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.

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

  • A detailed description of your problem or question, including instructions for re-creating any problem that you are encountering. Screenshots are often helpful.
  • 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.

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Showing: limited to 100 requests
Exclude counting of decoys from prot, pept, precurs, transitions numbers in target list
(2 responses) dkueltz 2020-08-06

Hi Brendan et al.,
When constructing DIA assay libraries from raw spectral libraries by sequentially applying QC filter criteria my lab is routinely recording the numbers of proteins, peptides, precursors, and transitions remaining after each filter step. We get these numbers from the display on the lower right hand corner of the skyline app. However, these numbers currently include decoys and we have to delete the decoys and then paste them back tp the target list after every filter step. It would be nice to not have to do that. So I am asking if you could program Skyline such that it excludes decoys automatically from the display in the lower right hand corner of the screen.

view request
(Loss of) resolving power when importing mzXML files from HRAM QTOF
(8 responses) anders honore 2020-08-04

Working with small molcules in MS1 from Bruker QTOF instrument. Data is stored as centroided and converted to mzXML (by Bruker converter) prior to upload. Resolution is typically +40,000.
After import resolving power is reduced to <10,000 and otherwise obvious peaks when reviewed e.g. in MZmine2 becomes severely distorted. Appears as though resolution is reduced to <<10,000
Transition Settings: precursor as monotopic, full scan count, peaks 3, precursor mass analyzer TOF, resolving power 50,000
Include all matching scans

Probably error-40 by a novice user - but please advise...

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PTM annotation
(2 responses) rajirathore3 2020-08-06

Hi Skyline Team

I am working on Glycation (a PTMs) of selected peptides of Albumin protein where I look for different types of glycation modifications of each peptide but when I add them in workflow it sums up the modifications shown in attachment. I have tried first with one of the representative peptide KVPQVSTPTLVEVSR where I specified 3 different glycation modifications on first lysine site. I added them separately in modification list and I was expecting it to be annotated separately but it has summed up two different lysine modifications and which does not meet my experimental workflow.
I want it to be annotated like K(58)VPQVSTPTLVEVSR and K(72)VPQVSTPTLVEVSR not K(130)VPQVSTPTLVEVSR. Please suggest me how this can be performed.


 modification issue_SKY.pptx 
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Assigning peak colors for small molescules
(3 responses) sarah williams 2020-08-06


I am working on a project with tmt labeled small molecules. I would like to assign colors for the MS1 peaks I have assigned for each of the small molecules and their tag. I noticed there is a tutorial for this for peptides, but I can not seem to make it work for my small molecules.

 Colors Peaks skyline support.JPG 
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Prosit - error
Ihor 2020-08-06

1) I am trying to build a Prosit library in Skyline v. and I get the error message (copied below). This error message also pops up on diff. computer systems and Skyline files. Please note that when I click on Options-->Prosit it does mention 'Server Unavailable' ( - is this related to the error?
2) Prosit is able to predict optimal (N)CE based on synthetic peptide library - is Skyline able to use that information from Prosit and suggest the best NCE for each peptide?

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")
   at System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
   at System.Runtime.CompilerServices.TaskAwaiter.HandleNonSuccessAndDebuggerNotification(Task task)
   at Grpc.Core.Internal.AsyncCall`2.UnaryCall(TRequest msg)
   at Grpc.Core.DefaultCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   at Grpc.Core.Interceptors.InterceptingCallInvoker.<BlockingUnaryCall>b__3_0[TRequest,TResponse](TRequest req, ClientInterceptorContext`2 ctx)
   at Grpc.Core.ClientBase.ClientBaseConfiguration.ClientBaseConfigurationInterceptor.BlockingUnaryCall[TRequest,TResponse](TRequest request, ClientInterceptorContext`2 context, BlockingUnaryCallContinuation`2 continuation)
   at Grpc.Core.Interceptors.InterceptingCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   at Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, CallOptions options) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:line 96
   at Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, Metadata headers, Nullable`1 deadline, CancellationToken cancellationToken) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:line 86
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 230
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 234
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.PredictBatches(PredictionServiceClient predictionClient, IProgressMonitor progressMonitor, IProgressStatus& progressStatus, SrmSettings settings, IList`1 inputs, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 314
   at pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibraryOrThrow(IProgressMonitor progress, IProgressStatus& progressStatus) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:line 113
   at pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:line 69

Thank you
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Software crashing while importing result files
(3 responses) alaine garrett 2020-08-05


I am currently working through the tutorials and have had the software crash during the import of results steps in both the Targeted Method Refinement and Processing Grouped Study Data tutorials. I am able to open the raw data files in Qual Browser so I believe that it is not a function of the raw data files being corrupted. We did have a difficult time getting the software installed on our off line system.

Any assistance would be greatly appreciated.

Thank you

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Max LOQ bias calculation
(2 responses) Zac 2020-08-06

Is there any documentation on how the Max LOQ bias is determined, or can you it explain it briefly? Thanks

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Manuscript on Biotin Acceptor Peptide
akulyyasov 2020-08-06

Dear Skyline/Panorama team and users,
I have uploaded the graphical abstract of the manuscript which I prepared during lockdown time.
In this paper, I summarized the application of Biotin Acceptor Peptide 1070 which is used in proximity utilizing biotinylation method.
The paper contains the description of its design, construction, and quantitation by Skyline software on the example of DNA dependent interaction of Sox2 and Oct4.
If someone would like to read the text of the article, please send a message to my e-mail
I will be very grateful for any help in improving the text, remarks, and comments on this manuscript.

With best regards,

view request
Crosslink search from Mascot to create library for Skyline
(2 responses) sandberg 2020-08-05

Is it possible to use crosslinking results from the Mascot search engine to create a library to search the files for crosslinked peptides in Skyline?


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Issue downloading skyline daily
(1 response) wlstutts 2020-08-05


I receive an error message when trying to download Skyline Daily. Any suggestions? I have tried two different computers. Thanks!

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Ion Mobility Window Type settings with Bruker timsTOF data
(1 response) Sebastian Winter 2020-08-05


I have experienced trouble with the latest Skyline Daily version when exporting a method for Bruker timsTOF. Regardless of the parameters entered for the "Window Type" under "Ion Mobility" in the Transitions Settings, the exported targets are always isolated with a 1/K0 window of 0.4. Setting different values for "Fixed Width" or"Resolving Power" have no effect on the exported window size.
Does anyone have an idea what could be wrong?



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Waters HDMSE Raw Data Visualization
(14 responses) brad williams 2020-04-13

I have been testing the latest Skyline builds (20.1) and Skyline Daily ( and they both do not allow the raw data to be visualized for MS1 precursors. The raw data plot shows up blank and the MS/MS information is there. Has this been reported before? I have attached a screen capture to show an example. The datafile type is HDMSE.

 MS1 Spectrum Viewer_041320.pptx 
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Time - course analysis: data export
(1 response) david morgenstern 2020-08-04


I'm running a time course analysis of a certain compound - and i have a quantification trace for that compound through time.
how can export the intensity value (i.e. height) for each sampled time point? is it possible to generate a smoothed trend line and export those values?


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Skyline truncating small molecule peaks that appear to have more data...
(8 responses) jrenders 2020-07-17

Hi There,
I am running a small molecule calibration curve using skyline. On my instrument, I am acquiring data via an inclusion list, so MS1 data matching to my list triggers an MS2 event for that mass. This inclusion list is also scheduled but my RT's are set correctly with 1.5 min of buffer on each side of the peak (on a 10 min method - so a pretty wide window).

When I import the data into skyline I see all expected peaks but often times the leading edge of the peak is highly truncated. This truncation often throws an error which can prevent proper integration and calibration ("MS1.PNG") - "The selected replicate has missing or truncated transitions". I am able to "rescue" these peaks using a trick Brendan mentioned in a similar thread (by bumping the integration boundary inward), however, this truncation is an error as far as I can tell because when I interrogate the data in a vendor spectral viewer I can see that there is data that doesn't seem to make it into skyline. I have attached an image from the vendor software ("Truncated_Peak.PNG") which shows that this mass appears (within the mass tolerance) in the MS1 dimension at a RT that is not properly tracked by skyline in the MS1 dimension. I am thinking I may have some setting set too strictly which is causing the peak to not be picked up until part way through it's elution?

I have attached the skyline document .zip here. In this document I am quanting using both MS1 and MS2 data. Most of the truncation occurs in the MS1 dimension (see for example the 1000 cal point which throws an error). I also see some strange integration in the MS2 dimension ("MS2.PNG") where it appears skyline is trying to "connect" data points that are separated by a long stretch of time (see for example the 500 cal point) and therefore not properly allowing the peak to discontinue or return to baseline. This also appears to me to be related to peak truncation and an improper parsing of data that I believe is actually present in the raw file and which would make these peaks look more normal...

Thanks for any help you can provide!  MS1.PNG  Truncated_Peak.PNG  MS2.PNG 
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SMILES InChi in Reports
(14 responses) Christina lucas 2020-07-27

Hi there,

maybe I am to lost in all the report possibilities...:)

In SM Mode, you can enter SMILES and Inchi/InchiKey, HMDB and all this...

If I want to add it to my Report, where can I find it in the Report Editor?
I had several guesses, thing what it might be in "Proteinlanguage" , like Proteinname for Molecule Name e.g., but Proteinsequence and so on did not work...

I would be very grateful for a tip :)
Best, Christina

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LC-MS/MS data file input for Skyline
(3 responses) avaldiviezo 2020-07-29
I've been using Skyline for a few months to analyze data files generated by Ion Mobility - Mass Spectrometry and the software works very well. Recently, I have been trying to use Skyline to analyze data generated by Liquid Chromatography - Tandem Mass Spec, but I cannot see any peaks when I upload the data files after importing my transition list. Is there a particular way I need to set up the molecule/transition settings or transition list settings in order to analyze LC-MS/MS data?

Any help with this issue is greatly appreciated!

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Skyline Access Error
(2 responses) diana saggese 2020-07-29

I cannot access Skyline documents made using a previous version. Could I please get access to these files?


 Skyline error.PNG 
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Applying manual integration windows when adding new transitions/fragments for an existing compound in Targets
(3 responses) Emmanuel 2020-07-27

Dear Skyline team,

I've manually adjusted the integration of a targeted small molecule across a dozen of samples and I'd like to add few more transitions/fragments for that compound (additional fragments found in another database than the first ones).

Is there a simple way to apply the manual integration boundaries to the new fragments without using the workaround of exporting/re-importing the peak boundaries as a report (as suggested by Tobi some years ago)?

When re-importing the results, it seems that the new transitions are integrated according to the default integration settings without taking into account the manual integration from the previous transitions. Therefore, I need to manually re-integrate all the samples ....

Thanks in advance for your help.


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ERROR: illegal character _ found in sequence [xxxxxxxx].... (line xxx)
(2 responses) siljebt 2020-07-26

I´m experiencing a problem using MaxQuant v1.6.14 and Skyline v20.1.0.155, trying to build a library in Skyline using msms.txt files from MQ. The message I get states "ERROR: illegal character _ found in sequence [ ].... (line xxx)". I see that the message refers to a "_", but I cannot find it.

Skyline import /was/ working nicely with the most conventional PTMs (oxidation, carboxylation, etc), but when I built on with additional PTMs in my MQ search the problem arose. It at first seemed like the problem arises when deamidation and amidation occur in the same peptide, but after manually deleting the lines that are causing problems - one by one, it seems like my initial assumtions about the cause for the error is not the case.

 Annotation 2020-07-26 184546.png 
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histone quantification
(8 responses) mariette matondo 2020-07-22

I like to do some quantification of modified peptides with the Skyline software.
For identification I use the MaxQuant software v1.6
To quantify my data, I import the msms.txt from the MaxQuant search into Skyline I also paste the modification.local.xml file from MQ.
But I cannot import my peptides
in attached files the msms files and modification.local. xml file

Could you please help me

Many thanks,

 msms.txt  modifications.local.xml 
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Intermittent Error: Could not connect to Skyline
(1 response) mmarx 2020-07-22

I am running the Agilent Automation tool and intermittently I am getting the error message when I click 'Create Project':
Error: Could not connect to Skyline.
Make sure you have a valid Skyline installation.

It usually occurs after Skyline and the Automation tool have been idle for a period of time.
Sometimes, if I just try again it will work. Other times, the error continues to occur.

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How can I select endogenous peptides not spiked-in iRT peptides for RT nomalization in Skyline?
andyzcq 2020-07-23

I have acquired diaPASEF data, but I forgot to spike iRT peptide into the samples.

So how can I select endogenous peptides for RT normalization?

CiRT peptides in the CiRT calculator are not applicable.

view request
Proteomics data processing into sample vs protein intensity matrices
(3 responses) saharak 2020-07-19


I am interested in downloading and processing proteomics data into sample vs protein intensity matrices for various disease types that include healthy control samples.

For the datasets that have been processed with skyline, I found that there is a viewer for windows, however, I wasn’t able to find any library to parse them. How do you recommend that I process them into sample vs protein intensity matrices?

I also found "MSstats" package::SkylinetoMSstatsFormat and lipidr::read_skyline, however both require sky csv files and I am only able to download .sky file. Where can I find the csv format and if I can't directly download it, how do I process the .sky files into csv files?

Also, where would I be able to obtain information on each sample, e.g. whether diseased or normal?

Thank you

view request
AutoQC upload on Panorama issue
(2 responses) Cedric 2020-07-21


I am using AutoQC for uploading my Waters file on Panorama (local installation). For most of my analysis it is working well, but for some project, AutoQC cannot update the results in Panorama. I got the log error message attached. What should I do to fix this?

Thanks in advance for your help. Kind regards


view request
small feature improvement
(1 response) Tobi 2020-07-13

Hi Skyline Team,

hope you had a good start into the week.

The M-1 chromatogram feature is so valuable but heavily underutilized as the user needs to click several times for each single precursor to enable it. Also, its quite a hidden feature.

Can you please add the option on having it enabled for all Precursors, for example by writing "M-1" or sth. else in the window next to p, y, b under Transition filter settings?

With best wishes,

view request
failed attempting to check for an update
(4 responses) andyzcq 2020-07-21

The details about the failure are shown below.

System.Deployment.Application.DeploymentDownloadException: 下载 file:///C:/Users/zcq/Downloads/Skyline-64_20_1_0_76/Skyline.application 未成功。 ---> System.Net.WebException: 未能找到路径“C:\Users\zcq\Downloads\Skyline-64_20_1_0_76\Skyline.application”的一部分。 ---> System.Net.WebException: 未能找到路径“C:\Users\zcq\Downloads\Skyline-64_20_1_0_76\Skyline.application”的一部分。 ---> System.IO.DirectoryNotFoundException: 未能找到路径“C:\Users\zcq\Downloads\Skyline-64_20_1_0_76\Skyline.application”的一部分。
at System.IO.__Error.WinIOError(Int32 errorCode, String maybeFullPath)
at System.IO.FileStream.Init(String path, FileMode mode, FileAccess access, Int32 rights, Boolean useRights, FileShare share, Int32 bufferSize, FileOptions options, SECURITY_ATTRIBUTES secAttrs, String msgPath, Boolean bFromProxy, Boolean useLongPath, Boolean checkHost)
at System.IO.FileStream..ctor(String path, FileMode mode, FileAccess access, FileShare share, Int32 bufferSize, FileOptions options, String msgPath, Boolean bFromProxy)
at System.Net.FileWebStream..ctor(FileWebRequest request, String path, FileMode mode, FileAccess access, FileShare sharing, Int32 length, Boolean async)
at System.Net.FileWebResponse..ctor(FileWebRequest request, Uri uri, FileAccess access, Boolean asyncHint)
--- End of inner exception stack trace ---
at System.Net.FileWebResponse..ctor(FileWebRequest request, Uri uri, FileAccess access, Boolean asyncHint)
at System.Net.FileWebRequest.GetResponseCallback(Object state)
--- End of inner exception stack trace ---
at System.Net.FileWebRequest.EndGetResponse(IAsyncResult asyncResult)
at System.Deployment.Application.SystemNetDownloader.DownloadSingleFile(DownloadQueueItem next)
--- End of inner exception stack trace ---
at System.Deployment.Application.SystemNetDownloader.DownloadSingleFile(DownloadQueueItem next)
at System.Deployment.Application.SystemNetDownloader.DownloadAllFiles()
at System.Deployment.Application.FileDownloader.Download(SubscriptionState subState, X509Certificate2 clientCertificate)
at System.Deployment.Application.DownloadManager.DownloadManifestAsRawFile(Uri& sourceUri, String targetPath, IDownloadNotification notification, DownloadOptions options, ServerInformation& serverInformation)
at System.Deployment.Application.DownloadManager.DownloadDeploymentManifestDirect(SubscriptionStore subStore, Uri& sourceUri, TempFile& tempFile, IDownloadNotification notification, DownloadOptions options, ServerInformation& serverInformation)
at System.Deployment.Application.DownloadManager.DownloadDeploymentManifest(SubscriptionStore subStore, Uri& sourceUri, TempFile& tempFile, IDownloadNotification notification, DownloadOptions options)
at System.Deployment.Application.DeploymentManager.BindCore(Boolean blocking, TempFile& tempDeploy, TempDirectory& tempAppDir, FileStream& refTransaction, String& productName)
at System.Deployment.Application.DeploymentManager.Bind()
at System.Deployment.Application.ApplicationDeployment.CheckForDetailedUpdate(Boolean persistUpdateCheckResult)
at pwiz.Skyline.UpgradeManager.AppDeploymentWrapper.CheckForDetailedUpdate() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\UpgradeManager.cs:line 339
at pwiz.Skyline.UpgradeManager.updateCheck_DoWork(Object sender, DoWorkEventArgs e) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\UpgradeManager.cs:line 103

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Isotope ([M+1], [M+2])
(2 responses) dbergmann 2020-07-22

What could be the issue for precursor isotopes ([M+1], [M+2]) not to show up at all? Filter is set up okay as well as full scan in my opinion; could there be any other setting that might be missing or a missing column whenever transition list is uploaded? 
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Avoidance of missed cleavages, methionine, cysteine
(2 responses) ddickerson 2020-07-20

I understand that proteases with high numbers of missed cleavages are to be avoided if using Skyline. Also, peptides which contain methionines or cysteines, which can be post-translationally modified, are also to be avoided. I was asked to work on a project which involved trypsin+Glu-C digested peptides (with high numbers of missed cleavages), and also many peptides which contained methionine and/or cysteine. A lab mate had already done some work on samples of such peptides using DDA, and he asked me to do the PRM. The samples were in quadruplicate. My results gave similar results to his, and many of the peptides detected by PRM had coefficients of variation <.05 for the 4 repeats, and the differential expression volcano plots showed many peptides had significant p-values. I used 9 maximum missed cleavages for digestion and included a background proteome with 9 max missed as well. The results seem plausible, and the corresponding trypsin-only digests gave far fewer results. Can you please confirm whether I need to throw away this data.

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Base Peak not showing in but visible in
(2 responses) H.Schmidt 2020-07-20

Dear Skyline community,

I have FullScan-PRM data from a QExactive.
Importing those to Skyline let me see the TIC but give me back a "No base peak chromatogram found" when selecting Transitions-->Base Peak.
However, Skyline version shows me the Base Peak of the very same data with Transitions-->Base Peak.
MS1 filtering is setup for both Skyline files in the same way.

I would like to know how I receive the Base Peak in the actual Skyline version.

Best regards.

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Peak area of molecular contaminant during specified time window
(1 response) lbergluecke 2020-07-17


Is is possible to look at peak areas (in the replicate comparison view) for a specified time window ? I am interested to see what a molecular contaminant's peak area is during the gradient not the wash.

Let me know if my question does not make sense and I will try to explain better.


view request
Skyline document with cross-linked peptides
(9 responses) danielacgranato 2020-06-27

Sorry, I forgot to attach the documents in the prior message (pLINK output and prox.xml converted). Thank you very much. Best, Daniela 
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Transition export for small molecule CE optimization not working
(2 responses) stolltho 2020-07-14

Hi sky team.

I followed the CE opt tutorial, but the transition export does not seem to work for me. It's only exporting transitions for one (the lowest) CE.
Could you please check?


PS.: It would be nice if you could also adjust MRM and sMRM transition export templates for Agilent QQQ, this would save time re-formatting each export
(Agilent QQQ export template attached)  PGD2 tran.csv  Agilent QQQ export template.xlsx 
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Export Peak Areas light and heavy separate
(1 response) camiel aarents 2020-07-15

Dear operators,

For my experiment i spiked heavy labelled isotope peptides in all my experimental samples to quantify my peptide of interest.

i found that via export-> report -> Peptide Ratio Results i get the ratio between sum of heavy and light transitions in my sample which is very usefull. However i would also like to have the sum of light and heavy transitions separate (thus not a calculated ratio). Is this also available in an export file?

Best, Camiel

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Top 5 quantitation
(1 response) swg42 2020-07-15

I'm new to using skyline and was hoping to get some help on how best to output the top 5 intensity peptides for label-free quantitation? Any suggestions?

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Identifying proteins
(1 response) p pereira79 2020-07-14

Dear ones,

I am using Skyline to identify proteins from a TOF analysis.
Well, I'm using a UniProt FASTA file as a library (75,000 proteins, and countless peptides), and the upload of RAW files takes too long time (it's taking more than 24 h just one file of size 1.32 Gb!).
Then, I would like to know:

  • Does Skyline work without internet?
  • Why is the upload taking so long?
  • Is it possible for the skyline to connect with a library (like Uniprot) online?

Best regard.

view request
Error MSConvert demultiplexing
(12 responses) wendy white 2020-07-09

Hi Skyline team!

My goal is to acquire staggered DIA using a Thermo Exploris 480 ( Garcia Lab Histone methods on QE or HF). Unfortunately, the Exploris is missing features that allow it to upload an inclusion list from Skyline. The method I made on Exploris looks similar but I can't get MSConvert to demulitplex. Could you please take a look at error and let me know if there is a problem with the method, or can I change the MSConvert settings to process my data?

Thank you!

C:\Skyline Documents\2020_0708_histone_std_combinedList_01.raw

Opening file "C:\Skyline Documents\2020_0708_histone_std_combinedList_01.raw" for read...
Calculating SHA1 checksum...
Writing "C:\Skyline Documents\output\2020_0708_histone_std_combinedList_01.mzML"...
Failed - System.Exception: SpectrumToIndices() Number of demultiplexing windows changed. Minimum window size or window boundary tolerance may be set too low.
at pwiz.CLI.msdata.MSDataFile.write(MSData msd, String filename, WriteConfig config, IterationListenerRegistry iterationListenerRegistry)
at MSConvertGUI.MainLogic.processFile(String filename, Config config, ReaderList readers, Map2 usedOutputFilenames) at MSConvertGUI.MainLogic.Go(Config config, Map2 usedOutputFilenames)

view request
Maintaining Integration Windows When Re-importing Data
(8 responses) meyermr 2014-02-28
Hi Skyline Team,

I'd like to know if Skyline can maintain integration windows manually set by the user when re-importing a data file? For example, when I manually set the integration window for a given peak, add additional fragment ions to the peptide tree, and then re-import the data...Skyline will shift the integration windows slightly. Is there a way to override this so I don't have to manually go through and reset the integration windows?

Thanks for the help,
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iRT regression based failure to import DIA runs
(3 responses) kguehrs 2020-07-09

Dear Skyline team,

I know that similar issues were already discussed but I am somewhat frustrated and not sure how ti further proceed.

I did 6 DIA runs for 2 preparation schemes of 3 samples similar to 3 biological replicates for 2 conditions. I added the Biognosys 1 peptides iRT reference to all sample and also used the Biognosys 11 calculator in Skyline in the peptide settings.

Four of the six runs were imported without problems. The remaining two run were not imported with the failure message shown in one of the screenshots in the attached zip archive. I added 6 additional figures that show the regression line for each of the samples together with the extracted peaks for one of the Biognosys 11 reference. We use for some special reason rather complex gradients that are optimized for the main samples that are often measured and that might be one possible explanation for the deviations of the retention times of some of the iRT peptides, but this behaviour is shared wth all samples.

I have tried to use different iRT calculated based on the Biognosys 11 with several samples peptides added but without success. I have also tried to use a Biognosys reference with only 9 or 10 peptides but this did not result in successful import. I also reduced the transitions to 3 product ions only without success of reimport.

I have asked a colleague to use the dataset with Biognosys software and he managed to import the sampled without any problem. Therefore, I assume that the problem is in Skyline and you might think one more time about the idea of r*r>=0.99 for the iRT regression being the most appropriate setting for import of DIA data that are more noisy and not that simple as PRM chromatograms.

Another suggestion relates to the import procedure. As far as I understood from previous discussions of the import issue the r*r>=0.99 for the iRT regression is calculated at the beginning of the import process but the error message is only shown after finalizing data import. In my trials I always had to wait some 10 minutes (rather old computer) until I had to realize the failure. If my assumption of the process timing is correct it should be possible to give an early message and possibility that the user can cancel the process to change settings and retry more early.

Perhaps, you can supply some more ideas how to progress as I am running out of ideas for further steps.

Thank you for your good work to offer Skyline for the community. 
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Making libraries for synthetic peptides and packaging them into single file in skyline
(4 responses) SRS 2020-07-07

Hi There,

I have been trying to make a spectra library of about few thousands synthetic peptides from a DDA run. I was able to import the data using "mziden" and "mzxml" files from the search and mass spectrometry data, respectively. But I have two questions here, 1) how can I export all the spectra libraries into a single file in skyline?

  1. How could I do the peak picking for over thousand peptides, as they are so many, it is not possible for me to open them to make sure the RT is aligned.

Do I need to make sure the alignment of the RT is correct then package them?

I can send you the skyline file if that help,

Thank you, looking forward to hearing from you.


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Quantifying on multiple fragment ion isotopic peaks
(3 responses) johan gobom 2020-07-08

Dear Skyline Team,

I’m setting up a PRM method for large peptides, around 4.5 kDa, +4-charged, on an Orbitrap. For many of the fragments, the monoisotopic peak is not the strongest by far, so I would like to quantify on up to four isotopic peaks for each fragment. Is this possible to do in Skyline?

All the best,


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Bug in document grid reporting
(4 responses) jmeyer 2020-07-08

Dear Skyline team,

I believe I found a bug in skyline reporting identifications in runs where the peptide was in fact not identified.

Please see the two attached screenshots where there is clearly no identification line in the chromatogram, but the document grid reports "TRUE" for an identification in that run (top line in the document grid). These are two separate peptides. When I manually drag the chromatogram to re-integrate anywhere in that run, even the same area, it goes to FALSE.

Example 1 is R.SEQEDEVLLVSSSR.Y [27, 40] ++

Example 2 is R.YPDQWIVPGGGMEPEEEPGGAAVR.E [41, 64] +++

I also attached a minimal skyline share with these two peptides, and the other peptides in the same protein.

Any help fixing this or providing a workaround would be greatly appreciated.

Best regards,

 Inkedwrong ID 1 skyline_LI.jpg  wrong ID 2 skyline.JPG 
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Failed to export method for CE optimization
(10 responses) benoit fatou 2020-06-08

Dear Skyline Team,
I tried to export a MRM method to perform CE optimization and I have got this error message (see attached file).
Could you please help me solve this issue?

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TMT0 Labeled Peptide as Internal Standard for TMT10 Targets
drothenberg 2020-07-07

Instead of using a heavy labeled synthetic peptide as an internal standard, I'm interested in using a TMTzero labeled peptide to compare against TMT-10plex labeled targets. I know ratios can be calculated using the Surrogate Standard option, but is it possible to have synchronized zooming and transition matching similar to heavy-labeled peptides? This would be similar to the TOMAHAQ approach, but without MS3. Thanks.

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QuaSAR Error
msj0506 2020-07-06

Dear Skyline Team,

I have a problem using QuaSAR tool in Skyline.

Now, my desktop system is window 10 and QuaSAR tool is connected R 4.0.0 ver in Skyline.

But, When i used QuaSAR for calculating LOD and LOQ, the error occurred continuously. i attach the related file.

Reinstallation does not solve the problem, and the same problem occurs in my laptop. Can you tell me how to solve this?

 Error(1).PNG  Error(2).PNG 
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Calibration curve calculations problems.
(2 responses) monika lysakowska 2020-07-06

I have a problem calculating the calibration curve. The calculations Skyline does are different from my calculations in Excel.​I have entered all the appropriate values for settings and grids
and I got a good calibration curve. R square and intercept is identical to the calculations in the excel spreadsheet, slope differs by two decimal places (I think it is related to the Y-axis conversion). However, the calculated values by Skyline Quan differ slightly from heavy normalized to light values from the excel worksheet.

The data should match the normalized skyline. Currently they are about a percentage point off, which is substantial. It’s about 20% difference between the two. What can explain this?​

Monika Lysakowska

 1.png  2.png  3.png 
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Install issue
(3 responses) Brett Phinney 2018-01-30
Hey everyone, not sure if this is just my messed up win10 computer but I tried to install

And win10 would not let me install it even as the admin.

This seemed to fix it

just an FYI if someone else has this issue


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White screen on Collision Energy Regression
(1 response) benoit fatou 2020-07-03

Hi Skyline Team,

I am doing some CE optimization on a Shimadzu instrument.
After editing the Collision Energy Equation, I would like to visualize the corresponding graphs but I have a white screen after clicking on "Show Graph...".
I have the same issue when I select default equations from other instrument vendors.

Let me know if you need more information and I am happy to send you the Skyline document.
Stay safe!

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Failure opening
(1 response) Flora.Sh. 2020-07-03

Dear Skyline Team

At my workplace I work a lot with skyline. If I want to work at home at my laptop there is a problem. I can not open the files because it says that my document format version is 20.13 and newer than the version 20.1 supported by skyline (64bit) (a0e7323e3), which I have in my laptop. How can I change that or what can I do? There is not a newer version at your homepage.

Thanks in advance for the answer

Best wishes


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Lipid isomer data import problem in scheduled MRM
(4 responses) stolltho 2020-07-02

Hi sky team.

A similar problem was reported earlier (Small Molecule MRM extraction with isomers) but it seems the problem persists.

I've got a long list of lipids, some of which have the same transition pairs (774.6>184.1).
Following scheduled MRM data import, skyline is showing only one / the same sMRM window for all the lipid isomers.

I already changed product ion m/z by 0.01 (774.6>184.10, 774.6>184.11) to circumvent the problem and it worked, but unfortunately the QQQ we are using is not acquiring identical peak profiles for this slight shift.

Hence I changed product ion m/z by 0.0010 (774.6>184.1000, 774.6>184.1010, 774.6>184.1009 etc.), but now I face the same import issue again, showing only one / the same sMRM window. I changed match tolerance to 0.0001, but then no chromatograms are shown after import.


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different heavy IS concentration
(3 responses) Wael 2020-07-02

Dear Skyline team,
as we do multiplexed analysis we might need to adject the amount of spiked IS standard depending on the endogenous peptide level to be within a defined (SIS/EN) range. Is it possible to feed the results grid with different IS standard concentration for each peptide individually, or we should create a different skyline folder for each analyte to be able include the particular SIS concentration?

The issue also applies when creating a standard curve using range of different EN concentrations since the dilution curve concentration range vary depending in the analyte.

Best regards

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Import of Mascot error tolerant searches in Skyline
ingo.wohlgemuth 2020-07-02

Hey Skyline team,

I try to import error tolerant Mascot searches with a focus on amino acid substitutions. While many PTMs can be imported, amino acid substitutions which were found in Mascot are often lost in Skyline (e.g L to V mutations, see picture). Is the a rational for that? How Sykline handles cases is which the delta mass could not unambiguously assigned (e.g: D to E substitution vs D methylation) when both interpretations are available in Mascot?

Thanx a lot!

 Amino acid substitutions.jpg 
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collision energy prediction for QTRAP 6500+
(1 response) mark adams 2020-07-01

Hey Brendan,
We are currently using a SCIEX QTRAP 6500+ platform for protein-based work. Prior to transition list export, would a collision energy prediction setting of 'SCIEX' or 'ABI 5500 Q Trap' be more appropriate for our instrument platform?
Thanks for any input!

 Annotation 2020-07-01.png 
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SAV recognition by speclib builder
(1 response) dkueltz 2020-06-27

Hi Brendan et al.
I am using SPIDER within PEAKSsuiteX to identify allelic variants that manifest themselves at the protein level as single amino acid substitutions (SAVs). These can be exported from PEAKS to pep.xml + mzxml files that Skyline uses to built spectral libraries from DDA data. However, unlike unmodified peptides and regular PTM peptides, the SAV peptides are not recognized by the spec library builder. I was not able to open the mzxml files but the structure of SAVs is a bit different from that of regular PTMs in the pep.xml files (see below). I would be happy to send you a pair of pep.xml + mzxml files that can be used for speclib construction with Skyline and contain umnmod. PTM, and SAV peptides. Is there any way that I could import these SAV peptides into Skyline spectral libraries such that I can quantify the stoichiometry of allele-specific expression (ASE) in different biological contexts? If this is not possible yet, do you have any plans to implement this capacity into Skyline in the future?
Below is part of a pep.xml file that shows 3 entries, one each for a regular PTM, SAV, and unmod. peptide... in this particular example the PTM and SAV are actually the same - deamidation of Q to E - but there are many other instances with unique SAV mass changes that do not match a conventional PTM and can be interpreted unambiguously...
<spectrum_query spectrum="DK0020-B1B_2-A,1_01_2365.d" start_scan="2626" end_scan="2626" precursor_neutral_mass="1515.7789" assumed_charge="3" index="1303">
<search_hit hit_rank="1" peptide="LSNGTTKPQTNGVAK" calc_neutral_pep_mass="1515.7893" massdiff="4.7" num_tot_proteins="1" protein="XP_033982937.1" protein_descr="mucin-5AC [Trematomus bernacchii]" protein_mw="124905.71">
<modification_info modified_peptide="LSNGTTKPQ(+.98)TNGVAK">
<mod_aminoacid_mass position="9" mass="129.04259"/>
<search_score name="-10lgP" value="44.82"/>
<search_score name="confidence" value="0.99"/>
<search_score name="PeaksScore" value="99.8"/>
<analysis_result analysis="reconstruction">
<tags denovo_tag="LSNGTTKPQTNGVAK" reconstructed_tag="LSNGTTKPQTNGVAK"/>
<spectrum_query spectrum="DK0020-B1B_2-A,1_01_2365.d" start_scan="2935" end_scan="2935" precursor_neutral_mass="1515.7789" assumed_charge="3" index="1304">
<search_hit hit_rank="1" peptide="LSNGTTKPQTNGVAK" calc_neutral_pep_mass="1515.7893" massdiff="4.7" num_tot_proteins="1" protein="XP_033982937.1" protein_descr="mucin-5AC [Trematomus bernacchii]" protein_mw="124905.71">
<modification_info modified_peptide="LSNGTTKPE(sub Q)TNGVAK">
<aminoacid_substitution position="9" orig_aa="Q"/>
<search_score name="-10lgP" value="41.80"/>
<search_score name="confidence" value="0.95"/>
<search_score name="PeaksScore" value="95.8"/>
<analysis_result analysis="reconstruction">
<tags denovo_tag="LSNGTTKPETNGVAK" reconstructed_tag="LSNGTTKPETNGVAK"/>
<spectrum_query spectrum="DK0020-B1B_2-A,1_01_2365.d" start_scan="2596" end_scan="2596" precursor_neutral_mass="1088.5857" assumed_charge="3" index="1309">
<search_hit hit_rank="1" peptide="YTRPTPVQK" calc_neutral_pep_mass="1088.5978" massdiff="0.4" num_tot_proteins="1" protein="XP_034007142.1" protein_descr="ATP-dependent RNA helicase DDX3X-like [Trematomus bernacchii]" protein_mw="80330.54">
<search_score name="-10lgP" value="41.08"/>
<search_score name="confidence" value="0.97"/>
<search_score name="PeaksScore" value="96.3"/>
<analysis_result analysis="reconstruction">
<tags denovo_tag="YTRPTPVQK" reconstructed_tag="YTRPTPVQK"/>

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Issue: The following modifications could not be interpreted
(3 responses) Mark Athanason 2020-06-29

Hello Brendan,

I saw another post on your forums from someone with a similar issue, but mine is different. Long story short I performed a search using Comet with a few variable modifications on K,R,S,T,C (drug adducts) along with oxidation of M. When I import my data into skyline to build a spectral library (using the wizard) the Add Modifications window lets me pick one of my adducts and the oxidative M. These are rounded to the nearest one's place. The modifications that cannot be interpreted have 2, 4, and 5 decimal places. In my comet parameters file I have the mass going out to 4 or five decimal places for all variable mods. If you can let me know how to circumvent this using the search data rather than manually entering the variable mod in skyline (future up-scaling of the project), I would appreciate it.


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Different retention times for heavy and light from
(2 responses) Martin Eisinger 2020-06-23


I know the topic has been discussed several time but I didn't find a conclusive answer. I work with small molecules and use fully deuterated versions as internal standards. The deuteration leads to a considerable RT shift between heavy and light form. However if I define them as heavy and light in my transition list, skyline uses the same integration boundaries for both forms. The only solutions I found to overcome this problem is to set very wide integration boundaries, so that both peaks heavy and light are covered. It's ok but no perfect solution as in that case a lot of baseline is also integrated.

Is there any good way of telling Skyline that heavy and light RTs don't have to match and allowing it to use different integration boundaries for both forms?

Thanks in advance!



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Quantifying labelling efficiency
(2 responses) sudip ghosh20975 2020-06-24

Hi I have a set of reference peptides which I labelled with TMT0. I ran both the labelled and unlabelled samples in Fusion and wanted to evaluate the labelling efficiency in Skyline. How do I proceed?

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SkylineRunner.exe to export mProphet Features
(6 responses) s26guan 2020-06-25

Dear Skyline team,

Can you show me the export option for mProphet features? I tried --report-name="mProphet Features" and it was not working.

Got error: "Error: The report mProphet Features does not exist. If it has spaces in its name, use "double quotes" around the entire list of command parameters."

Many thanks.

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Average peptide masses instead of monoiso
(1 response) Zac 2020-06-25

Is there a way to work with average peptide masses instead of monisotopic masses in skyline

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Reprocessing with new parameters
(2 responses) thomas boegl 2020-06-24

I have following problem:
When I change any parameters e.g. adding new molecules ( Insert --> Transition List... ) I do not get any results until I do "Manage Results.." and Re-Import the files. Is there a simpler way to do reprocessing only for the new molecules?
I am currently starting to work with skyline.

Thank you!


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Problem performing iterative CE optimization with explicit collision energies
(6 responses) vincentroyrichard 2020-06-10


I'm currently trying to perform iterative CE optimization for an MRM method on a QTRAP 6500+ instrument using the latest version of skyline daily. What I would like to do would be 3 rounds of CE optimization with successively decreasing step sizes (starting with the CEs derived from the linear equation). What I'm noticing is that for the second pass CE optimization, I can only export a transition list for CE optimization with the optimized CEs (ie not derived from the linear equation) when entering these CE values in the explicit collision energy tab in the documents grid. The problem is that when I'm reimporting the data from the 2nd pass optimization it doesn't "center" around the correct CE.

For example, after optimization of a single MRM transition, the optimal CE was determined to be 18.6. I then reanalyzed the sample with a method to optimize the transitions with 3 steps +- 2 (ie 12.6 , 14.6, 16.6, 18.6, 20.6, 22.6, 24.6 -with 18.6 still remaining the most sensitive) - however when I reimport the data from this into skyline it changes the center CE to 24.6.

Seems to be a bug? Any assistance with this would be greatly appreciated!

Thanks very much!

Vincent Richard

 troubleshoot trans list.csv 
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dotp / rdotp between samples
(2 responses) Juergen 2020-06-23

Dear Skyline team,

I currently use PRM in the identification of several peptides. My reference peptides are, however, not labeled and hence I measure them in separate runs. I know it is possible to get dotp values for each measurement in comparison to the corresponding library spectrum but I would prefer the dot product of the selected transitions in comparison to my control run (similar to the rdotp value for labeled and unlabeled peaks within one run). Is it possible to get this value?

I would like to use this value instead of the dotp against the library spectrum because I guess that than all the MS2 spectra below the peak would count for the calculation, while in the library only those spectra are retained that are nicely identified.

Best regards,

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DIA method for Exploris 480
1kubo f 2020-06-23

I would like to ask whether Skyline has a function to export a DIA method (MSX) for Exploris 480 operated by Xcalibur Thank you, best regards Jakub

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Interesting error: TIMSTOF and Mascot
(4 responses) Jason Held 2020-06-12


I'm trying to build a library from TIMSTOF data search with mascot. I'm using the DAT files to build the library and getting this error:

System.IO.IOException: ERROR: Maximum limit of 2000 spectrum source files was exceeded. There was most likely a problem reading the filenames.
ERROR: reading file F013408.dat
ERROR: Maximum limit of 2000 spectrum source files was exceeded. There was most likely a problem reading the filenames.
ERROR: reading file F013416.dat

Command-line: C:\Users\lab_held.pcl-held1\AppData\Local\Apps\2.0\NRM38HKN.2B5\OM37C28G.N7K\skyl..tion_e4141a2a22107248_0014.0001_1e739a267632795e\BlibBuild -s -A -H -o -c 0.99 -i Bowman005_TIMSTOF -S "C:\Users\lab_held.pcl-held1\AppData\Local\Temp\tmp2696.tmp" "C:\Skyline\SpectralLibraries\Bowman005_TIMSTOF.redundant.blib"
Working directory: S:\Users\Jason\Bowman005-New2CysMutantForPaperTIMSTOF
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.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\pwiz_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Mode\Lib\BiblioSpecLiteBuilder.cs:line 155


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Issue uploading a Skyline Document to Panorama with a library file (.clib)
(2 responses) hogan 2020-06-18


I have encountered an issue uploading a Skyline document to panorama that includes a .clib file. When I remove the .clib file using the peptide settings -> Library tab I am able to upload the file to panorama without issue. I would like to be able to include the .clib file in the document as the library dot product is data we would like to keep in the doc and it is not available if the library file is removed. Here is the error I receive from panorama when I attempt the upload. Thanks for your support!

18 Jun 2020 17:18:19,750 INFO : Starting to run task 'org.labkey.targetedms.pipeline.TargetedMSImportTask' at location 'webserver-high-priority'
18 Jun 2020 17:18:19,762 INFO : Starting to import Skyline document from
18 Jun 2020 17:18:19,772 INFO : Expanding Popeye Chromatogram Library 20min_rev7.clib
18 Jun 2020 17:18:19,876 INFO : Expanding 20200615_H9407_1280777_1280778.skyd
18 Jun 2020 17:18:21,030 INFO : Expanding
18 Jun 2020 17:18:21,059 INFO : Expanding
18 Jun 2020 17:18:21,367 WARN : The version of this Skyline document is 20.1. This is newer than the highest supported version 4.1
18 Jun 2020 17:18:21,390 INFO : 1% Done
18 Jun 2020 17:18:24,287 INFO : Failed to complete task 'org.labkey.targetedms.pipeline.TargetedMSImportTask'
18 Jun 2020 17:18:24,314 ERROR: SqlExecutor.execute(); uncategorized SQLException for SQL []; SQL state [25P02]; error code [0]; ERROR: current transaction is aborted, commands ignored until end of transaction block; nested exception is org.postgresql.util.PSQLException: ERROR: current transaction is aborted, commands ignored until end of transaction block
Caused by: org.postgresql.util.PSQLException: ERROR: current transaction is aborted, commands ignored until end of transaction block
Caused by: org.postgresql.util.PSQLException: ERROR: insert or update on table "spectrumlibrary" violates foreign key constraint "fk_spectrumlibrary_librarysourceid"
Detail: Key (librarysourceid)=(0) is not present in table "librarysource".
18 Jun 2020 17:18:24,378 ERROR: Uncaught exception in PipelineJob: (DONE) Skyline document import -
org.springframework.jdbc.UncategorizedSQLException: SqlExecutor.execute(); uncategorized SQLException for SQL []; SQL state [25P02]; error code [0]; ERROR: current transaction is aborted, commands ignored until end of transaction block; nested exception is org.postgresql.util.PSQLException: ERROR: current transaction is aborted, commands ignored until end of transaction block
Caused by: org.postgresql.util.PSQLException: ERROR: current transaction is aborted, commands ignored until end of transaction block
Caused by: org.postgresql.util.PSQLException: ERROR: insert or update on table "spectrumlibrary" violates foreign key constraint "fk_spectrumlibrary_librarysourceid"
Detail: Key (librarysourceid)=(0) is not present in table "librarysource".

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Case sensitivity for .mzID files
(2 responses) hayes.mcdonald 2020-06-19

When I am trying to import .mzID files from Morpheus searches, I end up having to change the suffix to all lower case mzid before Skyline will process. It isn't a huge deal, but it is annoying as the set of files gets larger.

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Metabolite ID Workflow for Oligonucleotides
(1 response) Richard Lam 2020-06-17

Can Skyline perform metabolite ID for oligonucleotides? If yes, any tutorials or webinars available. Thanks!

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Peak extraction window shifting
(4 responses) rmolden 2020-06-18

My retention times shift very little from run to run, so I would like to use a stringent retention time filtering (0.5 minutes of MS/MS IDs) when chromatograms are extracted to reduce noise. When I import my results I notice that for some peptides the window in which XIC are extracted shift from run to run (see attached), even though the library is built just from one run and I have not applied any iRT calibration. Is there any way to make sure that that extraction window stays fixed across runs?

 RT shift.PNG 
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Change the scale of the peptide intensity
(1 response) benoit fatou 2020-06-18

Hi Skyline Team,

I was wondering if it is possible to do a log2 transformation of the peptide intensities to then export the chromatogram as an image.
The idea is to uniform the intensity and make the chromatogram much nicer to visualize.


view request
Retention time Prediction
(5 responses) scv01958 2020-04-23


I had a question regarding the retention time prediction tool of Skyline. I understand that the tool is capable of using imported data to calibrate a calculator and build up an iRT database. This iRT database is then very useful in validating retention times recorded in future trials. However, I am confused on the "prediction" capabilities of this tool. Let's say I have a calibrated calculator already made consisting of an iRT database of related peptides/molecules. I have a new peptide/molecule that is related, but slightly different in some way, the difference is a change in structure or charge. Will this calculator be able to predict the retention time of my new compound based on the database I have already built?


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PRM - Unstable signals
(5 responses) melanieH 2020-06-15

Dear all,

I would like to have your advices regarding my issue.
We are working with a newly acquired Q-exactive HF-X and I am trying to setup a PRM method to quantify one bacterial protein (matrix : bacterial culture supernatants).
My issue is the signal that I obtain in scheduled PRM method, it seems really unstable but I am not sure if it comes from my method (actually pretty basic, following Thermo recommendations) which could be too much for just a low number of peptides [PRM R=30000, AGC t = 1e5, IT max = 60 ms, isolation window = 1.6m/z ) or if it could come from the instrument itself.
I attached a pdf file with some screenshots. I have more data but the results are similar for all the runs operated in scheduled mode, with or without FullScan. As you can see the signals seem "smoother" when the method is unscheduled.
I am asking if it could come from the instrument because we had once a problem with a lens in the quad that fell down during a cleaning session by the Thermo technician...

All advices are welcome.


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Exporting resolution values
(4 responses) sstoychev 2020-06-16


I would like to export the resolution values for both precursors and fragments in MRM-HR and DIA data but can't find the metrics in the report templates. There are FWHM and Max FHWM values but I think these are related to chromatography rather than mass spectra?



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Probem in creating library from Maxquant results
(6 responses) nicolas pierre 2020-06-10


I want to create a library from Maxquant results but I did not find the modifications.xml file.
So, I have an error message. Where to find the modifications.xml file ?
Thank you for your help,

Nicolas Pierre

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Issues with removing the display of compound names in skyline
(1 response) julien faugere 2020-06-16

How to remove compound names when overlaying multiple MRM transitions ?

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(1 response) ingo.wohlgemuth 2020-06-16

Hey Skyline Team,

can I export individual prosit scores via the document grid?

Thanks a lot!


view request
Special SIL peptide standard library
(7 responses) lyjgbb 2020-06-10

Hi Skyline Team,

I wish Skyline could have a function to allow us to build a special SIL peptide standard library, which includes the customized stable-isotope labeled peptides as standards for our target LC-MS quantification on the unlabeled peptides from either cells or tumor samples. Because we are focusing on the HLA peptides, which are not typical tryptic peptides but they have a kind of diverse peptide sequences, we have a very large SIL peptide library with peptides labeled on diverse amino acids. When we run different samples, we have to choose different SIL peptides from the library, input the corresponding light partners first, and then manually modify the amino acids into SIL version one by one. So this process is very time-consuming and has risks for many human errors. It will be greatly helpful if we can set up a SIL peptide library first in Skyline and then choose the selected SIL peptides per unique sample efficiently from the library, by just inserting the light partners into Skyline. That means Skyline will give users a chance to choose which library (typical protein database or SIL peptide library) they would like to match the peptides with when they insert peptide sequences into the program.

Thank you so much for your great efforts and kind consideration!

view request
How to import amino acid substitutions from ProteinPilot 5.0 search results
(1 response) christof.lenz 2020-06-16

Dear all,

I am looking for a systematic way to import peptide search results from SCIEX ProteinPilot 5.0 including amino acid substitutions. In 'thorough' mode, ProteinPilot allows to search peptide sequences including all amino acid substitutions. Scoring is then adjusted using an underlying BLOSUM substitution matrix and goes into scoring, so we could use a peptide score cutoff here.

I am stumped, however, on how to import the search results including the 180 or so possible AA substitutions into Skyline to look for these peptides e.g. in HR-MS1 or DIA data. Is there any good way to do this, except 'declaring' each potential substitution as a variable PTM?
Data are from a SCIEX TT5600+ with Analyst TF 1.7; PP is version 5.0 rev. 4769; Skyline is on the latest version.

Any hints or experiences much appreciated!!


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Document Grid
Tobi 2020-06-15

Dear Skyline team,

hope you are doing well. I would like to drop some inspiration below.

Could you please add the Acquisition cycle time in seconds to the document grid? I know that people can calculate that themselves, but it would be very helpful to have the option for QC to check it directly in Skyline without having to export first and calculate by hand. The cycle time in seconds would be ((End Time - Start Time) / Points Across Peak * 60s) on the Transition level.

Another really important value was highlighted by the Heidelberg CF, which is the Tailing Factor. It is as old as chromatography itself and crucial in QC for observing tailing which requires hardware maintenance as well as indicating interference for reviewing transitions. It would work extremely well together with peak picking by avant-garde DIA as the boundaries could be set in an X% max intensity fashion. Please see below.

Page 27: User Group Meeting at ASMS/%40files/Presentations/1-01-Sakson.pdf

With best regards,

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Is it possible to use the Skyline program for the quantitative analysis of various modified forms of the same peptide in different samples with known relative ionization coefficient k?
(1 response) akulyyasov 2020-06-12

Dear Brendan,
I have a question for you regarding the use of the Skyline program for the quantitative analysis of various modified forms of the same peptide in different samples.
According to the results of the experiment, I got two samples. In one control tube, a small amount of the biotinylated form of BAP is present, and basically there is a propionylated form of BAP.
In another tube, the amount of biotinylated form of BAP is much higher than propionylated BAP.
Does the Skyline program provide for the possibility of calculating the level of biotinylation between samples taking into account the known value of the relative ionization coefficient k of two modified forms of peptides?

With best regards

This Biotin Acceptor Peptide (BAP) is a non-naturally occurring peptide and is absent in the NCBI and Uniprot database.
BAP is the result of a design in which the amino acid sequence is modified to reduce substrate activity compared to the known natural AviTag peptide.

Biotin Acceptor Peptide (BAP) - tryptic peptide for LC-MS/MS
BAP biotinylated - ILEAQK(Bio)IVR, m/z value of (M+2H)2+ (648.4)
BAP propionylated - ILEAQK(Pro)IVR, m/z value of (M+2H)2+ (563.2)

The relative ionization coefficient k between the biotinylated and propionylated BAP peptides was estimated as k = (HP - LP)/(LB - HB), where HP corresponds to the area of TIC of heavy propionylated, LP of light propionylated, LB of light biotinylated, and HB of heavy biotinylated BAP. K were calculated for two independent experiments, giving an average value 11.9 (Kulyyassov A, et al. PUB-MS: a mass spectrometry-based method to monitor protein-protein proximity in vivo.//J Proteome Res. 2011 Oct 7;10(10):4416-27. doi: 10.1021/pr200189p).

I also uploaded one experiment in Panorama database Center for Biotech Kazakhstan – Mass Spec Lab/BAPSx2_9hrbiotpulsonNidigest1_RA3_01_562/project-begin.view?

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Intact proteins+MRM
(2 responses) z1854947 2020-06-12


I am new to the skyline family.

My current project is specifically focusing on Top down proteomics. I am separating and quantifying intact proteins using Shimadzu Triple-Quadrupole 8045 by optimizing MRM methods.

Can anyone help me by listing the specific tools/setting which will be helpful for my kind of work? Most of the tools on Skyline that I learnt are for tryptic digested peptide.

Also, on the instrument, my files are generated with .mzxlm extension. How can I upload these files?

Thank you.

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Full scan with MS1 filtering--- the intensity of precursor doesn't show up in the chromatogram
(4 responses) yusyuanluo 2020-06-08

Dear Skyline team,
I imported IM-QTOF data (Agilent) with a full scan setting as attached. I couldn't see the "peak" in intensity versus retention time plot.
However, the MS spectra do show up with intensity if I click on a time point in the intensity versus retention time plot. Is there any way that I can resolve this problem?



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Installation on Windows Server 2019
(5 responses) s26guan 2020-06-10

Dear Skyline Team,

Would you recommend a version to install on Windows Server 2019?

Thank you.

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Cross-link Transition Calculator - Problem/input
(4 responses) tatianibl 2020-06-10
Dear Skyline Team,
How are you?

I tried to use cross-link transition calculator tool and I am having a problem during the input process (error message below).

System.Reflection.TargetInvocationException: Exception has been thrown by the target of an invocation. ---> System.Exception: System.Runtime.Serialization.SerializationException: Type 'pwiz.Skyline.Model.LineColNumberedIoException' in Assembly 'Skyline, Version=, Culture=neutral, PublicKeyToken=null' is not marked as serializable.
   at System.Runtime.Serialization.Formatters.Binary.WriteObjectInfo.InitSerialize(Object obj, ISurrogateSelector surrogateSelector, StreamingContext context, SerObjectInfoInit serObjectInfoInit, IFormatterConverter converter, ObjectWriter objectWriter, SerializationBinder binder)
   at System.Runtime.Serialization.Formatters.Binary.ObjectWriter.Write(WriteObjectInfo objectInfo, NameInfo memberNameInfo, NameInfo typeNameInfo)
   at System.Runtime.Serialization.Formatters.Binary.ObjectWriter.Serialize(Object graph, Header[] inHeaders, __BinaryWriter serWriter, Boolean fCheck)
   at System.Runtime.Serialization.Formatters.Binary.BinaryFormatter.Serialize(Stream serializationStream, Object graph, Header[] headers, Boolean fCheck)
   at SkylineTool.RemoteBase.SerializeObject(Object o) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\SkylineTool\RemoteBase.cs:line 60
   at SkylineTool.RemoteService.ProcessClientThread(Object streamArg) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\SkylineTool\RemoteService.cs:line 104
   --- End of inner exception stack trace ---
   at SkylineTool.RemoteClient.RemoteCallName(String methodName, Object[] arguments)
   at SkylineIntegration.Program.Main(String[] args) in c:\Users\Yuval\Documents\Skyline\3-27-17-tool\pwiz_tools\Skyline\Executables\Tools\PRMCalc\c#\SkylineIntegration\Program.cs:line 50

I am using as input:

0.0 0.0
NVVHQLSVTLEDLYNGATRKLALQK KLALQK 837.532@7,156.079@20 3103.692@1 2
GGKKGAVECCPNCR CKSCNGR 1018.4325@3,156.0786@4,57.0215@9,57.0215@10,57.0215@13    57.0215@1,1885.8485@2,57.0215@4    2
GGKKGAVECCPNCR ISPKDRCKSCNGR 1870.903@3,156.0786@4 156.0786@4,57.0215@7,1885.8485@8,57.0215@10 2
GGKKGAVECCPNCR KLALQKNVICDK 1665.9375@3,156.0786@4 156.0786@1,1885.8485@6,57.0215@10 2
GKNVVHQLSVTLEDLYNGATR CKSCNGR 1018.4325@2 57.0215@1,2451.2758@2,57.0215@4 2

Thank you!
Best wishes,
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Instrument setting issue - Document grid bug
(3 responses) Tobi 2020-06-08

Dear Skyline team,

I noticed the Instrument settings/ Min Max m/z applies to both fragments and precursors at the same time. Did skyline always behave like this? I find it a bit troublesome as fragment measurement ranges are nearly all the time much wider than precursor measurement ranges. If I use this setting to properly filter precursors I loose lots of fragments, and if I properly filter fragments I spend alot of time on loading sometimes thousands of nonsense precursors into the target list.

Do you have a good tip or workaround on how I can differently filter precusors/fragments when building a target list?
Do you consider having separate settings for MS1 and MS2 in the near future? (just asking if by chance it makes sense for me to wait a bit).

Looking forward to a short reply.

[Skyilne-daily, add peptide PEPTIDER++, filter for 400 to 500 mz yields only 3 fragments]

Best wishes,

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Hide from View
(1 response) 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.


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DIA-NN .speclib support
(17 responses) Tobi 2020-05-27

Dear Skyline Team,

could you please consider implementing support for DIA-NNs .speclib spectral libraries? Its a highly convenient tool for predicted libraries and much faster than Prosit.

With best regards,

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Shimadzu data file not loading
(3 responses) egauth 2020-06-08

I'm encoutering a problem when loading a specific MRM Shimadzu data file inside Skyline The error is:

Failed importing results file 'E:\Shimadzu Joe\20200601002_Repro_Cocaine_Hexane_003.lcd'.
The sample 20200601002_Repro_Cocaine_Hexane_003 contains no usable data.

I attach the Skyline method as well as the problematic .lcd data file. I also included another data file from the same MS but with a different MS method that loads correctly with the same Skyline method.

Can someone spot where the problem is?
Thanks!  DO_NOT_LOAD_20200601002_Repro_Cocaine_Hexane_003.lcd  LOAD_OK_20200129001_opt_012.lcd 
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Quantification of unlabeled peptides using SIL calibration curve
david schmidt 2020-06-08


once again thanks for this great piece of software!

We are trying to quantify light (native) peptides using their isotope-labeled synthetic counterparts as standards. In contrast to the example in the "Absolute Quantification" tutorial, our calibration curve is generated with heavy peptides in a comparable matrix (and not with different light/heavy ratios). We do, however, spike a fixed concentration of heavy peptides into each sample to compensate for recovery differences.

The goal is to calculate the concentration of the light peptides using the labeled peptide's calibration curve and then to normalize that concentration value to the recovery (measured conc. heavy/spiked conc. heavy). My problem is, how to apply the heavy peptide-based calibration curve for the quantification of light peptides. Is there a way to specify the "internal standard type" separately for the calibration curve and the samples?

Thank you,


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His-tag peptides
(2 responses) jung165 2020-06-04

Hi Skyline team.
I always appreciate for the fascinating tool that is applicable for targeted protemics!

I have one quick question about selecting peptides.
I prepared the his-tag purified and enriched proteins which are transfected in Ecoli.
Therefore, I would like to insert modification about His-tag on my target peptide but, in modification tap I could not find such modification.
Is it applicable to just put 6H(histidine) after my target peptide and inset on Skyline??

I really appreciate for your help

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Support for RDkit isotope aware
(3 responses) landerson 2020-05-26

Does Skyline support RDkit isotope aware molecular formulas? RDKit uses nomenclature that a heavy carbon is [C13]. For example, if 2 carbons in glucose were heavy you might use C4[C13]2H12O6.

I know of the apostrophe " ' " method e.g. C’C5H12O6 that is embedded in Skyline. The RDkit nomenclature feature would make the synergy between a postgresql database more streamlined.

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Peptide Uniqueness
(1 response) Hui 2020-06-02

Hello Skyline Team,

Hope all is well.

I am trying to create an SRM list where we have human samples with mouse heavy standard. Is there a function that works the opposite of "Exclude Background Proteome" under "Edit -> Unique Peptides..."? The goal is to choose peptides that are shared between both human and mice genomes.

Thank you!

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Fix for QuaSAR download error
(2 responses) jung165 2020-03-02

Hello, Skyline team

I have one question about downloading QuaSAR.
I am currently using Skyline 20.1.

When I try to download QuaSAR, it automatically downloads R 3.0 and continue to download QuaSAR related package.
However, the error message appealed with mentioning I need R 3.5 or higher version to download the packages.
So, I downloaded R 3.5 but, error message still appeals and it seems that those packages were still trying to be downloaded in R 3.0 folder.
May I ask your help for this issue??

I appreciate for your help


The following packages failed to install:


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Import PRM results from Xevo QTof
(9 responses) taleb sedighi 2020-05-29
Dear Skyline Support Team, I have a problem in importing PRM result files from Masslynx (acquired on a Waters Xevo G2-XS QTof) into the Skyline. The PRM data includes one MS scans and 5 MSMS scans. Importing completes without any error but Skyline only shows the precursor chromatograms, not the product ions. Because it only reads the MS scan, not the MSMS ones. Here, I have attached the selected transition settings as a ppt file. In the full-scan tab, "Targeted" is selected as the acquisition method; so Skyline should expect multiple MSMS scans but it does not read any of them. I would appreciate your help! Taleb
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Boxcar support
(13 responses) DiegoB 2020-05-13

Hi Skyline team
I was wondering if Skyline support MS1 filtering on data acquired using Boxcar on the Exploris.
Thanks a lot

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Unable to Import Result Files due to multiple chromatograms
(3 responses) stephanie 2020-05-28


I am trying to import results (from a byonic search) for full scan filtering. I created my library and a background proteome. I have a custom labeling system with labeled R/M and methyl groups. My samples are fractionated. I have tried adding samples as multiple replicates with multiple injections, single injection, and one new replicate but I get a similar error message (see below).

I was working with three libraries and importing multiple replicates. I am going to go back and re-build my library as a single library. Not sure if that will help.

Do you have any suggestions? If there is one peptide like this, there will be more.


Error Message:
At 8:44 PM:
Failed importing results file 'C:\Users\slynn\Documents\3Plex_Fraction_Skyline\ADMA Fraction\20200113_SML_ADMACombo_HILIC_10.raw'.
Unable to process chromatograms for the molecule 'YMHR[+18]NR' because one chromatogram ends at time '23.7843837738037' and the other ends at time '25.4307174682617'.

Inner exceptions:
Exception type: System.InvalidOperationException
Error message: Unable to process chromatograms for the molecule 'YMHR[+18]NR' because one chromatogram ends at time '23.7843837738037' and the other ends at time '25.4307174682617'.
Unable to process chromatograms for the molecule 'YMHR[+18]NR' because one chromatogram ends at time '23.7843837738037' and the other ends at time '25.4307174682617'.
at pwiz.Skyline.Model.Results.PeptideChromDataSets.AddDataSet(ChromDataSet chromDataSet) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 838
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Add(PeptideDocNode nodePep, ChromDataSet chromDataSet) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 794
at pwiz.Skyline.Model.Results.ChromCacheBuilder.AddChromDataSet(Boolean isProcessedScans, ChromDataSet chromDataSet, PeptidePrecursorMz peptidePrecursorMz, IDictionary2 dictPeptideChromData, ICollection1 listChromData, ChromFileInfo fileInfo) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 670
at pwiz.Skyline.Model.Results.ChromCacheBuilder.CalcPeptideChromDataSets(ChromDataProvider provider, List1 listMzPrecursors, HashSet1 setInternalStandards) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 510
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 371
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252

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Selective peak integration based on idotp
(4 responses) Fabian 2018-05-29

Dear all,

i would like to have the option to integrate only peaks above a certain idotp threshold.
At the moment, the option [refine_advanced_results] Min idotp: decides if a peptide is kept or not.
I would like to have the same option based on the peaks not on the peptide.

Did I miss sth. or are there workarounds (peak training model did not convinced me).

Best regards


view request
LOQ CV requirement iterates from low to high concentration, but high to low concentration would be better
(1 response) philip remes 2020-05-28

Hi guys,

I'm trying to use the Calibration Curve functionality, and have set the LOQ CV requirement to 20%. I notice some strange stuff going on, where sometimes a compound will have crap CV's at low concentration, then randomly have a level with a CV less than 20% when there's just noise. This level will get picked as the LOQ, even though some higher concentrations have worse CVs. Looking at the data, it's clear that the true LOQ should be at much higher concentration. To me, this looked as though the algorithm was proceeding from low concentration to high concentration, and taking the first level that meets the CV requirement as the LOQ. I would suggest that the LOQ would be more accurate if you proceeded from high concentration to low concentration, and picked the last level that meets the CV requirement. The peptide IFYNQQNHYDGSTGK is one example of this.


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Moel Method for Scheduled PRM
z-proteomics 2020-05-28

Can any skyline experts with experience of using QE HF machine provide me a model PRM (Scheduled) method with detailed parameter setting?
Greatly appreciate your helpn in advance.
--- OmicSky

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unable to import HD-MRM result files into skyline in the last release version/daily version
(2 responses) simon daled 2020-05-28
i am unable to import my HD-MRM runs into skyline. It works fine in the 19.1 release version, but not in the newest version, there must be some bug somewhere. The runs are partially imported but the shown results are completely wrong.
Is it possible to download the previous version somewhere?
Thank you!
view request
Some small molecules don't support calibration curve
(1 response) philip remes 2020-05-27

Hi guys,

I have a small molecule document that is being used to analyze a dilution curve. Of 79 molecules, 2 of the calibration curves give the message "Error: All of the external standards are missing one or more peaks". This would appear to be similar to other reported issues.

Other support requests that mention this error, but don't seem to help me

Similar Issue 1
Similar Issue 2
Similar Issue 3

However, there is no internal standard normalization, the Analyze Concentrations are set, and the Sample Types are set to standard. Additionally it's kind of weird that just two of them do this, when they both have reasonable looking Peak Area plots vs Analyte Concentration.


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method export error
(3 responses) dawn dufield 2020-05-22

do you know what would cause this error. We have the analyst configuration in simulation mode and seems to be able to open the methods properly. This same skyline file can be exported on a different computer with analyst. So it feels like an analyst config issue, but seems like it should work.

any suggestions

An error occurred attempting to export.
ERROR: Unable to cast object of type 'Sciex.MultiQuant.AnalystImprs.ApplicationObject' to type 'Interop.Analyst.ApplicationClass'.

Command-line: Method\AbSciex\TQ\BuildQTRAPMethod -s -m "X:\Analyst Data - BioPharma\Projects\Peptide\Acquisition Methods\Trap Pepcal 10 min.dam"
Working directory: X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods
Standard input:
X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods\~SK14E.tmp
X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods\test.dam
1752.439942,1930.069112,5,sequence1.xremoved this sequence info....
OK More Info
System.IO.IOException: ERROR: Unable to cast object of type 'Sciex.MultiQuant.AnalystImprs.ApplicationObject' to type 'Interop.Analyst.ApplicationClass'.

Command-line: Method\AbSciex\TQ\BuildQTRAPMethod -s -m "X:\Analyst Data - BioPharma\Projects\Peptide\Acquisition Methods\Trap Pepcal 10 min.dam"
Working directory: X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods
Standard input:
X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods\~SK14E.tmp
X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods\test.dam
1752.439942,1930.069112,5,sequence removed....

---> System.IO.IOException: ERROR: Unable to cast object of type 'Sciex.MultiQuant.AnalystImprs.ApplicationObject' to type 'Interop.Analyst.ApplicationClass'.

Command-line: Method\AbSciex\TQ\BuildQTRAPMethod -s -m "X:\Analyst Data - BioPharma\Projects\Peptide\Acquisition Methods\Trap Pepcal 10 min.dam"
Working directory: X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods
Standard input:
X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods\~SK14E.tmp
X:\Analyst Data - BioPharma\Projects\Ext\Acquisition Methods\test.dam
1752.439942,1930.069112,5,sequences removed.....

   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 57
   at pwiz.Skyline.Util.Extensions.UtilProcess.RunProcess(ProcessStartInfo psi, String stdin, String messagePrefix, IProgressMonitor progress, IProgressStatus& status) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Util\Extensions\UtilProcess.cs:line 44
   at pwiz.Skyline.Model.MethodExporter.ExportMethod(String exeName, List`1 argv, String fileName, String templateName, Dictionary`2 dictTranLists, IProgressMonitor progressMonitor) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 3906
   at pwiz.Skyline.Model.AbiMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 2397
   at pwiz.Skyline.Model.ExportProperties.<>c__DisplayClass119_0.<ExportAbiQtrapMethod>b__0(IProgressMonitor m) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 523
   at pwiz.Skyline.Util.ProgressWaitBroker.PerformWork(ILongWaitBroker broker) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Util\UtilUI.cs:line 125
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 246
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1907
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 194
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 131
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2022
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