comparision_result(sample).sky  routine_1transition_1st(sample)  routine_1transition_2nd(sample)  routine_1transition_3rd_comparision_result(sample) 
view request
chromatogram files disappearing
(4 responses) Christian F 2021-01-25

Opening previous experiments doesn't open chromatogram for processed data, and results in the file for chromatographic data being erased completely. The file is not in the recycling bin (indicating it wasn't deleted), but seems to have been wiped from the computer all together. This happened for three experiments in a row.

view request
FDR control for DIA data
(3 responses) Z. Jiang 2021-01-13

Hi Skyline team,

I am currently using Skyline to analyze my DIA data. I was wondering if there is any way for Skyline to calculate protein and peptide FDR rate? I found there is a "Reintegratie..." function under the "Refine" menu. I was wondering if this one does the FDR calculations? If so, how does it return the results of 1% FDR? If not, what's the function of this "Reintegrate"?

Thank you,

view request
CE optimization
(1 response) hjl 2021-01-20

Dear, Skyline team

First of all, I wanted to send a huge thanks for you to support us by this far useful software.

On the way i am using a CE optimization option in Skyline, I got a question which turned out to be misterious for me.

In my study, I targeted 8 compounds for collison energy optimization and the process was described below.

  1. Specified 3 transition per compound by direct infusion
  2. Based on specified transition, set methods for 1st CE optimization( step size 4, step count 6) = 1st analysis
  3. Analyzed and clarified the best CE for each transition
  4. On the basis of 1st result, set methods for 2nd CE optimization( step size 1, step count 3) = 2nd analysis
  5. Analyzed and acquired the final optimized CE for every transition each
  6. Compared two methods( one = original method earned by direct infusion ; the other = obtained by CE optimization)

My question is on the 1st optimization, I could clearly see that the CE optimization is needed for every compounds
then, after setting the optimized method( on the comparison step) Original data showed far better results than the other.
If the original data is better than modified one, what made the optimization result (especially that of 1st optimization) different ?

Thank you for your help as always.

 1st optimization.PNG  comparison.png 
view request
MS1 Quant with t-SIM partially solved, still questions on skyline
(5 responses) Tobi 2018-06-22

Dear the whole Skyline Crew,

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

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

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

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

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

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

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

Best regards,
view request
Installing/enabling external tool in Administrator install of Skyline
(7 responses) m j noga 2021-01-14

Dear Skyline Team,
We are using Skyline in a very restrictive environment where Administrator install is the only way to make it run for regular users. I am currently exploring the possibility to also use external tools to better integrate it in our workflows. I see I can install and run a tool with the Administrator account but this tool is not showing up in the Tools menu for regular users.
Is there any way to make Skyline discover tools installed by admin?
I see I can enable the tool manually by filling in the form through Tools->External Tools...->Add...->Custom and changing the command path for absolute path to the executable in Tools folder within Skyline installation directory. So while it allows me to achieve the goal, this is a rather complex workaround considering what my users need (it also requires manual installation of report and annotations). I wonder if there is a simpler way.
I see there was also a similar call in 2018: and I wonder what changed since then as Nicks suggestion was not very encouraging.
I will very much appreciate your help!

view request
Bar chart for total protein intensity
tomasz gozdziewicz 2021-01-26
Hi Skyline Crew! I just love Skyline and I'm using it for peptide/protein relative quantitation. One minor issue I notice is that in the replicate comparison plot I cannot show total (summed) intensity for a particular protein as a bar chart. The only visible Graph style is Line. The Bar Graph is perfectly present when I select any peptide but for whole protein, it changes for line style. I'm not sure it is a real issue or maybe it is a reason for such plot style for protein selection. Thanks for a comment. Best Regards, Tomasz Gozdziewicz Skyline Daily; Windows 10
 PeakArea_Peptide.png  PeakArea_Protein.png 
view request
RT data export isn't showing consistent values with the chromatogram
(2 responses) Christian F 2021-01-25

Anytime I go to export results from the experiment after processing the chromatogram data, the retention time listen for each peak doesn't correlate with the RT of the actual peaks. I'm not sure why this is, but each peak in the data is significantly off (almost 0.3 min).

view request
IMS Filter Error with version
(10 responses) Juan C. Rojas E. 2021-01-25

Dear support,

I am having some issues with applying the IMS filter windows generated from the in-built predictor for DDA data with IMS separation. The data was generated with a Synapt G2-Si.

The steps I usually follow are:

  • Load DDA data
  • Filter reliable peptides
  • Use IMS Predictor
  • Re-import results to apply IMS filter
  • (New due to change in settings) Set Window type as Resolving Power and set value of "15"; this was the optimal value found for my data in the past. BTW, why was this setting changed out of the library generation window? Shouldn't the resolving power settings be set prior to generating the IMS library?

This worked fine in version However, I recently updated to the newer version and every time I try to do the re-import I get an error message "An item with the same key has already been added.". Additional text (in German) attached.

I also tried removing the file, save, set IMS library created previously, set IMS filter settings, and then import the file again. This time the file loads, but no IMS filter is applied.

Can you give me some insight for this problem? What else could I share to help?


view request
Overlaying same peak across different samples
(4 responses) victor gonzalez 2018-02-05
Dear all,

I would like to know if there's a way to get a graph in which I could plot an overlay of the profile of all the peaks corresponding to the same transition, across all of the available result files. I have checked the tutorials and webinars, but I was unable to find an example in which such feature was employed.

Thank you so much in advance, and best regards,

view request
To get started
(1 response) fliabaldo 2021-01-23

Hello! I´m writing for you in order to ask some questions. I am starting to use skyline and I want to know if by entering the sequence of a protein and telling the software which digestion enzyme we use, I can have the information about the cleavage peptide sequence? and If it possible to learn about the use of skyline with the tutorial reading alone? I´m from Argentina and I'm interested in the Skyline.
Sorry for my english y for consuming your valuable time. Thank you very much.

view request
5MRM not shown at the same time
(10 responses) xin huang 2021-01-20

Hello Skyline team,
I am working on peptide quantification with Water QToF-MRM method, I have five peptides and five MRM methods for one injection. From Masslynx, I could see the 5 peptides are detected, and they have been divided into two function channels (four MRM in Function channel 2, and one MRM in Functional channel 3). However, when I import the raw. file into Skyline, I could see the four peptides MRM very well, but the one peptide in Function channel 3 behaved strangely (it did not pick up the peptide at retention time 3.27 min). I was playing with the fdc. file to change the function number to the same, it did not work. I attached the chromatogram I obtained from both Masslynx and Skyline, appreciate if someone could help with the data import. Thanks!


 5 peptide MRM from Masslynx.pdf  Peptide in Function channel 3 from Skyline.png 
view request
Optimal analysis workflow for 500 sample DIA experiment?
(1 response) becky carlyle 2021-01-19

Hi everyone,

Not necessarily a specific Skyline request here, but a hope that members of the Skyline community can help guide our workflows. We have 500 biofluid samples from a neurology clinic that are currently undergoing DIA. We are using an in-house generated fractionated library.

The Core running these samples uses Scaffold DIA but it's not capable of handling large sample sets. I also dislike the “black box” aspect and would prefer an open source solution. I was wondering if this community would be able to offer thoughts on an optimal pipeline for analysis of this data - preferably something with the potential for parallelization (I think ideally we’d use AWS to spin up some clusters to do this work - we have plenty of experience doing this with other 'omics pipelines). We are a biomarkers group and have moderate experience with MQ, Skyline, and X!Tandem, but we are definitely not experts in this particular field. We're also completely over-run with working from home and childcare, so it's very difficult to find the time to thoroughly research the huge number of developments in this field over the past couple of years. I hope the community can help point us in the right direction!

Thank you in advance!
Becky Carlyle (Buck course attendee from a few years ago)

view request
Libraries and targeted analysis
(1 response) Babak 2021-01-19

Hi Skyline Team,

I would like to ask you to answer my questions please.

  1. In the Proteome Discoverer, I uploaded raw data from MS, divided into three types and from these three types I created three libraries – each type had its own library. Is there any difference to upload all three libraries into Skyline, in which I evaluate the data of PRM analysis, or to upload them as one common library?
  2. We did targeted analysis on MS and the samples were measured in triplicates. Is it better, during the process of evaluation in Skyline, to do the average (total ratio) of all three replicates or just to select the most suitable replicate?

Thank you

view request
Error importing Percolator v3.05 xml
(5 responses) damon barbacci22098 2021-01-19
I receive the following error when I try to import Percolator pep xml output into SkyLine.

ERROR: boost::filesystem::directory_iterator::construct: The system cannot find the path specified: "../sequest/"
ERROR: reading file 2021012_MSB50697A.perc.xml

Command-line: C:\Users\dbarbacci\AppData\Local\Apps\2.0\LY2EZ3ML.RV9\JVPER9HM.HDE\skyl..tion_e4141a2a22107248_0014.0002_2f1cb11a037aa924\BlibBuild -s -A -H -o -c 0.95 -i MSB50697_98A -K -S "C:\Users\dbarbacci\AppData\Local\Temp\tmpFB5A.tmp" "S:\Process Dev\Analytical Team\Damon Barbacci\MSBioworks_HUD01_HCP\MSB50697_98A.redundant.blib"
Working directory: S:\Process Dev\Analytical Team\Damon Barbacci\MSBioworks_HUD01_HCP
OK More Info
System.IO.IOException: ERROR: boost::filesystem::directory_iterator::construct: The system cannot find the path specified: "../sequest/"
ERROR: reading file 2021012_MSB50697A.perc.xml

Command-line: C:\Users\dbarbacci\AppData\Local\Apps\2.0\LY2EZ3ML.RV9\JVPER9HM.HDE\skyl..tion_e4141a2a22107248_0014.0002_2f1cb11a037aa924\BlibBuild -s -A -H -o -c 0.95 -i MSB50697_98A -K -S "C:\Users\dbarbacci\AppData\Local\Temp\tmpFB5A.tmp" "S:\Process Dev\Analytical Team\Damon Barbacci\MSBioworks_HUD01_HCP\MSB50697_98A.redundant.blib"
Working directory: S:\Process Dev\Analytical Team\Damon Barbacci\MSBioworks_HUD01_HCP
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 156
view request
paired comparison in Skyline
(6 responses) zdarula 2021-01-15

I have a SureQuant dataset for 50 paired normal-tumor tissue samples and I would like to ask your advice how to set up a paired comparison (like "nested experiment" in Proteome Discoverer). As the biological replicates are not independent I should have the tumor/normal ratio per sample pairs instead of annotating the samples into a normal (50 sample) vs tumor (50 sample) groups. Can I do this in Skyline? Thanks a lot.

A minor issue: unfortunately I set up the Skyline experiment on the peptide level and now that I want to assign these to proteins so as to get a protein-level fold-change I cannot do it without losing my corrected peak boundaries, e.g. skyline reintegrates peaks if I associate proteins to the peptides. Can I somehow bypass this?

Thanks a lot,

view request
Small molecule (non-global) standard concentrations
(4 responses) jrenders 2020-09-01

Hi there,
Thanks so much for adding the ion ratio features to skyline - we are currently rebuilding all of our small molecule methods in skyline and have unfortunately hit a snag. I think you previously made mention of (in the future) adding the ability to assign calibration curve regression and weighting on a per-analyte basis. Right now, via the "Document grid --> Reports --> Replicates" report we can assign analyte concentrations, but only globally. However, we have methods where our calibrators do not have all analytes at the same concentration. Is there plans to allow for a per-analyte concentration assignment as well in the future? Or perhaps there is already a way to handle this via the document grid or pivot table? Thanks for any guidance you can suggest.

view request
DIA analysis; unable to remove precursors
(10 responses) narjis fatima 2020-12-25

Hi ,
I have recently done a DIA analysis of whole proteome CLL cells after drug treatments. I could not resolve the issue of precursor removal in my analysis. I have unchecked the include DIA precursor window in transition settings --> filter and added "Start by precursor" in the document grid as recommended by one of the expert here.
I am still getting this error (mentioned below)
I am analysis DIA results for the first time, and therefore I think I am having some issues with the processing of results. Can you let me know how to fix this issue.

I am also observing precursor and product ions both in the target section. I saw you mention some one that either precursor or product ions should be there. Could you let me know if this is the issue, how to fix it ?
Thanks a lot.

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

** Reading the data for MSstats.....
** Peptides, that are used in more than one proteins, are removed.
** Truncated peaks are replaced with NA.
Error in SkylinetoMSstatsFormat(raw, removeProtein_with1Feature = TRUE, :
** Please check precursors information. If your experiment is DIA, please remove the precursors. If your experiments is DDA, please check the precursor information.

Can't finish analysis.

view request
Peak Scoring with heavy peptides
(1 response) tilman werner 2021-01-08

Dear Skyline Team,

Happy new year - thank you for this amazing tool!
I have a question regarding peak scoring. I have run a PRM experiment with spiked in heavy peptides to detect several cytokines. Some we could clearly detect, but with others the results are shaky and we are not really sure if we should count the peptides as detected in our sample or not. The main issue is which peaks to already count as a fragment pattern and which ones to discard as noise.
I was wondering if there is a tool in Skyline that allowed us to calculate something like a Q-value for the peaks by using the heavy peptide peaks as input/template instead of a seperately generated peptide library? Or is there another way to discern signal from noise in the presence of heavy peptide standards?

Thank you for your great work: Skyline is by far the fastest way to go from measurement to shiny result!
Best wishes,

view request
Protein abundance issue
(1 response) francescodemetrio lofaro 2021-01-13

Hello there,

I'm a new user of Skyline. I imported my DDA search from Mascot and I'm able to quantify proteins in two different conditions using MSstats. However, using this tool i can't quantify protein inside samples, but I obtaine only log of fold chande between conditions. So, is it possible to obtain protein quantification for each sample using MSstat integreted in Skyline?

Furthermore, i tried to quantify protein using protein abundance report integrated in Skyline. However, there are different missing values (#NA) for proteins that show peptide intensity in Skyline interface. How can resolve this issue?
I hope to find a solution to quantify in MS1 my samples thanks to your help.


view request
Importing results does not show peaks (which I know are there)
(4 responses) barryh 2021-01-12

Hello there,

I am creating a very simple project for absolute quantitation, I selected my one and only peptide then I imported the file with the results (and while uploading I can visualize the peaks at around 2.5 min) and then when it is done importing the results the chromatogram window shows from 0 to 1.2 min and shows that there are NO peaks (See attached screenshot). I have tried everything to zoom out of that region and see the 2.5 min peaks (LC method is 6 minutes long) and I can't seem to find a successful way. Any advice would be greatly appreciated.

Thank you very much!

view request
Modifying Molecule Name in Target List
(1 response) davis 2021-01-12

Hi Skyline team,

When I try to rename ( right click on molecule in target list >modify>name) a molecule in my target list, the chromatographic vanishes following name change. Is there work around to avoid this?

Best! SSD

view request
PRM-quantitate with precursor or fragment?
(1 response) sallym8 2021-01-10

Hi Skyline Team!

Your software and resources are amazing- thank you for all your hard work! I recently ran my first PRM experiment on an Orbitrap Fusion and have been analyzing my data with Skyline. In the past I have run DDA experiments, but my protein is in a buffer with albumin and my peptides of interest have been getting drowned out. I have been able to use a large amount of protein and the DDA method in order to generate a list of peptides that I could include in my PRM experiment, but moving forward I will be limited in the amount of protein I can use. The goal of my experiment is to quantitate peptides in two regions of the protein in order to monitor a cleavage event. Currently, I am trying to compare the PRM and DDA experiments in order to determine how much the new method improved my signal. I am confused about whether I quantitate the PRM data using the precursor ions or the fragment ions and why. If you could offer some clarification that would be amazing!

view request
Different results from raw data that are nearly the same in MassLynx
(3 responses) anna walke 2021-01-07

Dear SkyLine Team,

I'm working with a simple method on a waters Premier Pro QToF to weekly check system performance by analyzing a standard of angiotensin II. Measurement is performed by Full Scan (function 1) and MS/MS scan (function 2) in parallel over the entire time range of the method.

Unfortunately when analyzing my raw data with Skyline (version I get very different results compared to manually analyzing the same data in MassLynx. I added screenshots of this and also attached the corresponding information of my LC-MS experiment. Within the experiment report I highlighted the different paramters in yellow, but there are only little differences.

I do not understand why the same SkyLine Document (also attached below) with same molecule and transition settings gives that different results after integration.

Could you please help me or give a hint towards a possible solution?

With kind regards,

 AGII Std 01_Experiment Report.pdf  AGII Std 16_Experiment Report.pdf  SkyLine AGII  Screenshots for SkyLine Support.pdf 
view request
Not able to install Skyline software on my computer (Windows 7)
(1 response) jeon 2021-01-07

Skyline suddenly stopped working a day ago.
We re-installed all skyline software and tried re-installing but it didn't work.

I've attached a word file with the error details and out computer operating system.

Please help up resolve this issue.


Marlene Thaitumu on behalf of Ju Eun (

 Error Message when installing Skyline software.docx 
view request
Analysis Strategy
(4 responses) klemens froehlich 2021-01-05

Dear Skyline Team,
First of all happy new year and thank you for your great work !

Yesterday a collegue approached me and asked about detection qValues with Skyline for some peptides he wanted to quantify and now wanted to have some numbers for his collaboration partners how sure we can be numerically that we detected the peptides with our PRM experiments.
I only had experience with peak scoring with DIA data, so I did some reading in the forum and the advanced peak picking tutorial: Similar problem I think is described here:

I came up with some general workflow and I would really appreciate it if you could share your thoughts:

I would first of all generate independent spectral libraries and retention time libraries for all measured targets. For this I would use the integrated PROSIT feature (awesome integration btw!)
Then I would go to reintegration and build an mPROPHET model based on second best peaks
However, when I try to do that, Skyline gives me an error saying I do not have enough targets (0 and 374 decoys), which is ironic because when I check the use decoys then Skyline says I do not have any decoys and I should uncheck the use decoys box......

Since I cannot measure the samples again and we are stuck with the PRM masses isolated during the measurements I was wondering whether I can get Skyline to accept decoys without the 10m/z mass shift usually wanted by mPROPHET. I would not like to use that in the final analysis but I would at least like to look at the comparison between this decoy scoring model and the second best peak scoring model.
I therefore generated decoys, exported them in a transition list, subtracted 10m/z from the precursor, added a "decoy" column filled with "true" and reimported it. But Skyline does not recognize the peptides as decoys but only adds them as normal targets (at least they are not red like my decoys normally are).

I think I screwed up the document somehow, but before I continue deeper into this rabbit hole I wanted to ask whether this is the right direction or whether I should try something else.

I will attach the skyline document.

Best Klemens 
view request
iRT on non linear gradient
(6 responses) benoit fatou 2021-01-04

Dear Skyline team,
I was wondering how I can use the iRT peptides for retention time adjustment when using a non-linear gradient.
Thanks for your help and Happy New Year !

view request
Grey dotted lines?
(18 responses) henrik molina 2020-12-16

I am using Skyline to extract MS1 signal of small molecules (Q-Exactive +).
Once in a while I come across cases where what I think is an obvious peak has not returned an area THOUGH Skyline have clearly marked - with grey dotted lines - what the correct integration should be. What does the grey dotted lines mean?
I have attached an example: the same sample injected 2x. For sample B, the area was ok integrated. No area for sample A. Both sets of dotted lines appears to have marked the correct peak.

I can easy manually mark what should be integrated - but I am curious why the software only integrated this molecule in Sample B.

I am running Skyline

Cheers, henrik

 Auto-Skyline -Dotted grey lines - no signal.pdf 
view request
(4 responses) ziyang zhang 2020-12-22

Hi again skyline gods,

I recently had an issue when importing a DDA search (done by MaxQuant). The peptides carry a custom modification "ARS", which is defined in the modifications.xml file. When importing, Skyline prompts that there is no matching modification for "ARS".

I did the following:

  1. adding modifications.xml file to the data folder containing msms.txt
  2. adding modifications.xml file to the spectral library folder under the user home folder
  3. adding modifications.xml file to replace the skyline installation folder (with new App installation tool, this is now at C:\Users[user]\AppData\Local\Apps\2.0\\\skyl..tion_xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
  4. adding a modification in peptide settings named "ARS" with the same formula as in modifications.xml

But still cannot import the search result - the error persists. Could I please get some help...

Many thanks,

 Skyline_Mod_NoMatch.png  Mod_Def.png  modifications.xml  msms.txt 
view request
transition detection issues
(3 responses) clemence balty 2020-12-17


I am trying to quantify peptides in a biological sample using the light / heavy ratio (using the Sure quant method by thermo). I have designed heavy peptides corresponding to the peptides that I want to quantify in the biological sample. First, I created a spectral library with heavy peptides. Then I added these heavy peptides to all samples (potentially containing the light version of each heavy peptide). When I opened the raw file in Skyline, I detected heavy peptides and their transitions except for two heavy peptides (SGYSSPGSPGTPGSR and TPSLPTPPTR). These peptides are present when I manually check the raw file. I don't understand why skyline can't detect the transitions of these two peptides.

Can you help me or give me advice to solve these problems?
Is the library required to perform quantification?

I can share my raw files and the library I created with you.

Thank you,

 peptides list.xlsx  SQ_Method_Development_20201120.skyd  transitions issues.png  Tau_Aqua_skyline.blib 
view request
Must have MS1 scans in PRM methods?
(4 responses) ziyang zhang 2020-12-16

Hello fantastic people at skyline,

I am quite new to quantitative mass spec and have very limited sources to learn - hope you don't mind these very basic questions...

  1. Is it mandatory that MS1 scans be include in PRM methods for skyline to perform quantification? My current method only has an inclusion list for precursor masses to cycle through. When I analyze the data acquired this way, Skyline is able to extract the ion chromatograms but cannot quantify the results. Also giving red tags for all the transitions and dotp=0. Screenshot attached.
  2. I have gone through the tutorial ( without much issue. I do wonder if there is a way for plot the extracted chromatograms for a particular transition on the same plot (overlaid)?

Again, I know these questions may be due to simple lack of understanding of skyline features or mass spec fundamentals. If there are tutorials somewhere that can save you some frustration with my ignorance or time explaining simple concepts, please do point me to them!

Many thanks in advance!


view request
Target non-monoisotopic ion
(1 response) Zac 2020-12-17

I want to target a non-monoisotope peptide ion. I have used mono and average before, is there a way to work with ions outside of these two options. The isolation width will be close to 0.4 m/z

view request
Cannot load Daily installation file onto any pc's that do not already have it installed
(1 response) jgb2 2020-12-16

Every time I try to download a daily installation file onto a pc that does not already have it installed, I am prevented from completing the installation and shown the attached prompt. Sent email to Brendan as prompted then redirected to support board.

 Screen Capture Daily install 202012161.PNG 
view request
Peptide Atlas Download for human library build
(1 response) daraghob 2020-12-16

I am attempting to generate a new spectral library to achieve better target peptide coverage for a transition list I am generating for a future MRM experiment.

My issue is trying to discern which file to download on the peptide atlas builds page (
There are many options for the human build but I can't seem to find the appropriate data format (.pep.xml) for the library build.

Any help with this would be greatly appreciated.
Many thanks,
view request
Issue With Retention Time Selection
(3 responses) ed3 2020-12-11


I am trying to import a pepXML file from PEAKS Online into Skyline to generate a peptide library. However, I am having an issue when doing this as for some reason Skyline is not selecting the appropriate retention time for the peptides in the pepXML file. I have attached a picture of this issue to illustrate what I am talking about. In the attached picture, Skyline has selected the retention time to be 0.02 minutes for the selected peptide; however, the actual retention time of this peptide is 15.20 minutes. Is there a way to resolve this issue?

Thank you in advance for your help, and please let me know if any clarification is needed.


view request
Error when importing iproph.pep.xml
(1 response) ztflaten 2020-12-07

I am trying to build a spectral library with an iproph.pep.xml document I've generated and I keep running into various forms of this error
ERROR: index 17017 out of range in mzXML file '.\P2.mzXML'

Command-line: C:\Users\pc\AppData\Local\Apps\2.0\5PYCWJGV.XX2\M6EMW1MM.TJV\skyl..tion_e4141a2a22107248_0014.0002_be619ba2d44825a4\BlibBuild -s -A -H -o -c 0.99 -i fragpipe_dda_2 -S "C:\Users\pc\AppData\Local\Temp\tmp79D2.tmp" "C:\Fragpipe_Skyline\fragpipe_dda_2.redundant.blib"
Working directory: C:\Fragpipe_Skyline
OK More Info
System.IO.IOException: ERROR: index 17017 out of range in mzXML file '.\P2.mzXML'

Command-line: C:\Users\pc\AppData\Local\Apps\2.0\5PYCWJGV.XX2\M6EMW1MM.TJV\skyl..tion_e4141a2a22107248_0014.0002_be619ba2d44825a4\BlibBuild -s -A -H -o -c 0.99 -i fragpipe_dda_2 -S "C:\Users\pc\AppData\Local\Temp\tmp79D2.tmp" "C:\Fragpipe_Skyline\fragpipe_dda_2.redundant.blib"
Working directory: C:\Fragpipe_Skyline
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 156

I am not sure I understand what the error is telling me but I think it has to do with the location of the files it's trying to find (which are located in the same directory in a sub folder). I've tried moving files around in my directory but keep having issues, even when they are all in the same folder as the saved skyline document.


view request
"resolving protein details" problem AND lost library problem upon install of newer Skyline version
(3 responses) cabarnescabarnes 2020-12-05

Hi all,
Thanks for the great software. I am using the EncyclopeDIA work flow to get DIA data into Skyline. I imported an .elib library out of EncyclopeDIA that seemed to be working and everything was working great until one day I got the "resolving protein details" problem. I was using Skyline, so I uninstalled that and then downloaded Skyline and installed that. Now, I'm getting a library problem. Everything else seems to be working. Here's the error code:

Skyline version: (64-bit)
Installation ID: 5b133551-e4fe-4406-b30c-a9e78485df9a
Exception type: Exception
Error message: Failed loading library 'C:\TPP\data\smORF_Experiments\Mouse_plasma_inflammation_young_old_smORF\Mouse_plasma_inflammation_young_old_smORF_spectral_library_w_plasma_fractionations_Joan_chromolib_quant_report.elib'.
SQL logic error or missing database
no such table: LibInfo
Format: BiblioSpec
FileRevision: 0
SchemaVersion: 0

System.Exception: Failed loading library 'C:\TPP\data\smORF_Experiments\Mouse_plasma_inflammation_young_old_smORF\Mouse_plasma_inflammation_young_old_smORF_spectral_library_w_plasma_fractionations_Joan_chromolib_quant_report.elib'.
SQL logic error or missing database
no such table: LibInfo
Format: BiblioSpec
FileRevision: 0
SchemaVersion: 0 ---> System.Data.SQLite.SQLiteException: SQL logic error or missing database
no such table: LibInfo
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.CreateCache(ILoadMonitor loader, IProgressStatus status, Int32 percent) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLite.cs:line 605
at pwiz.Skyline.Model.Lib.BiblioSpecLiteLibrary.Load(ILoadMonitor loader, IProgressStatus status, Boolean cached, Exception& failureException) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLite.cs:line 949
--- End of inner exception stack trace ---
at pwiz.Skyline.Model.Lib.BiblioSpecLiteLibrary.Load(ILoadMonitor loader) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLite.cs:line 927
at pwiz.Skyline.Model.Lib.BiblioSpecLiteLibrary.Load(BiblioSpecLiteSpec spec, ILoadMonitor loader) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLite.cs:line 150
at pwiz.Skyline.Model.Lib.LibraryManager.LoadLibrary(LibrarySpec spec, Func`1 getMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\Library.cs:line 295
at pwiz.Skyline.Model.Lib.LibraryManager.LoadBackground(IDocumentContainer container, SrmDocument document, SrmDocument docCurrent) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\Library.cs:line 124
at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
Exception caught at:
at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
at System.Threading.ExecutionContext.RunInternal(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state)
at System.Threading.ThreadHelper.ThreadStart()

view request
Trouble reading wiff files. Does not contain SRM/MRM chromatograms.
(1 response) stephan kamrad 2020-12-05

I am trying to quantify peptides using our Sciex QTOF 6600. I have made a document of my targets in Skyline and exported an acquisition method. The acquisition method looks good to me and contains multiple 'Product Ion' experiments, one for each of the precursors. There is no MS1 full scan experiment as this was not included in the template. I think this shouldn't be an issue as I only want to quantify on the MS2 level. When I try to load the data in Skyline, I get the following error. This persists after converting to mzML with MSConvert.

The file xxxxx.wiff does not contain SRM/MRM chromatograms. To extract chromatograms from its spectra, go to Settings > Transition Settings > Full-Scan and choose options appropriate to the acquisition method used.

Does anyone have any clue where the issue could lie? Is there any reason why Skyline shouldn't be able to deal with these data files in principle?
The same seems to work when my acquisition method also includes an MS1 experiment (as I have done previously)

Thank you,

view request
Is there any availiable tool for export skyline results and deal with the results?
(1 response) 16622080827 2020-12-02

I find the exported results format is just liake this:
Protein Name Protein Accession Peptide Modified Sequence Point Count Protein Gene Replicate Name
sp|A0AVT1|UBA6_HUMAN A0AVT1 YQC[+57]VVLTEMK 0 UBA6 500ng_1
sp|A0AVT1|UBA6_HUMAN A0AVT1 YQC[+57]VVLTEMK 0 UBA6 500ng_2
sp|A0AVT1|UBA6_HUMAN A0AVT1 YQC[+57]VVLTEMK 0 UBA6 500ng_3

But actually,I want the result like this:
Protein Name Protein Accession Peptide Modified Sequence 500ng_1 500ng_2 500ng_3

This format is more friendly for my data analysis.So is there any .skyr file for exporting the quantification in both protein and peptide level?I know that skyline support exporting the results mannuly.But there're too many informations here.So I want some useful information by default. And for the data format transformation,is there any avalible tools here?

view request
Hide from View
(2 responses) Jan Sklenar 2020-06-09

Hi Brendan,

I would like to be able to hide some chromatograms from view, without completelly removing them from document. Is it possible? The reason is, I like to keep all data for future purposes, but wish to compare only some runs in my QC data document.


view request
MS1 filtering mode for lipids
(6 responses) Eric Merkley 2020-12-01

Is there a way to use Skyline in an MS1 filtering mode similar for what you would use for peptide untargeted DDA data?

view request
Issue with Skyline transition list export
(7 responses) Shalini Aggarwal 2020-10-27
Dear Skyline team,

I am facing an issue with the export of the transition list from skyline daily software. It is showing the below shown error. I cross-check each step but still unable to figure out the issue. My colleagues tried the same thing on their PC and it worked. Kindly guide me with the error rectification.

thank you in anticipation,
view request
Specifying Integration Boundaries
(20 responses) jrenders 2020-11-09

Hi there,
My small molecule analysis methods produce some pretty ugly peaks for a few analytes. Skyline does its best, but I do not expect any integration algorithm could figure out some of my data. What I have been searching for to fix the problem is some place to specify explicit integration boundaries. In reading back on support, I understand that "explicit retention time" and "window" will get me close but these are more "suggestive" to skyline (and in fact do not work for my peaks). Integrating a peak and then rt-clicking and hitting "Apply Peak to All" also seems more suggestive than explicit and does not work. I would like to have skyline integrate all peak area between two specified RT's without any consideration to inflection points or anything else and then apply this setting across the whole batch for that particular molecule. Is that possible at this time? Thanks!

view request
Sequential protein extraction analysis
(1 response) francescodemetrio lofaro 2020-11-30


I would like to performe a DDA analysis on three sequential protein extraction on each sample comparing two different conditions. My idea is to import DDA search from Mascot engine merging all technical replicates of all fractions together and import the dda search on Skyline.

  1. How i can specify that .raw files are fractions of the same sample?
  2. Is it possible to consider the total amount of the same protein combaning peaks of the three fractions of the same sample?

Thank you for your help


view request
Adding peptides manually in spectral library
(1 response) mdshadman abid 2020-11-30

I am using software PEAKS for the peptide search in my samples and also using these database search from peaks as the spectral library in skyline. However there are some peptides present in the sample but peaks could not detect somehow. That is why when I am generating a spectral library, I am missing those peptides. Is there any way to add/input peptide sequence manually in the spectral library?
I tried by going to Edit- Inset-peptide- then just manually typed the peptide sequence to add the peptide in the library, but I think it's not proper way of what I want to do.
In case I am unable to ask my question, I am giving an example. I know the peptide LYQNKPRRPYIL is present in my sample but PEAKS software couldn't detect it, so it is also missing in spectral library as I am generating library from PEAKS. How can I manually add this peptide in my spectral library?

view request