### Welcome to Skyline Support

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.

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.

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 .sky.zip 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.

### Requests

Showing: limited to 100 requests
(5 responses) laura corveleyn 2021-09-27

Hi,

I am having some troubles when importing SWATH data as results. I want to compare histone peptidoform expression across several conditions (cell lines) using peak areas. To do this, I made a QC sample (mixture of all conditions) as a baseline. However, when importing the SWATH raw data of these QC samples into Skyline, it seems like something goes wrong when reading it. The XICs look a bit strange. I tried reimporting results several times and made a new Skyline project, but nothing helps. You can find a screenshot of some XICs in the attachment (the QC samples are in the bottom left). Hope you can help me out :)

Kind regards,

Laura

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XIC_QC.PNG  XIC_QC_2.PNG  XIC_QC_3.PNG
view request
Skyline cannot import result file
(1 response) guo xue 2021-09-27

Hi, I installed skyline on my laptop. but I am not able to import raw files. the error info is pasted below. I tried to copy all the raw files to local drive it also failed. Can you please help with this issue?

At 3:23 pm:
Failed importing results file 'Y:\Collaboration_Services\Raw_files_FPL\Fusion Lumos\2021\Guo Xue\Ub\210923_Whole_titration_A549_Raw\210923_GX_Ub_A549_W_M2_100pg_spike.raw'.

pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'Y:\Collaboration_Services\Raw_files_FPL\Fusion Lumos\2021\Guo Xue\Ub\210923_Whole_titration_A549_Raw\210923_GX_Ub_A549_W_M2_100pg_spike.raw'.
---> pwiz.Skyline.Util.AssumptionException
at pwiz.Skyline.Util.Assume.Fail(String error) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 2055
at pwiz.Skyline.Model.Results.SpectrumFilterPair..ctor(PrecursorTextId precursorTextId, Color peptideColor, Int32 id, Nullable1 minTime, Nullable1 maxTime, Boolean highAccQ1, Boolean highAccQ3) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectrumFilterPair.cs:line 57
at pwiz.Skyline.Model.Results.SpectrumFilter..ctor(SrmDocument document, MsDataFileUri msDataFileUri, IFilterInstrumentInfo instrumentInfo, Nullable1 maxObservedIonMobilityValue, IRet/entionTimePredictor retentionTimePredictor, Boolean firstPass, GlobalChromatogramExtractor gce) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectrumFilter.cs:line 267
at pwiz.Skyline.Model.Results.SpectraChromDataProvider..ctor(MsDataFileImpl dataFile, ChromFileInfo fileInfo, SrmDocument document, IRetentionTimePredictor retentionTimePredictor, String cachePath, IProgressStatus status, Int32 startPercent, Int32 endPercent, IProgressMonitor loader) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 121
at pwiz.Skyline.Model.Results.ChromCacheBuilder.CreateSpectraChromProvider(MsDataFileImpl dataFile, ChromFileInfo fileInfo) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1264
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 233
--- End of inner exception stack trace ---

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view request
Peak Integration Issues
(6 responses) sa825 2021-09-19

Hi,
I am looking at some peptides over multiple replicates and I find that Skyline is not constant in its assignment of the peak range of each peak from replicate to replicate.

Please have a look at the images attached. For replicate 4 in the image attached, skyline selects from 25.8s to 26.27s but for replicate 5, it only selects from 25.8s to just over 26s. Is there a way to make Skyline more constant in the way it does this?

I could manually go around adjusting each peak but that would introduce a lot of bias in my work. I also plan to use Skyline for a method involving 116 peptides across 85 samples, so manually adjusting each peak would be a lot. Moreover, if you look at the "Peak Area- Replicate Comparison" graph, you could easily make the mistake in thinking that a peptide is not being properly detected in a particular replicate (e.g. replicate 5) as its peak area appears so small in comparisons to the other replicates but that's not actually the case, a smaller peak area is just being selected by Skyline.

Final question, is there a way to change the dwell time in Skyline when I export a method? I am exporting methods for a Xevo TQ_XS.

Thanks,
Shimon

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Replicate 4.png  Replicate 5.png
view request
DIA-NN .speclib support
(34 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,
tobi

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view request
MIDAS format missing
(7 responses) taperez 2021-09-21

Hello,

When I save a file, skyline usually creates and saves a MIDAS format as well. For some reason the files are no longer saving this data format. Is there something Ive accidentally clicked off? Im running Skyline version 21.1.0.146

Thanks!
Tatiana

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view request
points per peak in DIA data
(5 responses) danielacgranato 2021-09-21

Dear,
We have been starting to optimize DIA method in Exploris 240, and we are evaluating the gain or loss in points per peak based on different isolation windows.

We would like to know:
1-) how does Skyline calculate points per peak of a peptide. Does it add the points per peak given by the MS/MS spectra and the MS1 full scan acquisition? Or does it account only for the MS/MS spectra?
2-) In addition, can you share with us the best parameters to extract the chromatograms from DIA acquired data?
Thank you very much in advance. Best, Daniela

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view request
mProphet report and redundant library
(6 responses) heyang 2021-09-22

Morning Skyline team,

I have some questions want to get help.

1. How can I export mProphet p value and q value in report? I tried, but can't find p value. Only find detect Q value, is it the same with q value in mProphet ?
2. what redundant library have? only contain redundant peptide info? could we delete them?

thanks,
Heyi

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view request
Small Molecule Method Development and CE OptimizationPrint
(4 responses) carlos penno 2021-09-16

Dear all,

I follow the tutorial Small Molecule Method Development and CE OptimizationPrint
and it does not work. I cant get the information needed.

I just wanted to report this.

Best regards,

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view request
Skyline Batch use of sample list
(4 responses) Juan C. Rojas E. 2021-09-21

Hi all,

I was wondering if there is a way to import a sample list (e.g. csv file) to Skyline Batch with the exact files I would like to download? Sometimes a general expression won't solve everything (?) or perhaps I just don't know how to use it fully.

Or maybe an interactive box that allows selecting which files are desired after visualizing the contents in a server space (e.g. PXD project)?

Sincerely,
JC

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view request
Can we gave multiple modification on one amino acid
(1 response) Bo An 2021-09-22

I have a peptide started with K on the n-terminal, when biotinylated the peptide, this n-terminal K will be double labled with biotin, is there a way we can put multiple modification on the same amino acid like this situation? Thanks.

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view request
proteome discoverer
(3 responses) wangyan2 2021-09-21

Hi,
I'm new to Skyline. Trying to use quantify a handful of peptides identified in PD but can't seem to figure out how to do that. Also, because it was a targeted analysis, for some peptides I have >1000 PSMs. What do you recommend importing as library? Thanks.

Yan

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view request
RT Calibrators for SWATH
(2 responses) taperez 2021-09-21

One of my coworkers is working on SWATH data with skyline and says you're able to process data on skyline without needing to use retention time calibrators. Are retention time calibrators necessary, and if so why?

Thanks,

Tatiana

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view request
Proteome Discoverer mzid file could not be imported
(3 responses) mmr6q 2021-09-20

The error message stated that the mzid file contains an unsupported score type.
Thank you.

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view request
How to show the entire data acquisition/retention time range
(3 responses) mmr6q 2021-09-20

Nick,
Attached is the zipped file with Skyline files with two raw files, both of which are DDA LC-MS analyses of the Pierce PRTC mixture (plus two peptides). The first file has peptide RTs ~20-30 min., and the second has RTs ~10 min. longer. I cannot get Skyline to show the full RT range. Thank you.
Mark

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New PRTCAV Skyline.zip
view request
Peptide Mapping with Skyline
(3 responses) RuMa 2021-09-20

Hi Skyline team,

I am trying to study the coverage of a biopharmaceuticals with Skyline. The goal is to digest the recombinant protein present in formulation and see if a full coverage can be obtained. The instrument used is a xevo qtof g2-xs and the data is acquired in MSE mode.

I would like to steer away from Biopharmalynx and use Skyline instead, but I find it hard to set parameters that can give me confidence in peptide identifications since I do not have a spectral library. I thought I could gain additional confidence with a predicted library using Prosit, but since the MSE mode does a ramp up of collision energy, a predicted library with a fixed CE was not helpful.

Do you have any suggestion, tutorial or paper I can refer to?

Thanks,

Ruma

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view request
"Chromatogram information unavailable" message
(1 response) DanCam 2021-09-20

Hi Skyline team

I am new to using Skyline for data analysis and am trying to generate PRM calibration curves, only using heavily-labelled versions of my peptides of interest. I have been following the data processing and analysis outlined in Skyline Webinar 17 in the first instance.

I generated a spectral library of my peptides of interest pooled together, and with data acquired using DDA mode on a QE-HF. I then spiked in mixtures of my 22 peptides of interest into a complex background at different concentrations and acquired PRM data using the appropriate inclusion list with the QE-HF, with the same LC parameters as the DDA run.

I imported the raw data, and filtered for my peptides of interest using Edit>Insert>Peptides. There are transitions showing up for the majority of the peptides I am interested in, apart from two, for which I get a message saying "Chromatogram information unavailable". These two peptides have the same primary sequence aside from the fact that one contains deamidated asparagine and the other contains asparagine linked to N-acetylglucosamine. These two modifications are included as variable modifications in within the appropriate settings.

I can see that both of these peptides are present within the spectral library, and when I make XICs of my RAW files using FreeStyle, they also seem to be present at the correct m/z.

I wondered whether you might be able to help me figure out why I am unable to see the peaks using Skyline, please. I have had a look on your forum, but I can't work it out.

Is there a way I can send screenshots privately to you, if you require them?

Thanks,
DanCam

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view request
Error installing skyline daily unplugged version.
(2 responses) Niyatti 2021-09-17

Hey,
I need to use skyline daily and tried installing the latest version but was not able to install it so downloaded skyline daily unplugged version from the URL: https://skyline.ms/wiki/home/software/Skyline/daily/page.view?name=install-disconnected-64 but, getting an error something like this(mentioned below)
ERROR DETAILS
Following errors were detected during this operation.

• [9/17/2021 3:48:29 PM] System.ArgumentException
• Value does not fall within the expected range.
• Source: System.Deployment
• Stack trace:
at System.Deployment.Application.NativeMethods.CorLaunchApplication(UInt32 hostType, String applicationFullName, Int32 manifestPathsCount, String[] manifestPaths, Int32 activationDataCount, String[] activationData, PROCESS_INFORMATION processInformation)
at System.Deployment.Application.ComponentStore.ActivateApplication(DefinitionAppId appId, String activationParameter, Boolean useActivationParameter)
at System.Deployment.Application.SubscriptionStore.ActivateApplication(DefinitionAppId appId, String activationParameter, Boolean useActivationParameter)
at System.Deployment.Application.ApplicationActivator.Activate(DefinitionAppId appId, AssemblyManifest appManifest, String activationParameter, Boolean useActivationParameter)
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivation(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl, Uri& deploymentUri)
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivationWithRetry(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl)
--- End of stack trace from previous location where exception was thrown ---
at System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivationWithRetry(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl)
at System.Deployment.Application.ApplicationActivator.ActivateDeploymentWorker(Object state)

COMPONENT STORE TRANSACTION DETAILS

• Transaction at [9/17/2021 3:48:29 PM]
• Status: Set
• HRESULT: 0x0
• System.Deployment.Internal.Isolation.StoreTransactionOperationType (27)
• HRESULT: 0x0

could you please check and tell me what could be the issue?

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view request
How can I expand the RT range in the peak profile window
(6 responses) mmr6q 2021-09-17

How can I increase the RT range in the peak profile window to view peptides that have shifted in RT beyond the range that I guess is expected by Skyline. In other words, how I can make the RT range the full time range of the LCMS analysis and then pick the peak?

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view request
problem installing Skyline 21.2-64 bit
(2 responses) Niyatti 2021-09-15

Dear Skyline team,

I tried to install Skyline21.2 (64 bit) on a computer with win 7 and got an error.
could you please tell me what's wrong?
Thanks,
Niyatti

The following properties have been set:
Property: [InstallMode] = HomeSite {string}
Property: [NTProductType] = 1 {int}
Property: [ProcessorArchitecture] = AMD64 {string}
Property: [VersionNT] = 6.1.1 {version}
Running checks for package '.NET Framework 3.5 SP1', phase BuildList
Reading value 'SP' of registry key 'HKLM\Software\Microsoft\NET Framework Setup\NDP\v3.5'
Setting value '1 {int}' for property 'DotNet35SP'
The following properties have been set for package '.NET Framework 3.5 SP1':
Property: [DotNet35SP] = 1 {int}
Running checks for command 'DotNetFX35SP1\dotNetFx35setup.exe'
Result of running operator 'ValueGreaterThanEqualTo' on property 'DotNet35SP' and value '1': true
Result of checks for command 'DotNetFX35SP1\dotNetFx35setup.exe' is 'Bypass'
'.NET Framework 3.5 SP1' RunCheck result: No Install Needed
Running checks for package 'Microsoft .NET Framework 4.7.2 (x86 and x64)', phase BuildList
Reading value 'Release' of registry key 'HKLM\Software\Microsoft\NET Framework Setup\NDP\v4\Full'
Setting value '528049 {int}' for property 'DotNetFull_Release'
Reading value 'v4' of registry key 'HKLM\SOFTWARE\Microsoft\NET Framework Setup\OS Integration'
Not setting value for property 'DotNetFull_OSIntegrated'
The following properties have been set for package 'Microsoft .NET Framework 4.7.2 (x86 and x64)':
Property: [DotNetFull_Release] = 528049 {int}
Running checks for command 'DotNetFX472\NDP472-KB4054530-x86-x64-AllOS-ENU.exe'
Result of running operator 'ValueEqualTo' on property 'InstallMode' and value 'HomeSite': true
Result of checks for command 'DotNetFX472\NDP472-KB4054530-x86-x64-AllOS-ENU.exe' is 'Bypass'
Running checks for command 'DotNetFX472\NDP472-KB4054531-Web.exe'
Result of running operator 'ValueNotEqualTo' on property 'InstallMode' and value 'HomeSite': false
Result of running operator 'ValueGreaterThanEqualTo' on property 'DotNetFull_Release' and value '461808': true
Result of checks for command 'DotNetFX472\NDP472-KB4054531-Web.exe' is 'Bypass'
'Microsoft .NET Framework 4.7.2 (x86 and x64)' RunCheck result: No Install Needed
Launching Application.

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view request
Import Peptide Search - No spectra in library
(1 response) claire tonry 2021-09-15

Hi, I am trying to import peptide search data into Skyline for dia analysis. The library was generated from an 'open search' of dda files (timsTOF) that were converted to mzML. When I try to use the individual .pepXML files (15,000 - 20,000 KB) for each sample or the 'interact.pep' file (177,609 KB) Skyline tells me that it cannot find any spectra for the library. I also tried using the library.tsv.speclib file generated but this also caused an error as 'has version 3 bu BiblioSpec only supports up to version 2'. These dda and associated dia files were previously searched in Spectronaut software with no issues. Would you have any ideas as to why Skyline cannot read any spectra from the output files generated from the fragpipe search? The objective of this search is to identify PTMS which is why I wanted to use output from a fragpipe open search

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view request
Import Peptide Search: Search Failed
(2 responses) kmp95 2021-09-15

I have been searching thermo .Raw files to see proteins that were observed. I have been successfully doing this on two different computers, but I have failed every time on this computer no matter what. It is my desktop computer at my office and would look to be able to work on Skyline while in the office. It always fails after running the percolator. Any ideas on what is causing the search to fail? I have uninstalled and reinstalled skyline twice. I am using skyline 64 bit version and I am working on windows 10 64 bit version.

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Screenshot 2021-09-15 111101.png
view request
How to do normalization by 3 heavy compound for 3 more compounds
(1 response) xue shi 2021-09-14

Dear Skyline team,
I know how to do normalization just by one compound normalized by one heavy compound by skyline. However, Do skyline have the ability to normalize 17 compounds by 3 heavy compounds? Not every compound has heavy compound as internal standard. If yes, How should we process this in transition list? I attached my transition list in the attachment.
I really appreciate if you could help with this.
Thank you,
Katie

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transition list for skyline SCFA_ratio cal.xlsx
view request
Division of transitions when multiple methods exported
(1 response) Orla Coleman 2021-09-09

Dear Skyline team,

I am wondering how Skyline divides transitions when exporting into multiple methods? I have 740 transitions which I exported across 3 methods but I can't see any trend in each method to indicate how to decision was made.

Thanks!

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view request
Any plans for a Linux version of Skyline (even with limited capabilities)?
(8 responses) lparsons 2017-08-07
Skyline is a great tool for me, and I am wondering if there are any plans to make it available - even in a somewhat stripped-down form - for Linux. I have read that the vendor specific libraries that make it possible to open vendor native formats are often locked to Windows, but there are other functions that could be helpful to have that might not require them. If I could open an existing Skyline project and do basic editing of the project (for example deleting ions) and basic exporting (to text) that would be really helpful for me.
I don't know the ins and outs of the Skyline .sky file format (amongst other things), so I don't know if that is a request that would be too great to handle or not.

thank you!
Lee
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view request
Fragpipe tutorial hangs up
(3 responses) ani 2021-09-09

I have raised this issue with Fragpipe dev as well https://github.com/Nesvilab/FragPipe/issues/453 but maybe it is Skyline/timTOFpro setup issue? I am trying to follow the tutorial https://fragpipe.nesvilab.org/docs/tutorial_skyline.html where DDA-Search import seems to have worked (attached
image) but it gets stuck at building-molecular-search-library stage?

I have loaded the IO files at https://drive.google.com/drive/folders/1ILiqR2mvWeMWIpeM55qyj9jehPebWBrF which includes the logs, wondering if you can take a look/identify the issue and tell me how to proceed?

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Skjermbilde 2021-09-09 151410.png
view request
target per frame
(3 responses) Antoine 2021-09-07

Hello Skyline team,

For prm-PASEF
May I ask if the targets per frame graph corresponds to the max, the average or the median number of targets per frame within an MS cycle?

Thanks

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view request
Failed importing DIA-PASEF results and isolation schemes
(4 responses) msielaff 2021-09-02
Dear Skyline team,

I'm trying to set up a Skyline document for a AutoQC-Panorama pipeline in order to monitor some peptides as QC from HeLa DIA-PASEF runs. Unfortunately, Skyline fails to import the results from some of our DIA-PASEF methods with following error:

At 3:23 PM:
Failed importing results file 'O:\RAW_ttp\O210827_003_Slot1-1_1_3239.d'.
error reading spectrum merged=12 frame=5 scanStart=735 scanEnd=937
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'O:\RAW_ttp\O210827_003_Slot1-1_1_3239.d'.
error reading spectrum merged=12 frame=5 scanStart=735 scanEnd=937 ---> System.Reflection.TargetInvocationException: error reading spectrum merged=12 frame=5 scanStart=735 scanEnd=937 ---> System.Exception: error reading spectrum merged=12 frame=5 scanStart=735 scanEnd=937 ---> System.Exception: Scan number [936] out of range.
at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, DetailLevel detailLevel)
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetCachedSpectrum(Int32 scanIndex, DetailLevel detailLevel) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1155
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetSpectrum(Int32 spectrumIndex) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 893
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.ReadSpectrum(Int32& i) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1075
--- End of inner exception stack trace ---
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.ReadSpectrum(Int32& i) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1126
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.<RunAsync>b__33_0() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 810
--- End of inner exception stack trace ---
at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1940
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.NextSpectrum() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 916
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatogramsLocked() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 259
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatograms() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 239
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.SetRequestOrder(IList1 chromatogramRequestOrder) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 592
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 384
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252
--- End of inner exception stack trace ---

Skyline also fails when I try to import the isolation scheme from the raw file:

---------------------------
Skyline
---------------------------
Scan number [936] out of range.
---------------------------
---------------------------
System.Reflection.TargetInvocationException: Scan number [936] out of range. ---> System.Exception: Scan number [936] out of range.
at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, DetailLevel detailLevel)
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetCachedSpectrum(Int32 scanIndex, DetailLevel detailLevel) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1155
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetSpectrum(Int32 spectrumIndex) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 893
at pwiz.Skyline.SettingsUI.EditIsolationSchemeDlg.<>c__DisplayClass74_1.<ImportRangesFromFiles>b__1(IProgressMonitor progressMonitor) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\SettingsUI\EditIsolationSchemeDlg.cs:line 1045
at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action1 performWork) in C:\proj\skyline_21_1_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_21_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1940
at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 202
at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
at pwiz.Skyline.SettingsUI.EditIsolationSchemeDlg.ImportRangesFromFiles(MsDataFileUri[] dataSources) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\SettingsUI\EditIsolationSchemeDlg.cs:line 1045
---------------------------

Based on these error messages, do you have any idea what the problem could be and how to solve it?

Many thanks,
Malte
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view request
Error building spectral library from MSFragger Search with timstof data
(1 response) claire tonry 2021-09-07
Hi, I get the following error when I try to import the pep.xml file generated from an MSFragger search of timstof DDA data into Skyline

---------------------------
Skyline
---------------------------
ERROR: Could not find spectrum file 'A210120_ct_heart_FFPE_fracs_AF_F1_dda_881[.mz5|.mzML|.mzXML|.ms2|.cms2|.bms2|.pms2|_uncalibrated.mgf|_calibrated.mgf]' for search results file 'A210120_ct_heart_FFPE_fracs_AF_F1_dda_881.pepXML' in current directory, ../,../../.
ERROR:

Command-line: C:\Users\3053492\AppData\Local\Apps\2.0\5VYXQT5K.TNB\893P3P9W.TXB\skyl..tion_e4141a2a22107248_0015.0001_b2be94ac8cfe2cb4\BlibBuild -s -A -H -o -c 0.95 -i Skyline_Lib_Test_07092021 -S "C:\Users\3053492\AppData\Local\Temp\tmpEA60.tmp" "C:\Users\3053492\Documents\AA_FFPE_Jan21\Skyline_LibBuild\Skyline_Lib_Test_07092021.redundant.blib"
Working directory: C:\Users\3053492\Documents\AA_FFPE_Jan21\Skyline_LibBuild
---------------------------
---------------------------
System.IO.IOException: ERROR: Could not find spectrum file

'A210120_ct_heart_FFPE_fracs_AF_F1_dda_881[.mz5|.mzML|.mzXML|.ms2|.cms2|.bms2|.pms2|_uncalibrated.mgf|_calibrated.mgf]' for search results file 'A210120_ct_heart_FFPE_fracs_AF_F1_dda_881.pepXML' in current directory, ../,../../.
ERROR:

Command-line: C:\Users\3053492\AppData\Local\Apps\2.0\5VYXQT5K.TNB\893P3P9W.TXB\skyl..tion_e4141a2a22107248_0015.0001_b2be94ac8cfe2cb4\BlibBuild -s -A -H -o -c 0.95 -i Skyline_Lib_Test_07092021 -S "C:\Users\3053492\AppData\Local\Temp\tmpEA60.tmp" "C:\Users\3053492\Documents\AA_FFPE_Jan21\Skyline_LibBuild\Skyline_Lib_Test_07092021.redundant.blib"
Working directory: C:\Users\3053492\Documents\AA_FFPE_Jan21\Skyline_LibBuild
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 65
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 157
---------------------------
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view request
Exporting phosphopeptides from MaxQuant into Skyline to visualize phospho spectrum
(1 response) Niyatti 2021-09-03

Dear MacCoss Lab, I'm working on enriched phosphopeptides and would like to import the identified phosphopeptides (filtered) from MaxQuant to Skyline to visualize the phospho spectrum or phosphosite spectrum. so is there any way I could do that in the skyline? If, yes then could you please help me out with the same.

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view request
DIA Umpire error
(3 responses) Francis Beaudry 2021-07-08
Hi,

I am setting up a DIA analysis workflow using DIA raw to build a library. I follow the tutorial using your dataset or my dataset but I obtain the same error message. I tried on 2 different computers. Please advise ?

---------------------------
Skyline
---------------------------
Error
---------------------------
---------------------------
System.IO.FileNotFoundException: Le fichier 'C:\Users\franc\AppData\Local\Temp\gyo1ftie.mkn.mz5' est introuvable.
Nom de fichier : 'C:\Users\franc\AppData\Local\Temp\gyo1ftie.mkn.mz5'
à System.IO.__Error.WinIOError(Int32 errorCode, String maybeFullPath)
à System.IO.File.InternalMove(String sourceFileName, String destFileName, Boolean checkHost)
à pwiz.Skyline.Model.DiaUmpireDdaConverter.Run(IProgressMonitor progressMonitor, IProgressStatus status) dans C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\DiaUmpireDdaConverter.cs:ligne 148
---------------------------
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view request
Multiple peptides EIC chromatograms display value
(1 response) Juan C. Rojas E. 2021-09-03

Hi all,

A colleague has performed a scheduled PRM, including a full ms scan in each cycle. Then he has created a Skyline project to analyze the targets both at the precursor and fragment level. Now we have a question about the y-axis values displayed when the EIC chromatograms of multiple peptides are displayed (when you highight a peptide list or a protein) as in the image attached. Are the values the summed intensity of all transitions? Precursor or fragment transitions? Is it only the intensity of the highest transition?

I hope all is going well and hope to hear back from you.
Sincerely,
JC

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EIC Chromatogram.png
view request
Error exporting method for Waters Xevo from Skyline
(1 response) cmboot 2021-09-01

Hello,

I've created a transition list in Skyline 64 bit (v 21.1.0.146 (9b577b3b8)) that I would like to export as a method or transition list to use on my Waters Xevo TQD system. Working from the instrument computer, when I choose 'export' and 'method' and select the Waters file provided in the 2021 small molecule online workshop (VerifyETemplate.exp) as the template field I get an error: System.IO.IOException: ERROR: Method not found: 'Boolean CompoundDatabaseClassLibrary.CompoundDatabase.InsertNewIonDetails(Int32, Int32, TransMode, Int32, Int32, Int32, Int32, Int32, Int32)'.

I tried this with other .exp files and receive the same error, and the full text of the error is attached.

Claudia

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Skyline Method Export Error Waters Xevo TQD.pdf
view request
Using R script in Skyline Batch
(2 responses) schen19 2021-08-27

Dear Skyline Team,

I was just trying to set up a configuration for Skyline Batch. I added the path to the R script in "R script file path" then clicked "OK". However, I did not see the path appears afterwards. I have attached some screenshots to illustrate the problem.

Am I missing some steps? Is there anything I can do to fix this problem?

Shimin

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Rscirpt path.jpg  Configuration.png
view request
Retention time decimal place
(4 responses) muluneh 2021-06-14

Is there a way to set up retention time decimal places? I use a windows computer and it displays only one decimal place. ..like 14.3. Can I set up Skyline to display 14.32 or something like that?
I use RT +/- 0.02 min between the analyte and heavy labeled standard, and it's making the comparison a bit difficult. I know the skyline displays two decimal places when I export the report in Excel.

Thanks!
Muluneh

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view request
Automatically import transitions list from Waters triple quandrupoles RAW files
(15 responses) gioele visconti 2021-08-20

Good morning,

First of all, thank you for your work. I really appreciate the program and its multi-platform versatility.

I would like to suggest if the MRM transitions list that are contained into the RAW files of triple quadrupoles instrument (see attached pictures) can be automatically imported into Skyline. To my knowledge, this function is not present and the user need to manually create a transition list.
I am currently using a Waters instrument (TQ-XS) and exporting the MRM transitions is very laborious and not automatable.

Thank you and best regards.

Gioele Visconti

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1.jpg  2.jpg
view request
QUantification
(1 response) priscillia lagoutte 2021-08-31

HI,

I will contact you because I try to find the linearity range of my peptide for quantification assay. So I Injected 10 different quantities of synthetic heavy peptide and then I looked calibration curve (generated by Skyline). I also export total areas (from result grid) and analyse with excel.

I found that calibration curve is different because in case of skyline, it takes peak areas (Max height) and in my case I look total areas and linearity range is not the same between both calibration curve.

Can you help me to understand ?

Priscillia

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calibrationcurve.pdf
view request
Peak summary data outdated on ms data reimport
(2 responses) jfoe 2021-08-26

Dear Skyline team,

one major pain point for my workflows manifests as follows:
When one enables further transitions for a precursor that already has a peak picked, those transitions would not have any extracted data, yet.
So I then reimport the MS data (Orbitrap full MS2, centroided extraction) in order to extract the data.
The transition is then displayed in the XIC successfully.

Yet, none of the quantifications or dotp information for the peak will adapt to the new data. In my understanding, the (manually picked) peaks should stay in terms of positioning, but all the extracted area measurements, dotp etc should update according to the new XIC data.
The same (presumably) effect comes into play, when one adjusts the extraction ppm limit, which could have major effects on the XIC (as is visible), but the summary data again is not updated.

This forces me to either run full peak reintegration, losing all manually picked peaks, or delete the .skyd, which causes deselected peaks to be picked automatically again. Both of these options are quite burdensome with fully curated files.

I am using Skyline 21.1.0.146

I just tried to set up a minimal example using a simple small molecule setup.
I am attaching a minimal skyline and rawfile. There is only one transition with a manually picked peak (just some noise for this example).
If you open that file, you can take note of the transition area (17608) extracted at 1 ppm.
You can adjust the extraction ppm to 1000 and attempt reimport (via edit -> manage results).
This to me doesn't update the XIC at all (a separate bug?). But upon saving and reopening, the XIC is updated to reflect the 1000ppm.
The transition area now is still 17608 and only updates when playing around manually with the selected peak limits.

I feel that this is likely very much unintended behavior and hope that this description will be helpful.
Please let me know if any further info is needed.

Best regards,
Jonas

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210826 skyline reimport issue.zip
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Icon for Skyline 64 bit Admin version is not seen in Start->Programs
(1 response) shobhabr20 2021-08-27

Hi,

I tried to install Skyline Admin 64 bit 21.1.0.146 (current)

The installation happens and is seen in Control panel->Programs and Features.

However the icon for Skyline 64 bit is not seen in Start->Programs
Hence cannot open sky document.

I have tried to uninstall Skyline daily and Skyline Released version which was installed before. And also cleaned up the folders in the path C:\Users\Administrator\AppData\Local\Apps\2.0 before installing admin version of skyline.

Thanks,
Shobha

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view request
Failure attempting to modify the Skyline document with SWATH spectral library search data
(2 responses) Chinmaya k 2021-08-25
Hello,

I am trying to search SWATH data against a Prosit-generated spectral library. However, when I tried to reintegrate the peaks using a mProphet model, the Skyline is not able to reintegrate the all peaks with a decoy trained mProphet model.

What could be the reason for this failure? Can you please help me to resolve the issue?

---------------------------
Skyline
---------------------------
Failure attempting to modify the document.
Unexpected document change during operation.
---------------------------
---------------------------
System.IO.InvalidDataException: Unexpected document change during operation.
at pwiz.Skyline.SkylineWindow.ModifyDocumentInner(Func2 act, Action onModifying, Action onModified, String description, Func2 logFunc, AuditLogEntry& resultEntry) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Skyline.cs:line 786
at pwiz.Skyline.SkylineWindow.ModifyDocumentOrThrow(String description, IUndoState undoState, Func2 act, Action onModifying, Action onModified, Func2 logFunc) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Skyline.cs:line 755
at pwiz.Skyline.SkylineWindow.ModifyDocument(String description, IUndoState undoState, Func2 act, Action onModifying, Action onModified, Func2 logFunc) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Skyline.cs:line 732
---------------------------

Thank you,
Chinmaya
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view request
Peak Annotation with a selected/forced time frame
(1 response) Stephan Klatt 2021-08-25

Good Morning,

I am using Skyline for several years and mostly for metabolite analyses.
It often happens that peaks are automatically not correctly selected. In most cases this is the case when there are several peaks in the same time window. Even when I use the function 'Apply peak to all', it doesn't help me in most cases. Still the wrong peaks are annotated and often also different ones. This is a little bit frustrating, as I have to spend a lot of time in manual peak annotation.

I was wondering if you can implement the following function into Skyline-daily:
When you right-click into the intensity window, you get the function to manually enter a time frame which is used for peak annotation no matter what/if there is a peak. This function would force to use a selected time frame, e.g. 5.45 - 5.55 min. This would be fantastic and I can imagine that others have similar problems.

I would appreciate an answer. Thank you very much!

Kind regards,

Stephan

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view request
Integrate all MS files over a user defined exact retention time window (peptides
lucasm7 2021-08-26

Hello Maccoss lab!

My name is Lucas, and I am an RS3 in the hoofnagle lab. I need to integrate all chromatograms over an explicit retention time window for each peptide. Is this possible in Skyline?

Thank you very much!
Lucas

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view request
Easier way to edit IM windows for prm pasef method generation
(1 response) af1234 2021-08-26

I am doing prm-pasef with heavy peptides but the integration in the IM dimension is off (similarly to https://skyline.ms/announcements/home/support/thread.view?rowId=48885) for method generation from dda-pasef.
Is there any plan to have it adjustable like peak boundaries? How can I manually edit the IM windows?

Thanks!

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view request
pep identification in DIA analysis
(12 responses) heyang 2021-08-18

Hi,

We just started to use Skyline to process DIA data. One questions is how we can export peptides confidently present in the data.

thanks,
HY

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view request
Error Importing Transition List: # of fields not picking up?
(1 response) jana carpenter7 2021-08-25

# I'm hoping to see if there is a solution to a formatting issue with importing a CSV transition list for small molecules.

I will attach my working list to this message. The error messages that I've been receiving are:

"Failed reading the file...Line 'Organic Acid and Derivative' has 1 fields when 5 expected."

I am trying to calculate masses of metabolites with corresponding adducts, and have been able to in the past (for isotopically labeled molecules), but I'm not sure what has changed about how I'm formatting the document. Please let me know if there is a way to fix this issue. Thanks!

-Jana

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Working Transition List_Unlabeled_w_prec_charge.csv
view request
problems in creating new equation for CE
(4 responses) amanzoor 2021-08-23

Dear Skyline team,

Thanks a lot for your help. We just started to use skyline for targeted proteomics. Everything is fine till we reach where we have to calculate the linear equation for CE. Although we can see nice peaks with good intensity but the graph is not up to the mark. I have attached the screen shots of our skyline and the graph. Please find it attached. We are using Skyline (64-bit) 20.1.0.155 version on windows 7. Please let me know if you want to have some more informations.

The other thing I would like to ask that there is no internet in our computer. Do you have some system to upgrade our skyline version offline?

Looking forward hearing from you
Best regards
AMK

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SKYLINE.docx
view request
Regarding the output of PREGO for a list of peptides from skyline

Dear Skyline,
I got some trpysin digested peptides for a protein for which I used PREGO. I got directed to a webpage which has given scores and ranks for each of my peptides. Can you tell me what the scores and the ranks are about? Are they giving any information about the detectability of the peptides in a mass spectrometer? I got the following output from PREGO.

Rank Sequence        Score
1       GGLDLR  0.16867737160352636
2       GDLSLR  -0.06754151848263762
3       GESLFQGR        -0.17631845487837808
4       GEPLQVER        -0.6208215989137332
5       TLIYYLDEIPPK    -0.9140796672874998



I am new to proteomics and it would be really helpful if you guys could help me out :)

Thank you.

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Failed to install the Skyline
(1 response) xguo 2021-08-23

Hi There,

I had a difficulty in installing Skyline to my office laptop. It always says that "Application installation did not succeed. Cannot locate application files on the server. Contact the application vendor or your administrator for assistance."

What would be the problem?

P.S I logged in with my personal email account, not my working email in the laptop.

Many thanks & best regards,

Xiaoxiao

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view request
Point mutations?
(15 responses) mnt 2021-07-28
Hi All

I'm curently working on a project where we want to use Skyline for some validation/relative quantification. I have used X!Tandem in The GPM to search the datafiles and am now attempting to import these into Skyline. However, as part of my search with X!Tandem, i deliberately engaged the "point mutation" option, and now skyline cannot import the data. The generated Error is attached below. So the question is if it is possible or not to to have this option engaged at all? Personally i would hope so since it seems to provide some quite interesting results, however at this point it seems as it is not.

Any suggestions?

ERROR:
---------------------------
Skyline
---------------------------
ERROR: GPM22200000640.xtan.xml(line 12641): Specified modification does not match sequence. Given a modified M at position 0 which is a S in SVQWCAVSQPEATK.
ERROR:
ERROR: GPM22200000641.xtan.xml(line 3397): Specified modification does not match sequence. Given a modified W at position 1 which is a M in VMPVLKSPTIPFFDPQIPK.
ERROR:
ERROR: GPM22200000643.xtan.xml(line 1085): Specified modification does not match sequence. Given a modified C at position 13 which is a Q in VKHEDQQQGEDEHQDK.
ERROR:
ERROR: GPM22200000644.xtan.xml(line 1067): Specified modification does not match sequence. Given a modified C at position 13 which is a Q in VKHEDQQQGEDEHQDK.
ERROR:
ERROR: GPM22200000645.xtan.xml(line 6883): Specified modification does not match sequence. Given a modified W at position 1 which is a M in VMPVLKSPTIPFFDPQIPK.
ERROR:

Command-line:
xxx
---------------------------
---------------------------
System.IO.IOException: ERROR: GPM22200000640.xtan.xml(line 12641): Specified modification does not match sequence. Given a modified M at position 0 which is a S in SVQWCAVSQPEATK.
ERROR:
ERROR: GPM22200000641.xtan.xml(line 3397): Specified modification does not match sequence. Given a modified W at position 1 which is a M in VMPVLKSPTIPFFDPQIPK.
ERROR:
ERROR: GPM22200000643.xtan.xml(line 1085): Specified modification does not match sequence. Given a modified C at position 13 which is a Q in VKHEDQQQGEDEHQDK.
ERROR:
ERROR: GPM22200000644.xtan.xml(line 1067): Specified modification does not match sequence. Given a modified C at position 13 which is a Q in VKHEDQQQGEDEHQDK.
ERROR:
ERROR: GPM22200000645.xtan.xml(line 6883): Specified modification does not match sequence. Given a modified W at position 1 which is a M in VMPVLKSPTIPFFDPQIPK.
ERROR:

Command-line:
xxx
ved pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) i C:\proj\skyline_21_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:linje 65
ved pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) i C:\proj\skyline_21_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:linje 201
ved pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) i C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:linje 157
---------------------------
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view request
Forward compatibility in Skyline versions
(6 responses) Liyan 2021-08-18

Hi Skyline team,

We are using Skyline for DIA. We have a few .sky documents complete with libraries and iRTdb that are used as master templates for any project with the same sample types. Most of these .sky templates were setup on version 19.1.0.193 (administrator installation)

We have a new project with over 200 files and thought to run the import on a new computer that has 21.1.0.146 installed. Unfortunately there is an issue with forward compatibility, our master templates are "broken" when opened on the new version and all imports fail. We have also tried to setup the templates from scratch on version 21 but this did not work either.

Our files were acquired on Thermo HFX. We have eliminated file corruption as the cause, as test imports on a few raw files with the .sky master templates work on other computers with version 19.

Will it be possible to give us access to the 19.1.0.193 installer file as a temporary solution?
Edit: I have found the link to the archive.

This is the error that we're getting on version 21.

At 2:46 pm:
Failed importing results file 'C:\Users\FPL\Documents\Liyan\to_import\20210722_Liyan_SFN_EvoSep_HCS001.raw'.

pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\FPL\Documents\Liyan\to_import\20210722_Liyan_SFN_EvoSep_HCS001.raw'.
---> pwiz.Skyline.Util.AssumptionException
at pwiz.Skyline.Util.Assume.Fail(String error) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 2055
at pwiz.Skyline.Model.Results.SpectrumFilterPair..ctor(PrecursorTextId precursorTextId, Color peptideColor, Int32 id, Nullable1 minTime, Nullable1 maxTime, Boolean highAccQ1, Boolean highAccQ3) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectrumFilterPair.cs:line 57
at pwiz.Skyline.Model.Results.SpectrumFilter..ctor(SrmDocument document, MsDataFileUri msDataFileUri, IFilterInstrumentInfo instrumentInfo, Nullable1 maxObservedIonMobilityValue, IRetentionTimePredictor retentionTimePredictor, Boolean firstPass, GlobalChromatogramExtractor gce) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectrumFilter.cs:line 267
at pwiz.Skyline.Model.Results.SpectraChromDataProvider..ctor(MsDataFileImpl dataFile, ChromFileInfo fileInfo, SrmDocument document, IRetentionTimePredictor retentionTimePredictor, String cachePath, IProgressStatus status, Int32 startPercent, Int32 endPercent, IProgressMonitor loader) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 121
at pwiz.Skyline.Model.Results.ChromCacheBuilder.CreateSpectraChromProvider(MsDataFileImpl dataFile, ChromFileInfo fileInfo) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1264
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 233
--- End of inner exception stack trace ---

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view request
Negative Polarity Export
(1 response) philip remes 2021-08-19

I was playing around with some small molecules, and a few are analyzed in negative polarity. They were imported to Skyline with -1 for the precursor and product charge states. When exporting the Isolation lists for Thermo Fusion, this polarity is not handled correctly. The charge state z field is set to -1 for these compounds, which the Thermo method editor highlights as an error. Instead, the z field should be written as a 1, and there should be a column with the Polarity header, with the string Positive for the positive compounds, and Negative for the ones with negative z. This is handled correctly for exporting transitions lists to the Thermo Altis, however.

I was trying to optimize some collision energies by hand, and running into the fact that one cannot optimize parameters for Isolation lists. This ability would be very useful for small molecule work.

Thanks
Philip

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ce_optimization.sky.zip
view request
Question New Protein Abundance Report Feature
(9 responses) roman sakson 2020-10-12

Dear Skyline team,

thank you for providing us with the possibility to get numbers representing protein abundance per replicate directly from Skyline, which is also supposed to be part of the next main Skyline release. I intend to use this option rather frequently in my research and would like to understand it a bit better. I am mainly interested in the normalization case against heavy (each light signal must have a heavy counterpart to be considered).

In the release notes, you state that this protein abundance value is the same that the Group Comparison (GC) framework uses. I thought that, as default, on protein level GC sums up all transition areas that belong to the same protein (also across different peptides) that are quantitative and have a heavy counterpart and then normalizes this sum to the sum of heavy signals. This is slightly different compared with calculating normalized ratios transition by transition and then averaging those ratios, since strong signals dominate the sums of values, as previously described on several occasions. However, the explanation in the reporting window for the "Protein Abundance" feature now states that "The Protein Abundance is calculated by taking the average of the normalized areas of all of the Transitions under the Protein." This now sounds to me exactly as the latter option to do it and the question is, whether protein abundance numbers really are exactly what the GC framework uses?

Thanks a lot,

Roman

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view request
no calibration curve shown for one analyte but others have it
(2 responses) laura.dubois 2021-08-16

Dear Skyline team,

One of my calibration curves won't show up (error is "The selected replicate has missing or truncated transitions") but all other calibration curves for all other analytes look fine. Any idea what is going on? Can I send you a shared file privately to see if you can pinpoint the problem file?

Thank you!
Laura Dubois

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view request
Different dotp value in peak area view and target window
(2 responses) meharunnissakohli 2021-08-13

Dear Support Team,
I was using Skyline for analyzing an MRM based data and also using a spectral library. During analysis, for some of the peptides, I noticed that the dotp value shown in 'peak area view' and that in 'target window' are different. However, for most of the other peptides, it is same at both the places.

Attached here is the screenshot for the same.

Regards
Mehar

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peak area.JPG  target window.JPG
view request
Ion mobility and mProphet scoring
(4 responses) hets 2021-08-11

Hello,
I am analyzing some FAIMS data to quantify heavy/light ratios but uncertain of the order of operations to obtain mProphet q-values.
When I apply ion-mobility settings to select the best CVs prior to adding decoy peptides, the mProphet model training does not fail and the decoy vs target distributions are clearly separated, but if I apply IM setting after adding decoys (followed by re-importing) it seems CVs are also picked for decoy peptides and the training model fails.

Any suggestions on what to do?
Thank you!

Using DDA peptide search with the latest Skyline release

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DIA Umpire error
(1 response) kongdezhi 2021-07-12

Hi,
I am setting up a DIA analysis workflow using DIA raw to build a library. I met a error message about MSconvert (error 1).
System.IO.IOException: Error occurred running process.
Command-line: H:\20210708-TPP-ZQN\msconvert -v --32 -z --mz5 -o "C:\Users\PC\AppData\Local\Temp" --outfile sz44e0ox.pqm.mz5 --filter "diaUmpire params=C:\Users\PC\AppData\Local\Temp\1afneglc.puq.params" "H:\20210708-TPP-ZQN\TPP-Huh7-1-0.raw"
Working directory:
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 65
at pwiz.Skyline.Model.DiaUmpireDdaConverter.Run(IProgressMonitor progressMonitor, IProgressStatus status) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\DiaUmpireDdaConverter.cs:line 139

Then I install Skyline in another PC, I met a new error (error 2) as followed:
System.ComponenModel.win32Exception (Ox80004005): The system cannot find the file specified
at sytem Diagnostics Process.StartWithCreateProcess (ProcessStartInfor startinfo)
at System Diagnostics Process.Start (ProcessStartInfor startinfo)

Please advise ? it is so difficult to solve the errors by myself. thanks

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error1.PNG  error 2.jpg
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Mirrorplotting measured MS/MS spectra against library spectra?
(2 responses) lackmann 2021-08-11

Hi,
I did a targeted analysis in Skyline using a library build using Prosit as a data source (Library --> Build --> Prosit). All worked out great, but as a final quality control and as a figure in a presentation, I want to compare MS/MS spectra from my runs against the library spectra, following the old View --> MS/MS spectra option. However, when I use View --> Library Match I can only generate plots between Prosit and my library (conveniently, I get a perfect dotp score as my Prosit-build library spectrum is compared with, well, Prosit).
Nevertheless, such a mirror plot with a measured MS/MS spectrum against the library would be great, although I would also settle for the old style without the mirror option, but it seems to no longer exist or I am just unable to find it. Search function sadly does not help as the old View --> MS/MS spectrum document is still online under "Tips", so maybe someone could point me in the right direction...?

Jan

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Statistics for lipids
(1 response) komal kedia15918 2021-08-11

Is there a plugin (similar to MSstats for proteomics) to help with downstream statistical comparisons for small molecules/lipids data analyzed within Skyline?

Thanks!

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"Peak Areas" graph
(4 responses) k valgepea 2018-05-23

Dear Skyline team,

I hope you can help me with two short questions:

1. I am double-checking if it is somehow possible to plot the "rdotp" value for a precursor above the bar showing its peak area in the "Peak Areas - Replicate comparison" graph? I can plot "dotp" in my Skyline version (4.1.0.11796), but not "rdotp".

2. Could I simultaneously have two "Peak Areas - Replicate comparison" graphs opened within one Skyline document? For example, one grouped by "Replicate" and showing non-normalised peak areas while the other grouped by "Condition" and showing CVs of pear areas normalised to heavy?

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MS Stats with heavy peptides
(1 response) io 2021-08-11

Hi all,
I am running MS Stats in a SRM experiment with heavy peptides but I think something is wrong because in the Comparison plot I see the log2 Fold Change instead of Log 2 Ratio (L/H). Maybe the label type is not correct (light instead of L, heavy instead of H) but I do not know how to change it.
I share the skyline doc
Irene

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Validación_2021_heavy_refined_STATS-2.sky.zip
view request
(1 response) laura.dubois 2021-08-10

We got a new error when trying to load in raw data:

At 3:10 PM:
Could not load file or assembly 'pwiz_data_cli.dll' or one of its dependencies. The specified module could not be found.
System.IO.FileNotFoundException: Could not load file or assembly 'pwiz_data_cli.dll' or one of its dependencies. The specified module could not be found.
File name: 'pwiz_data_cli.dll'
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache()
at pwiz.Skyline.Model.Results.ChromatogramCache.Build(SrmDocument document, String documentFilePath, ChromatogramCache cacheRecalc, String cachePath, MsDataFileUri msDataFileUri, IProgressStatus status, ILoadMonitor loader, Action2 complete) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromatogramCache.cs:line 618

Any ideas on how to fix this for import?

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Missing fragment results for precursors with fully permuted isotope labels which have the same mass

Hi,
I cannot figure out why my Skyline document is missing results for some fragments, and Skyline's behaviour appears somewhat inconsistent to me.

The setup is a bit unusual, but I hope the following makes sense.

• I am interested in peptides with two K residues and these can be either 13C or 12C. I have therefore applied a variable isotope label and used Refine -> Permute Isotope Modifications -> Complete. This results in 4 precursors per peptide (both lysines labelled, no lysine labelled, the first lysine labelled, the second lysine labelled).
• For other reasons, my cells were grown on 13C glucose, all other amino acids therefore carry fixed structural modifications to make them 13C
• Samples were measured on a Sciex QTOF 6600 with a targeted PRM method generated by Skyline.

The problem is as follows: The two precursors with exactly one labelled and one unlabelled lysine have exactly the same precursor mass, but they do have unique fragments (fragments which contain exactly one lysine). I would therefore like to use these for quantification to distinguish the two label states. When I import my .wiff data, Skyline shows no result for some fragments. And it seems to somehow recognise which fragments are shared and unique between the two precursors. Sometimes it shows no data for fragments which are shared (which is weird but OK for me), but in other cases it excludes all unqie fragments (which is bad).

Do you have any insight why it is not extracting results for some of these fragments? I realise this is a somewhat off-label use of Skyline but hopefully there is a workaround. I have attached a Skyline file with two peptides. The interesting thing is happening with the heavy2 precursor where some fragments are not quantified. For the first peptide the unqiue fragments are not quantified, for the second fragment, the shared fragments are not quantified.

Thanks a lot for your help,
Stephan

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PermutedLabels_missingFragments.sky.zip
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Optimization library for both collision energy and compensation voltage
(5 responses) chi nguyen 2021-07-29

Hello Skyline team,

I am currently doing targeted proteomics on Altis with FAIMS and Skyline Version 20.2
I was wondering if it is possible to have a optimization library with both information about the optimized CE and optimized CV values or if I can import two separate libraries, one for CE and one for CV

Currently I can only use either CE or CV optimization library but not both...

best,
Chi

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Capture.PNG
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one point quantification
(1 response) dms73 2021-08-05

Hi -

I plan to run a verification study of label free results and will be targeting 50 peptides. I have heavy labelled peptides that I plan to include in each sample. I would like to quantify with Skyline using a signal point curve. Does Syline have this quantification option?

Thanks,

Danie Schlatzer

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Using Skyline for target-directed DCC analysis
(2 responses) antoine lacour 2021-08-04

Hi all,

I am looking to use Skyline to speed up the analysis of a target directed DCC run. For those not familiar with the experiment, you take a library of reagents (in this case aldehydes and hydrazides) which react to form a library of products (here acylhydrazones). The reaction is an equilibrium which can be modified by Le Chatelier's principle, in this case by the addition of an enzyme. The idea is to find the products in the equilibrium which are amplified by adding the enzyme, which indicate potential binders to the target in question. See the following article for an example : https://pubs.rsc.org/en/content/articlelanding/2021/sc/d1sc00330e#!

I am wondering if I can use Skyline to help with the analysis of the data coming from these runs. We run a blank experiment with no enzyme and one with enzyme and compare the formation of the products in both.

I created a transition list (M to M+H (not sure if this is what I should be inputting but it seems to work as I intend)) with all possible products of the equilibrium, and imported the LC-MS data from both the blank run and enzyme run. Using this I can easily figure out the retention time of the product and compare its presence or absence in each run.

However I Have the following issues/questions

• Skyline seems to be only reading the MS channel from the .raw file imported from the HRMS machine we use to perform the runs. Therefore the integration of the peaks is done on the MS channel. In an ideal world, I would be able to use the integration from the UV trace that is measured in parallel during the run to compare amplification of certain compounds by comparing peak areas. I realise this could also be done with the MS channel, but due to greatly varying ionisation potentials within the library of compounds, I have my doubts whether this will compare well with the more well-established. Therefore, is there anyway to find the masses of the products in the MS channel but use the UV trace to do the integration analysis? Or would this have to be done in separate software?
• I don't seem to be able to find a way to export the peak areas to a table to compare the values for the two runs, I can only generate the chart using the "Peak Area - Replicate comparison" feature
• I was wondering if there is a way to subtract two chromatograms generated by Skyline using the transition list to only see the difference between the blank and protein-containing runs.

I attached a sample picture of the extracted chromatograms with all the compounds shown.

Thanks in advance for your help, and I apologise in advance for any incorrect terminology I use, MS is not my strong suit.

Cheers,

Tony

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Screenshot 2021-08-04 152118.png
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Max Height
(3 responses) minyg98 2021-07-15

Hi, Skyline team

Is the value of max height of a peptide coming from the highest point in the borderline I set?
I thought the max height in the picture attached was around 1.2e5, but when I checked in the csv file, it was 4.3e4

-Minyeong

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why.jpg
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Specifying Integration Boundaries
(29 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!

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Lipid Creator No longer Exporting to Skyline
(5 responses) wlstutts 2021-06-24

Hi,

After upgrading Skyline Daily, I have lost functionality in Lipid Creator for exporting to Skyline. See attached error message. Any help resolving this issue would be appreciated!

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Skyline_LC.PNG
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Feature Request: Background Colors
(3 responses) jdevitt 2021-04-07

Hello,
I am using the Skyline version 64-bit 20.2.0.343. I was wondering if in the future an option could be added to change the background color of the Skyline chromatography profile and peak area/retention time graphs from white to another color, especially a black or gray option. When I use Skyline for hours at a time it would be nice to have a background color that is a little less harsh on the eyes. Not urgent, just something that would be nice to have!

Best,
Julianna

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Orbitrap DIA search using converted Spectronaut library
(1 response) zhangfangfei 2021-08-02

Hi Skyline Support team,

I used a converted library from Spectronaut for the DIA analysis.,by importing the transition list, other steps referring the instruction here https://skyline.ms/_webdav/home/software/Skyline/%40files/tutorials/DIA-QE-20_1.pdf. The mProphet capture is in the attached. It seems to me a failed search with greyed RT difference and dot product. We got a very poor CV by replicates, low cross-correlations by retention time and quantification comparing with other DIA search results. The results are much better on other datasets.

What do you think could be the problem ? My guess would be 1) the neural loss ions or other ions are setting not properly from the converted Spectronaut library 2) retention time alignment failed (there displayed the RT in the library ) 3) the size of library is too large (300K precursors) 4)Is the Skyline Batch relevant for the DIA analysis?

As the library is very large, any operations took a long time. I would like to know your suggestions in how to quickly probe into the problem in quantification.

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Picture_B_skyline20210802.png
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MS stats error exporting the report
(6 responses) io 2021-07-28
Hi again,

I have another error with MS Stats: Error exporting the report

---------------------------
Skyline
---------------------------
Error exporting the report, tool execution canceled.
---------------------------
---------------------------
System.IO.IOException: Error exporting the report, tool execution canceled.
en pwiz.Skyline.Model.Tools.ToolMacros.GetReportTempPath(ToolMacroInfo toolMacroInfo) en C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Tools\ToolMacros.cs:línea 266
en pwiz.Skyline.Model.Tools.ToolMacros.ReplaceMacrosHelper(SrmDocument doc, IToolMacroProvider toolMacroProvider, ToolDescription tool, IProgressMonitor progressMonitor, String replacein, Macro[] macros) en C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Tools\ToolMacros.cs:línea 206
en pwiz.Skyline.Model.Tools.ToolDescription.RunExecutableBackground(SrmDocument document, IToolMacroProvider toolMacroProvider, TextWriter textWriter, IProgressMonitor progressMonitor, Control parent) en C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Tools\ToolDescription.cs:línea 492
en pwiz.Skyline.Model.Tools.ToolDescription.<>c__DisplayClass79_0.<RunExecutable>b__0() en C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Tools\ToolDescription.cs:línea 334
---------------------------
Can you help me?

Thank you

Irene
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Selection of razor peptides for Quantitative proteomics
jyoti 16541 2021-07-30

We are planning to develop a mass spectrometry based quantitative assay for the monitoring of certain proteins/ genes. As part of this, we are working on selecting the best proteotypic peptide for each of our target proteins using Skyline.

There are certain proteins which do not have any unique proteotypic peptide. But when we try to apply uniqueness at the gene level, we could see certain peptides considered as unique to a gene. So, I would like to know whether we can consider such peptides (razor peptides) for our quantitative proteomics?

For example, we are looking for a proteotypic peptide of p40 (deltaNP63alpha). However, the peptide sequence for p40 is present in its multiple isoforms. Hence we are not able to select specific proteotypic peptides for p40 isoform (i.e alpha).

--
Jyoti

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Number of scans from raw data
(4 responses) ulisse cucchi 2021-07-29

Hello Skyline Team,

I'm still trying to learn the basics of the software, in particular for the targeted analysis of peptides by PRM. I managed to perform all the steps well described by your tutorials, but I have an issue regarding the number of scans that are imported into Skyline from the raw data. To show you the issue I attached you 3 slides in a pdf file.

In the slide 1 you can see the peak from the raw file (two different visualizations of the same peak are showed). As you can see the peak contains over 40 scans of the same precursor (the instrument used was the Orbitrap Exploris 240 from Thermo).

In the second slide you can see how the same peak is imported into Skyline. As you can see, only few scans (less than 10) are used and the resulting peak has a lower resolution, that means also a lower accuracy when the peak is integrated.

In the third and last slide you can see the only parameter that I've found that should affect the number of scans that are imported, but the option "include all matching scans" was already selected. Since they are all very good scans from the raw file, I don't understand why Skyline is considering less than 10 of them. Is there any way to make Skyline import all the scans present in the raw file?

Best regards,

Ulisse Cucchi from Nerviano Medical Sciences (Italy)

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prm.pdf
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Agilent QQQ 6460 Data import showing " chromatogram information unavaliable"
(4 responses) xue shi 2021-07-29

Help! Help! Could you please help me out ?When I try to use skyline to process my Agilent QQQ 6460 LCMS data, It cannot show chromatogram correctly. When I click on the lysine or any other molecule, it cannot show chromatogram. I don't know what cause this.
I attached the screen shot for what I get from skyline and what I get from mass hunter.
I also attached my transition list.
Thank you,

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chromatogram information unavaliable.docx  transition list for skyline.xlsx
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Seperate MS/MS Filtering Settings for Light vs. Heavy Precursors
(6 responses) n t wamsley 2021-07-19

Hello, I am working on a PRM method with stable-isotope-labeled internal standards. Technically it is "SureQuant" where the SIL peptides are monitored to trigger acquisition of endogenous peptides. MS2 scans for the heavy SIL peptides are carried out in an orbitrap at a resolving power of 7,500 at 200 m/z. However, MS2 scans for the endogenous/light peptides are carried out at a resolving power of 60,000 at 200 m/z. Right now I am using 7500 at 200 m/z for both the light and heavy peptides. However, I would like to enforce a separate and more stringent threshold for the endogenous MS2 scans that reflects the different instrument settings used in my method. I believe that on account of the thresholds being too low for the endogenous (in skyline), some of the endogenous transitions appear to have a lot of interference. At the moment is it possible to change this in skyline?

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More proteins shown than library in SWATH analysis
(9 responses) heyang 2021-06-16

Hi Skyline team,

I just started to learn using Skyline to process SWATH data acquired by Sciex 6600. Library was created by multiple DDA runs searched by ProteinPilot software. Following your instruction, input FASTA files and spectra library. More pep/Proteins than number in library are shown in Skyline. How to explain this?

thanks,
Heyi

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CE optimization on a Sciex Qtrap
(1 response) alejandro.cohen 2021-07-15

Hi Skyline people

We are performing a CE optimization on a SMR experiment run on a QTrap5500 for small molecules, and following the Tutorials. Experimentally, everything worked fin.
We exported the Transition list into the .dam files with multiple the CEs, and reacquired data on known standards. When reimporting the wiff files into Skyline, we get the expected 'curve' for the CEs.

The Replicate graphs show a legend for the colors and CE Steps (-10,-9. etc).

Is there a way for Skyline to show the nominal CEs used by the instrument in the graphs instead of the Steps? Or, is there any parameter in the Document grid to show the Nominal CEs??? All the parameters with CEs I select show either calculated values, or Steps.

Cheers

Alejandro

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Skyline-daily (64-bit) 21.0.9.139 (6dbba07d9) cannot load the library.tsv file from EasyPQP
(8 responses) fcyu 2021-06-02

The library.tsv file from EasyPQP is in OpenSWATH format. But when loading it in Skyline, it says "the file F:\dev\msfragger\test_skyline\library.tsv is not a supported spectral library file format.". According to https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BlibBuild, it should support OpenSWATH tsv file.

I put an example of library.tsv in the attachment. Could you please take a look when you have time?

Thanks,

Fengchao

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library.zip
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Comparing Peak Scoring / Sample Specific Peak Scoring Models
(4 responses) peter r mosen 2021-06-17

Hello Skyline team, hello Skyline community,

Is there any tutorial etc. which deals with Skylines “Comparing Peak Scoring“ function? I would like to understand the plots (e.g. the different y-axis options) as well as its application better. The tutorials 14, 15 and 18 cover peak model building in general but I didn’t find anything on the “Comparing Peak Scoring” option.

Briefly about my situation: In my targeted MRM assay I am monitoring peptides across multiple organs (9x) and cell lines (5x). For data analysis I am using mProphet peak scoring models. I was now wondering if it is best to use for each organ type its own peak scoring model, or if a mixed-organ respectively mixed cell line peak scoring model can be used for the individual organs. Anyone with any experience in that? Any suggestions, any hints ?

Best, Peter

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MSstats installation error
(3 responses) io 2021-07-26

Dear all,
I am trying to install MSstats but it is not possible to intall de R packages.
I attached the error
Irene

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Error Skyline_210726.pptx
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Sciex 7500 Support
(1 response) dawn dufield 2021-07-26

Hi, We recently purchased a 7500 and were wondering what support there is for the 7500? Ie in method generation, parameter prediction, data review etc.

Thanks
Dawn

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FAIMS support
(1 response) sudip ghosh20975 2021-07-26

Dear Skyline team

I was wondering if Skyline support DIA files with FAIMS (multi CV) annotation acquired on the Exploris 480.

Sudip

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Using same molecules as iRT standard and surrogate internal standards?
(1 response) Will Thompson 2021-07-02

Hi Skyline Team,

I have a use case where i would like to use the same set of stable-isotope labeled molecules both as iRT standards as well as surrogate internal standards for the purposes of quantification. If i set the molecule as an iRT standard (i.e. the little "clock" icon comes up on the molecule) then when i go to the right-click menu to set that molecule as a surrogate standard the selections are greyed out. I can't think of any practical reason why this would not be allowed. Am I doing something wrong, is there a functional reason this is the case, or is this a bug?

Representative Skyline file attached. IF you look in the document grid, you do have the surrogate standards assigned to different molecules, but this is only because i did this part before applying the iRT correction.

Help as always is appreciated.

Cheers,

Will

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iRT_SurrogateQuant_Bug_forSKylineSupport.sky.zip
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Building a spectral library using msp
(7 responses) laura corveleyn 2021-02-02

Dear,

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!

Best,

Laura

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TEST_Prop.msp  Test_Predicted_msp.sky.zip
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Error to install skyline daily
(3 responses) Aline 2021-07-23

I'm trying to install in a windows 10 computer and I'm getting this error:

" Setup has detected that the file 'C:\Users\ADMINI~1\AppData\Local\Temp\VSDA0E1.tmp\DotNetFX472\NDP472-KB4054531-Web.exe' has changed since it was initially published.

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Importing peak boundaries via SkylineRunner
(1 response) l render 2021-07-19

Hello

Is this possible?

I was hoping to automate a workflow that involved it.

thanks

Lee

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Different types of skyline files
(6 responses) heyang 2020-02-27

Hi Skyline team,

Just want to know details about different types of skyline files, e.g document, view, skyl and chromatogram data. if I want to upload a result to panarama, I need to upload all 4 files, or just document file. thanks,

Heyi

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ambiguous PRM peptides don't show up as targets, but can be found in the library
(1 response) susanne matschi 2021-07-23

Hello,

First of all, I am quite new to the MS field so there might be some obvious error I just don't know any better about. I am working on Windows 10 Pro with Skyline 20.2.0.343.
I am trying to analyze PRM data measured on a Thermo QExactive Plus, including heavy peptides for quantification. Doing identification with Proteome Discoverer using our Mascot server, it can find the native peptides (unfortunately only 10-80 PSMs depending on the sample) as well as the labelled ones (more than 1000 PSMs per sample).
When trying to build a library with the msf or PD result files and importing the data to Skyline, the peptides do not show up as targets at all. I went back and tried to import some of the Mascot dat files, which then gave me the error "Peptides had multiple ambiguous peptide matches and were excluded". I redid the library build and importing, checking the box "include ambiguous peptide-spectrum matches", and now I can find the peptides in the library (only with the dat files, not the msf or PDresult), but it still doesn't show up as a target peptide. I could then manually add them as library matches to the target peptides, but I don't really understand why it would not do this automatically. I played around with a lot of peptide and transition settings but nothing worked to make them appear.
What could be an explanation for this, Is there anything I am missing?

Thanks,
Susanne

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dotp value after Prosit integration in Skyline
(1 response) meharunnissakohli 2021-07-22

Hello Support Team,
I want to analysis a data in skyline in which I want to match the spectral data obtained in an MRM experiment with the Prosit predicted library.
I could see that the Prosit is making predictions for my target peptides but dotp value is not shown.

However, if I monitor precursors ions also, then only dotp value is shown (but in my data, i have product ions for which I actually need the dot p value).

Looking forward to hearing from you.
Regards
Mehar Un Nissa

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Error when opening saved Skyline files
(1 response) antoine lacour 2021-07-22

Hi,

I've recently upgraded to Skyline 21.1 (downloaded on 22/7/21) and I have the following error when opening a saved .sky file. This happens with old files from a different Skyline version but also with newly created and saved files.

---------------------------
Skyline
---------------------------
Failure opening U:\KTGS\Skyline test sample\Skyline test sample2.sky.
The file contains an error on line 81 at column 9.
---------------------------
---------------------------
System.Reflection.TargetInvocationException: There is an error in XML document (81, 9). ---> System.InvalidOperationException: There is an error in XML document (81, 9). ---> pwiz.Skyline.Util.AssumptionException: error reading mz values - declared mz value 325.13691 does not match calculated value 324.129085450091
at pwiz.Skyline.Util.Assume.Fail(String error) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 2055
--- End of inner exception stack trace ---
at pwiz.Skyline.SkylineWindow.<>c__DisplayClass742_0.<OpenFile>b__0(IProgressMonitor progressMonitor) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 328
at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action1 performWork) in C:\proj\skyline_21_1_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_21_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1940
at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 202
at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 345
---------------------------



Any help would be appreciated!

Cheers,

Tony

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Screenshot 2021-07-22 154510.png
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Boxcar support
(16 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
Diego

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Skyline install problem
(3 responses) gemma hardmanfowler 2021-07-14

Hi,

I am getting an error when trying to install Skyline on my PC. Screenshot and details attached.

Have you seen this before? Can you offer any guidance? Thanks

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F170ATJM.log  Capture.PNG
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saving all transition image files at once and adjusting based on y-axis for selected peak
(1 response) jiwon hong 2021-07-20

Hello Skyline team,

My name is Jiwon and I am currently a graduate student at Korea University.

I have been using skyline for a few months, but couldn't find a solution for a very time consuming/repetitive job which lead me here.

I wish to save the image files of the transitions: 3 files (total, light, and heavy) for each peptide with a specific pattern name.
For peptide sequence XXXX,
I wish to save the total image file as XXXX_a, light as XXXX_e and heavy as XXXX_s in a designated directory (they can all be in the same directory) in .jpg format.

Currently, I was only able to save the files through right clicking > Save image as one by one, and was wondering if skyline is capable of saving all the images at once.

And also, would it be possible to leave the x-axis and only auto adjust the y-axis based on the selected region only?
An example file is attached below (skyline_adj : wanted scale, skyline_no adj: default image)

Sincerly,
Jiwon Hong

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DIA Umpire error
(1 response) Francis Beaudry 2021-07-20
Hi,
I am always getting this sort of message when trying to analyze DIA datasets without a spectral library. Any idea what is wrong ? I get the same message with the Tutorial files.
Francis

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Skyline
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Error
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System.IO.IOException: Error occurred running process.

Command-line: C:\Users\Francis Beaudry-LAB\AppData\Local\Apps\2.0\8NEPY2DK.Y0L\76REB5VE.A64\skyl..tion_e4141a2a22107248_0015.0001_b2be94ac8cfe2cb4\msconvert -v --32 -z --mz5 -o "C:\Users\Francis Beaudry-LAB\AppData\Local\Temp" --outfile djkspuzt.ic2.mz5 --filter "diaUmpire params=C:\Users\Francis Beaudry-LAB\AppData\Local\Temp\j0w24agx.41p.params" "C:\Users\Francis Beaudry-LAB\Documents\DIA Datasets_test\19ADL08-HELA-2.raw"
Working directory:
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 65
at pwiz.Skyline.Model.DiaUmpireDdaConverter.Run(IProgressMonitor progressMonitor, IProgressStatus status) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\DiaUmpireDdaConverter.cs:line 139
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How does Skyline treat shared fragment ions between precursors of same peptide from MSE data?
(2 responses) Juan C. Rojas E. 2021-07-07

Hi Skyline team,

I would like to confirm how skyline treats a fragment ion that is produced from multiple precursor ions of the same peptide when the data is acquired with MSE. Without the use of IMS it is not possible to discern from which precursor the fragment ion is being produced due to the lack of quadrupole isolation.

When the integrated fragment area is summarized for the peptide and a transition for the same fragment exists in two (or more) precursors, is the area of this "repeated" transition summed twice (or more) or does Skyline recognize it is a repeated value and only counts it once?

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

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Exporting all peaks and mass accuracy for chromatograms
(4 responses) thomas hopf 2017-07-24
Dear Skyline developers,

I am trying to set up an automated evaluation scheme for peak picking outside of Skyline. For this, it would be very nice to have access to all candidate peaks and their associated mass errors. While I see that information in the GUI, I wasn't able to fully access the underlying data so far.

1) When exporting the chromatograms for all of the precursors, besides the intensities is it somehow possible to get the corresponding mass error/accuracy for each time point? In the reports, I only seem to be able to get the error for the one chosen peak per precursor.

2) For each precursor, is it possible to get a list of all candidate peaks chosen by the Skyline heuristics?

3) I seem to be able to extract some of the above information by exporting the mProphet peak features; however the associated mass errors for the mProphet candidate peaks seem to differ quite substantially from nearby Skyline peaks. How does this occur?

4) Can the mProphet features be automatically extracted using a command-line parameter?

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export fasta of all verified proteins from Skyline
(5 responses) kguehrs 2021-07-15

Hello Skyline team,

Perhaps this matter was already asked but I could not find any answer after I drastically limited my search to "export fasta".

I have done a DIA analysis of some special tissues from an model organism. The basis for the analysis was a rather large fasta database of not so good quality which I used as the background proteome. The analysis of the run data was done with Umpire/Fragpipe and imported into Skyline for visualization and curation. I did quite a bit of manual refinement and came up with a status in which I erased a lot of identified by the search engines proteins that do not match my quality restriction. Besides the generation of a spectral library I would like to also create a fasta file of either all or a specified fraction of the proteins remaining after the curation of the data. This fasta coould be used for other purposes or as a core background proteome that could be further expanded by additional data.

Is there any chance to export the proteins remaining after curation as a fasta file from Skyline. If there is no direct way, can you propose an other tool or procedure to do so from any Skyline generated report.

Best Karl-Heinz

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Peak boundaries
(1 response) minyg98 2021-07-17

Hi, Skyline team

Can I get an explanation about how Skyline determines peak boundaries?

-Minyeong

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Filtering a Skyline document for protein length
(2 responses) cabarnescabarnes 2021-07-12

Hi. I have a Skyline document of something like 5000 protein entries and I'd like to filter it based on the amino acid sequence length of the proteins in the document. Specifically, I want it to only be the proteins under 150 aa. I can obviously export results to a table and do it there, but I'd like the Skyline document to only include this subset. Is that possible?

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MS/MS ion rank and type for full scan data
(13 responses) roman sakson 2021-07-12

Hi Skyline Team,

I am using the "show ion rank and type" feature for full scan targeted MS/MS analysis (=PRM). The feature makes my life a lot easier than before, thanks a lot! I have, however, a question: for a peptide that was detected quite confidently, I see a color code in the MS/MS that is perfectly matching my PRM XICs on the left (see first screenshot). When I switch to the show ion rank and type view, I am only getting a few matches for the MS/MS (see second screenshot). My library ion match tolerance is 0,5 m/z and what I have here are high resolution MS/MS orbitrap data. I wonder, why is my ion matching so poor when matching ion rank and type?

Thank you!
Roman

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I_Full_Scan_Conventional.PNG  II_Showing_Rank_and_Type.PNG
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Problem importing Brucker Tims-TOF Pro data in docker container
(9 responses) m j noga 2021-04-23

Dear Skyline Support team,

With a larger team of bioinformaticians and developers we are prototyping data processing and analysis workflow based on Skyline. In one of the use cases we performing targeted MS1 feature extraction using from the TimsTOF Pro data. We already successfully tested the workflow on Windows, both via GUI and command line and we are right now at the stage of transferring it to containerized environment on our Linux cluster.

As a first stage we are just using official docker image (https://hub.docker.com/r/chambm/pwiz-skyline-i-agree-to-the-vendor-licenses), but we experiencing a high number of import failures with the following exception:

Unhandled Exception: System.AccessViolationException: Attempted to read or write protected memory. This is often an indication that other memory is corrupt.
at System.Collections.Generic.GenericArraySortHelper2.Swap(TKey[] keys, TValue[] values, Int32 i, Int32 j)
at System.Collections.Generic.GenericArraySortHelper2.PickPivotAndPartition(TKey[] keys, TValue[] values, Int32 lo, Int32 hi)
at System.Collections.Generic.GenericArraySortHelper2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
at System.Collections.Generic.GenericArraySortHelper2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
at System.Collections.Generic.GenericArraySortHelper2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
at System.Collections.Generic.GenericArraySortHelper2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
at System.Collections.Generic.GenericArraySortHelper2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
at System.Collections.Generic.GenericArraySortHelper2.Sort(TKey[] keys, TValue[] values, Int32 index, Int32 length, IComparer1 comparer)
at System.Array.Sort[TKey,TValue](TKey[] keys, TValue[] items, Int32 index, Int32 length, IComparer1 comparer)
at System.Array.Sort[TKey,TValue](TKey[] keys, TValue[] items)
at pwiz.Skyline.Util.ArrayUtil.Sort[TItem](TItem[] array, TItem[][] secondaryArrays) in Z:\pwiz\pwiz_tools\Skyline\Util\Util.cs:line 945
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.SortSpectrum(SpectrumInfo spectrumInfo, Int32 i) in Z:\pwiz\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 997
at pwiz.Common.SystemUtil.ProducerConsumerWorker2.Consume(Object threadIndex) in Z:\pwiz\pwiz_tools\Shared\Common\SystemUtil\ProducerConsumerWorker.cs:line 185
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)



When attempting to import a single file multiple times it fails 30-40% of the cases, at random stage during import (see example import log attached).
We invoke Skyline by:

docker run -it --rm -v /Users/m/surfdrive2/Glycopeptides/data:/data \
--in=output/clean.sky --out=output/with_data.sky --timestamp --memstamp \
--import-file=Controle_16_Slot1-36_1_3174.d --full-scan-precursor-res=40 \
--log-file=output/import_log.txt > data/output/import_console_log.txt



Setting up options like '--import-process-count' or '--import-threads' doesn't seem to have any influence.

We already tested this using docker on Linux and OS X hosts with similar error rates.

We do not see this issue when running the same workflow on Agilent Q-ToF data, which may point out this is somehow linked to reading Bruker data.

Despite high failure rates we are able to import our 40-sample test dataset in the end (with up to 9 re-tries) and the final results are identical as on Windows.

I am wondering if you could help us with fixing this problem. Is this a Proteowizard issue or Wine? Should we contact proteowizard support directly? Our software engineer is eager to get involved, but would need some support and feedback to get started.