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.

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

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

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Showing: limited to 100 requests
Failed importing results- .wiff file from SCIEX triple Quad
(9 responses) sarahmacpherson676 2019-06-19

First, thank you for offering this software, I’m new to Skyline and I am excited to use it.

My data files were collected from another lab using SCIEX triple Quad (5500). I am looking at small molecule data (around 200 metabolites). I was given a .wiff file containing the multiple runs and the .wiff.scan. However I am unable to upload either(file-->import-->results). I’m not sure if this is a file issue, software or something on my side. I have tried using the newest version of Skyline and Skyline Daily.
Attached are the errors that I receive for the .wiff file and .wiff.scan
Also attached is the .wiff and .wiff.scan files
Thank you for your time

 error messages skyline.docx  cell_metabolomics.wiff  cell_metabolomics.wiff.scan 
view request
issue with MSstats installation
(3 responses) seoyoung park 2019-06-26
I would like to install MSstats, but it showed me an error message as below. Can you please help me to resolve it?
Download failed.
Check your network connection or contact the tool provider for installation support.
OK More Info
System.Reflection.TargetInvocationException: Download failed.
Check your network connection or contact the tool provider for installation support. ---> pwiz.Skyline.Model.Tools.ToolExecutionException: Download failed.
Check your network connection or contact the tool provider for installation support.
   at pwiz.Skyline.ToolsUI.RInstaller.DownloadR(ILongWaitBroker longWaitBroker) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\ToolsUI\RInstaller.cs:line 187
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 232
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1854
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 180
   at pwiz.Skyline.ToolsUI.RInstaller.GetR() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\ToolsUI\RInstaller.cs:line 137
Thank you,
view request
TIC not importing/displaying correctly
(5 responses) thicks 2019-06-26

Hi all,

I am trying to do a simple import of MSe Qtof data with lockmass correction. I've never had an issue before, but now I have several .raw file that import the last third of the chromatogram incorrectly. Up to 80ish minutes looks as expected but something bizarre happens after that. I've attached what the TIC looks like from the acquisition software. Is the file corrupted? Any ideas?



 skyline-import.PNG  skyline-import-zoomed.PNG  correct-chromatogram.PNG 
view request
Document Grid - Reporting Options for Peptide Ratio Result
(1 response) davis 2019-06-25

Hi Skyline team!

I have a quick question regarding reporting options for peptide ratio results in the Document Grid

In the customizable report is there a column for (1) name of acquisition method (2) If an analyte is the surrogate/global internal standard. If so I was having issues locating it using the Find Column tool. Thank you!

Best! Sonnet

view request
Quasar Error
(9 responses) danielacgranato 2019-06-19

I have just installed Quasar and I am getting an error message ( see attached) regarding some parameter that must be changed. Could you help me set the document so that I can run Quasar on Skyline. Thank you very much. Best, Daniela

view request
Request: rdotp filter
(1 response) roberthardt 2019-06-24

Dear Skyline-team,

is there any way to filter my results by the rdotp values within Skyline? If not I would highly appreciate such a feature beinig implemented in a future version.



view request
Retention Time Issues for DIA data
(1 response) timofiiva 2019-06-24


I encountered an issue with SkyLine recently and have not been able to sort it out:

  1. I imported the chromatograms from a phospho-enriched SWATH dataset and am analyzing the peak picking for my protein of interest
  2. One of the phosphopeptides of interest has a predicted retention time of 120 min
  3. For 5 of my 30 conditions, all is ok (screenshot1) where the peak is able to be picked by the expected retention time, and the chromatogram even extends further to 145min
  4. For the remaining 25, the chromatogram stops abruptly at 110 min (screenshot2). Even if you go through the Transition Settings/Full-Scan and then select "Include all matching scans", it will not allow you to see past the 110min.

I have tried the following so far:

  • To adjust the "explicit retention time" in the documents grid, and then reimport the file. What happens is that the "chromatogram information is unavailable.

Thank you in advance for the help!



 ScreenShot1.png  ScreenShot2.png 
view request
Peptide Transitions
(5 responses) iej4 2019-06-19

I am setting up an MRM peptide quantitation method but I need to input a non-standard transition. I have a short proline containing peptide that is producing what seems to be a strong y-18 ion but Skyline doesn't display that fragment as a possibility so I can't use it in the method. Is there a way to change the fragmentation rules of skyline so that the fragment is allowed or to manually add a transition to a peptide?


view request
Unsupported score in search output file generated from Peptideshaker and several different search engine output
(10 responses) weixiandeng 2019-02-11
Hi Skyline team,

I was trying to build spectrum library through Peptikdeshaker output which is a mzID file, however, it gives me an error report showed below.

Then I switch to comet raw output(pep.xml), tide-search and MSGF+(mzid), they were all given the same error.

Then I tried these files on both Skyline 4.2 and Skyline Daily, still same error.

Can you please help me figure out the problem?


ERROR: .mzid file contains an unsupported score type

OK More Info
System.IO.IOException: ERROR: .mzid file contains an unsupported score type

   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 59
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 171
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 137
view request
Filtering Peptide min and max length not updating
(2 responses) dawn dufield 2019-06-18


I'm trying to filter my peptides in skyline after a digestion and it does not seem to update after I change the filter length. ie if it says 5 and I change it to 10, it does not remove the peptides that are shorter than 10.

Also when I chose 5 initially, it did not eliminate the ones shorter than 5. Is there anything specifically I need to do to get it to take my changes?

view request
mProphet for DIA: Remove peptides to apply features & RT difference
(1 response) d o debets 2019-06-19

Dear Skyline team,

Recently I've performed a relatively big DIA experiment for the first time and I am experiencing some problems with applying the different features for the mProphet peak picking. I know some of these questions have been asked previously, but unfortunately I haven't been able to find an answer on this forum that could solve my isseus. I'd be very grateful if you could provide me with some insights!

First, some information about my experimental set up:

  1. I have made my own library by fractionating a pooled sample and running this in DDA mode on an Orbitrap, using the iRT peptides as well. This yielded a library of around 10.800 proteins and 208.000 peptides (including decoys).
  2. My DIA experiment consists of around 60 samples that I ran, again including the iRTs.
  3. I have used the command line version of Skyline to match my DIA files to the library and am currently using Skyline to look at the mProphet model and my peaks.

The first thing I noticed when looking at my model, is that it's not very good at separating my targets from decoys, but this is probably partly due to the fact that quite some important features were not used. First of all, I deleted all the peptides that didn't have a library dotp. These peptides made sense since in the majority of times there were not enough transitions matched to the library. However, there were quite a few of them and I had to remove them by hand, leading me to my first question: is there a smarter/more efficient way to remove these peptides?

After removal of these peptides, I looked at the peptides that had no information for the RT difference value feature. However there are loads of peptides that do not have this information, leading me to think that maybe something has gone wrong in the way that I set up my library/Skyline file. Leading me to my second question: Do you have any idea what could cause this? Maybe more generally, could you give me an idea why a peptide would not meet this criteria? And last but not least: how can I deal with this? There are way too many to delete them by hand, so I do not really know how to proceed from here.

As I said, it's the first time I am performing a DIA experiment, so some insights would be very much appreciated. I have attached the zipped Skyline file for you to have a look at if that's helpful. I have removed the majority of the raw files from the results there since otherwise the file would be way too big. Maybe leading me to my last question: is the file size that I have somewhat as expected? The output .skyd-file is around 380GB, which sounds like a lot to me?

Thanks a lot for your help in advance!

Best wishes,

view request
strange behaviour of unique peptides table checkmarks
(1 response) Tobi 2019-06-18

Dear all,

in skyline daily 19, I used a fasta file containing several isoforms of a heatshock protein. To check for shared and unique peptides I created a proteome file from the fasta and I imported the same fasta to create the target list. When selecting all targets with CRTL+A and looking at the unique peptides table yields some unexpected results. Could you please tell me if I have an error somewhere or what might be going on?

For example Peptide EEGTQQR from protein Q8N241. In the target list and even in the table it is clearly stated that this peptides comes from this protein, in the table however there is no checkmark. Do I misunderstand the default checkmarks? Because I would expect them to occur by default in every case where a peptide matches a protein, but this is not the case. All setting were just set to default.

When clicking on a specific protein for unique peptides I havent found an error so far.

Looking forward a short reply.

Best regards,

 protein digest .JPG  protein  HSPB7.fasta 
view request
DDA file without target peptide
(2 responses) gaohuanhuan 2019-06-18
Hi all,

I have used skyline to analyzed low abundance peptides which were gathered by PRM mode. The low abundance of those peptides couldn't find in DDA mode either, even if the peptides have been set fractions. For those peptides, I have no library, no MS2 information. How are these peptides in PRM data analyzed with skyline?

Best regard!

view request
Feature Request Sequence Dependent Precursor Charges
(1 response) roman sakson 2019-06-13

Hello everyone,

first of all, thanks again for the amazing piece of software that continues to be the highlight of my research days!

I work in a core facility, where we routinely develop MRM assays from crude synthetic standards without much prior knowledge, this means that for the first runs we have to "guess" the precursor charge state as well as suitable transitions based on common MS sense for relatively long target lists. In our experience, doubly charged precursor ions work best for tryptic peptides of average length, therefore this is the standard transition setting. However, if the sequence contains histidine, normally the more intensive precursor is triply charged. Is it possible to specify in the filter settings or elsewhere, that precursors should be doubly charged, unless there is a particular amino acid present in the sequence?

Thanks a lot in advance for your time!

Roman (Heidelberg, Germany)

view request
Importing result from TimsTOF (TDF forrmat)
(12 responses) m anna monika 2019-05-24

I was wondering if you could help me with a result importing issue. For some reason when I try to import results from a run performed on a TimsTOF instrument Skyline doesn't seem to recognise the TDF file. I'm using Skyline 4.2.

view request
DIA samples
(3 responses) geweigang 2019-06-16

I have used the skyline to run DIA. After exporting the results, why is the number of proteins detected in each sample the same?
What can I do to make the number of proteins or peptides detected in each DIA sample different?

view request
Displaying peptide mod in left target sequences
Erik de Graaf 2019-06-17

Hi all,

Currently I'm using skyline to quantify glycopeptides. However considering there are many glycan versions I end up with a big list of identical peptide sequences and it is hard to quickly see what glycan is on them (see attachment). Same goes for the peptide comparison plot that shows all the same sequence.

I now use note to write down the modifications, so when I hover over I can see the mod. However, this way I have to hover over all peptides to find the p.o.i.

Displaying the modified sequence in the list and graphs could greatly facilitate glycopeptide analysis.


view request
Using colors to mark peptide/precursors for review
Erik de Graaf 2019-06-17

Hi All,

While browsing through my results I'm marking peptides that give problems (interference, absence) in some samples.
Currently changing colors is not high-throughput, you have to rightclick->peptide note->color.
It would great speed-up my workflows if there was a shortcut to do this!

Kind regards,

view request
Keyboard Shortcuts
(6 responses) thomlau 2011-09-16
Hi all,

Is there a file or list somewhere with all the keyboard shortcuts for skyline? I'm going through a large batch of peptides and replicates and my hands feel like falling off!


view request
mzid file contains unknown score type ERROR
(8 responses) Shawn Rice 2019-06-12


I was trying to import an mzid file from a ProteinPilot search into SkylineDaily v19.0.9.149 and received and unknown score type ERROR. Do you have any suggestions to get this to work. The complete error message is below.



ERROR: .mzid file contains an unsupported score type

Command-line: C:\Users\srice\AppData\Local\Apps\2.0\1HWJB6KR.0MJ\D4N33RE7.E7A\skyl..tion_e4141a2a22107248_0013.0000_8870d1d2982721de\BlibBuild -s -A -H -o -c 0 -i proteinpilot -S "C:\Users\srice\AppData\Local\Temp\tmp513.tmp" "M:\06-2019\proteinpilot.redundant.blib"
Working directory: C:\Users\srice\Downloads

OK More Info

System.IO.IOException: ERROR: .mzid file contains an unsupported score type

Command-line: C:\Users\srice\AppData\Local\Apps\2.0\1HWJB6KR.0MJ\D4N33RE7.E7A\skyl..tion_e4141a2a22107248_0013.0000_8870d1d2982721de\BlibBuild -s -A -H -o -c 0 -i proteinpilot -S "C:\Users\srice\AppData\Local\Temp\tmp513.tmp" "M:\06-2019\proteinpilot.redundant.blib"
Working directory: C:\Users\srice\Downloads
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in C:\proj\pwiz_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 186
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 144

view request
how to import TraML file into skyline?
(4 responses) caixue 2019-06-11

Is there any way to import TraML file into skyline? I have failed to import TraML or csv which from TraML file into skyline.
Thank you in advance!

best regards.
Xue Cai

view request
Combining own DDA library with SWATHatlas library
(3 responses) SanL 2019-06-11

Good morning,

similar to a previous post submitted in 2016, I'd like to merge my own in-house DDA library with the Rosenberger library downloaded from here(phl004_canonical_s64_osw.tsv).

Now, I am trying to combine the two and align the retention times.

I know the process technically works the way it was suggested in the thread mentioned above. I did it before with a 'reduced' Rosenberger library similarly to the user in the thread.

Now, when I try to merge the in-house library with the 'full' Rosenberger library by importing the latter using the "import --> assay library" function, I get an "Index outside the bounds of the array" message (see screenshot attached with more details).

I cannot imagine the library being too big but I also could not figure out what the error message is telling me

Thank you in advance!


view request
Adding a water loss product ion
(4 responses) joe.palandra 2019-06-11


Apologies if this has already been asked but I am developing a targeted assay on a triple quad and wanted to know if there was a way to select a -H2O product ion? I have a peptide that gives very good sensitivity using such a transition and I would like to add this to my other regular y and b ions. Essentially it is like a parent to parent transition.


view request
Fail to install Skyline
(1 response) yueliangwias 2019-06-11


we are trying to install both Skyline daily and 4.2version but we failed to install it on the control computer of Sciex 5600 since we would like to do MRM analysis using Skyline. The error page is captured in the attach.

An error occurred trying to download ''.

See the setup log file located at 'C:\Users\5600\AppData\Local\Temp\VSD6E2D.tmp\install.log' for more information.
####install.log file
"The following properties have been set:
Property: [AdminUser] = true {boolean}
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'
Read integer value 1
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'
Read integer value 461814
Setting value '461814 {int}' for property 'DotNetFull_Release'
Reading value 'v4' of registry key 'HKLM\SOFTWARE\Microsoft\NET Framework Setup\OS Integration'
Unable to read registry value
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] = 461814 {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.
URLDownloadToCacheFile failed with HRESULT '-2146697208'
Error: An error occurred trying to download ''.

We are looking forward for your feedback. Thanks a lot

view request
SkylineRunner not importing RAW files
(2 responses) schen19 2019-06-07

Hi Skyline Team,

I was trying to use the latest version of SkylineRunner to import peptide search results from Proteome Discoverer version 2.3. Everything goes smoothly and a Skyline file was generated. However, the chromatogram information is missing in the Skyline file, so I had to re-import all the RAW file again in order to get the chromatogram displayed. Although things look fine after the reimporting, I'm still hoping to solve the problem because importing a large number of raw files takes a really long time and having to import RAW files manually sort of defeats the point of using an automated pipeline...

Here is the structure of the script:
SkylineRunner.exe --in= --dir= --import-search-file= --import-fasta= --import-threads=4 --import-process-count=4 --import-search-cutoff-score=0 --out=

I didn't have the script for importing the raw file because the location of the raw files are in the folder specified by "--dir". I also didn't see any error message after running the script.

Please let me know if more information is needed in order to understand/solve the problem.


view request
New to Skyline: Searching for specific MS2 ions
(1 response) irahman2 2019-06-10


I am a new user to Skyline, and would like some help to do some MS2 searching/filtering.

To explain the experimental setup: I have a bait peptide that has a reactive group that can covalently crosslink with C-H/C-X bonds on the target molecule. I would like to search through all the MS2 spectra for known daughter ions of my bait peptide so I can identify the precursor mass that corresponds to some adduct. I have a hunch that the peptide is actually binding to some lipid molecule if that is helpful.

Is Skyline an appropriate software to do this kind of searching, or are there other programs I should look into?

Thank you!

view request
Import ms2 ion transition list
(3 responses) davidz 2019-05-28

Hi there,

I know I can import a list of peptides with modifications specified for ms2 ion filtering, and refine which b/y ions to use in peak finding, but is there a way for me import a list of pre generated ions for each peptide with charges?


view request
Reintegration following model training fails
(1 response) matthew.r.russell 2019-06-07
Dear Skyline

I have an issue with a skyline document failing to re-integrate peaks and attribute q-values following successful model training.

The re-integration progress bar completes and then I get the error below. All other aspects of the document seem fine. I have tried closing skyline re-starting the PC and opening again with out solving the problem. A lot of work has gone into getting the analysis at this point and I would rather fix this issue than start again.

Is there any advise you can offer on how to fix the problem?

Failed attempting to reintegrate peaks.
The index 6 must be between 0 and 3
OK More Info
System.Reflection.TargetInvocationException: The index 6 must be between 0 and 3 ---> System.Reflection.TargetInvocationException: The index 6 must be between 0 and 3 ---> System.IndexOutOfRangeException: The index 6 must be between 0 and 3
   at pwiz.Skyline.Model.Results.ChromatogramInfo.GetPeak(Int32 peakIndex) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Model\Results\ChromHeaderInfo.cs:line 2508
   at pwiz.Skyline.Model.TransitionGroupDocNode.UpdateResults(SrmSettings settingsNew, SrmSettingsDiff diff, PeptideDocNode nodePep, TransitionGroupDocNode nodePrevious) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Model\TransitionGroupDocNode.cs:line 1361
   at pwiz.Skyline.Model.TransitionGroupDocNode.ChangeSettings(SrmSettings settingsNew, PeptideDocNode nodePep, ExplicitMods mods, SrmSettingsDiff diff) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Model\TransitionGroupDocNode.cs:line 1018
   at pwiz.Skyline.Model.PeptideDocNode.ChangeSettings(SrmSettings settingsNew, SrmSettingsDiff diff, Boolean recurse) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Model\PeptideDocNode.cs:line 903
   at pwiz.Skyline.Model.SrmDocument.<>c__DisplayClass134_5.<ChangeSettingsInternal>b__7(Int32 i) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1057
   at pwiz.Skyline.Util.ParallelEx.<>c__DisplayClass4_0.<For>b__0(Int32 i) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Util\Util.cs:line 2040
   at pwiz.Skyline.Util.ParallelEx.<>c__DisplayClass4_0.<For>b__2(IntHolder h, Int32 i) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Util\Util.cs:line 2044
   at pwiz.Common.SystemUtil.ProducerConsumerWorker`2.Consume(Object threadIndex) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Shared\Common\SystemUtil\ProducerConsumerWorker.cs:line 184
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Util\Util.cs:line 1854
   at pwiz.Skyline.Util.ParallelEx.LoopWithExceptionHandling(Action loop, Action`1 catchClause) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Util\Util.cs:line 2114
   at pwiz.Skyline.Util.ParallelEx.For(Int32 fromInclusive, Int32 toExclusive, Action`1 body, Action`1 catchClause, Nullable`1 maxThreads) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Util\Util.cs:line 2042
   at pwiz.Skyline.Model.SrmDocument.ChangeSettingsInternal(SrmSettings settingsNew, SrmSettingsChangeMonitor progressMonitor) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1045
   at pwiz.Skyline.Model.SrmDocument.ChangeSettings(SrmSettings settingsNew, SrmSettingsChangeMonitor progressMonitor) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Model\SrmDocument.cs:line 911
   at pwiz.Skyline.Model.MProphetResultsHandler.ChangePeaks(IProgressMonitor progressMonitor) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Model\MProphetResultsHandler.cs:line 180
   at pwiz.Skyline.EditUI.ReintegrateDlg.<>c__DisplayClass12_1.<OkDialog>b__1(IProgressMonitor pm) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\EditUI\ReintegrateDlg.cs:line 133
   at pwiz.Skyline.Util.ProgressWaitBroker.PerformWork(ILongWaitBroker broker) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Util\UtilUI.cs:line 123
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 232
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Util\Util.cs:line 1854
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 180
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 132
   at pwiz.Skyline.EditUI.ReintegrateDlg.OkDialog() in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\EditUI\ReintegrateDlg.cs:line 126
view request
using one peptide calibration curve to quantify another peptide
(4 responses) ana 2019-06-06

Hello Skyline team,

following up on some of the items of this previous discussion:

I am still having problems figuring out how to quantify a peptide for which I don't have standards using the calibration curve generated for another peptide. I'll try to describe my workflow as well as possible (attached step by step snapshots):

  1. I generate a calibration curve normalizing to an internal standard and using the Ratio to Global Standards normalization method
  2. I choose one of my peptides (FGLSLVR) to be used to quantify another peptide and mark is as Surrogate Standard
  3. I change the peptides for which I don't have standards for to be normalized by Ratio to surrogate FGLSLVR instead of Ratio to Global Standards\
  4. The result is a new calibration instead of using the one for peptide FGLSLVR. I tried excluding the calibration points for the samples identified as standards (since I do not have them for these peptides) but it doesn't make a difference.

I know that this is not best practice, but instead of reporting just areas, I would like to report a concentration (even if it's far off from the real value, it can be used for comparing different samples and give an idea of how much it's present). I can do this type of analyses with the software provided by our LCMS and would love to find out if I can also do it using Skyline (since I am quantifying all other peptides for which I have standards with it).

Thanks in advance!

view request
dotp label
(4 responses) stephen swatkoski 2019-06-06


Is there a way to turn off the dotp labeling of chromatographic peaks? For my dataset, it appears that some XICs have a dotp label along with the mass accuracy and retention time labels, but others do not. Please see attached slide.


 dotp label.pptx 
view request
How to edit peak picking in AutoQC?
(1 response) danielacgranato 2019-06-06

Hello Dear,

A System Suitability workflow is being implemented in the lab but we are experience an issue with the data uploaded to Panorama using AutoQC. A few peptides are being inaccurately picked over different runs (screenshot enclosed). We double checked opening the files into Skyline and the correct peak was always present. Is it possible to provide the correct peak boundaries or scheduling the acquisition is the best option? Can we edit peak picking on AutoQC online?

Best regards, Daniela

view request
reverse external calibration curves
(5 responses) chiva cristina 2018-04-26

I was wondering if Skyline supports reverse external calibration curves in which you inject different concentrations of heavy peptide and you use the light peptide (at fixed concentration) to normalize.


view request
Importing pepXML, mzid, or mzTab from OpenMS in Skyline
(5 responses) lparsons 2019-04-16

I am attempting to import Peptide Search results into Skyline ( that were obtained in OpenMS (in this case using the Comet search engine). The default output from OpenMS is idXML, which is not supported in Skyline. I looked at the options in File -> Import -> Peptide Search and found three possible formats for this though each returned a different error:

pepXML : "file is not from one of the recognized sources"
mzid : "No spectra found for the new library"
mzTab : "No acceptable score type found"

I checked to make sure these files are reasonable in size; they are all 10-25 MB with 34,000 - 271,000 lines. If I instead continue to work with the idXML file in OpenMS I see plenty of hits as well. If I use the same spectra (in their original raw format) and the same database but instead run it through MaxQuant, I can import the msms.txt file without a problem.

My goal is to import the Peptide Search results and then load a series of MS1-only spectra on top of the results so that peptide quantification can be done using the MS1 spectral data and mapped to the MSMS data of the search results. This works fine if I use the MaxQuant results here instead, though I'm looking to use the Comet (OpenMS) results at this time.

thank you

view request
Unable to select charge state 2+
(2 responses) melechco 2019-06-04

Dear all,
Although Transition Settings -> Filter are adjusted to accept precursor charges from 2 to 5, I am unable to see in the filter list the 2+ for several peptides (for example, in iRTs GILFVGSGVSGGEEGAR and ELGQSGVDTYLQTK). It is not a matter of turning off auto-select filter. Charge 2+ is just not available.
Is there another parameter to be set?
view request
Tutorial: MS1 Full-Scan Filtering problems
(2 responses) j dabrowski 2019-05-27


I wanted to do MS1 Full-Scan Filtering tutorial posted here: Unfortunatly after doing the very first step (adding two *.group.xml files), Import Peptide Search window just disapears, I'm using Skyline daily (right now:, 64-bit). Would you know what I am suppose to do, to make that tutorial work?

Kind Regards

view request
Error when importing raw file
(9 responses) davidz 2019-05-29

I got this error when importing the raw file from Q-Exactive:
At 1:41 PM:
Failed importing results file 'E:\Users\WeiZ\Downloads\qe\72828_90397.RAW'.
The time interval 1 to 0 is not valid.

What does it mean?


view request
Pepsin cleavage sites
(2 responses) stefan ehling 2019-05-31

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

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

Thank you.

view request
Which MS1 trace is imported in thermo data?
(4 responses) michelle dubuke17846 2019-05-31


I'm importing data that was acquired using lockmass correction on a QE-HFX, and in Xcalibur there are two profile traces - lockmass corrected, and non corrected. Which of these does Skyline import when it generats the XIC for the data?


view request
Error: Library File cannot be opened for transferring
(2 responses) tes1027 2019-05-30

I'm getting the following error when attempting to import a .blib file, exported from ProteinProspector, into Skyline. The directory includes the peaklist and .RAW files, with the same title as the .blib file.
I'm on Windows 10, running Skyline Any help would be appreciated, thank you!


ERROR: Library file 'BSA_0214.blib' cannot be opened for transfering

OK More Info

System.IO.IOException: ERROR: Library file 'BSA_0214.blib' cannot be opened for transfering

at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 59
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 171
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 137

view request
Peak Picking Model for Small Molecules
(1 response) hogan 2019-05-28


I am attempting to use skyline as the data analysis software for small molecule quant assays. I have had good success but have found that even slight shifts in retention/aging columns results in a deterioration of skylines peak picking ability and makes it very difficult to automatically process the data using skyline runner. I see that the peak picking model is used to train the system for peptide peak analysis. Has there been any advances in using this model for small molecules?

Is there a way to tell skyline to pick the most intense, first, last, etc. peak in a desired retention time window to increase peak picking robustness?

As always, I love working with the software and am very excited about all the new features that have come out since I started using it. Thanks!


view request
Not able to get report using SkylineDailyRunner
(6 responses) veerupaksh 2019-05-29

Hi Skyline Team,

I am trying to import mass spectrometry result using SkylineDailyRunner.

A brief description of the procedure:

  1. Have created a template, (attached below)
  2. I open that file and import a transition list (attached below)
  3. Then I import results, there are 143 of those. Each named SHRSP_xxx.d where xxx varies from 001 to 143. (have attached a .zip containing SHRSP_001.d)
  4. Export the report using a custom PythonTemplate.skyr (attached below)

I am able to do this flawlessly using the Skyline Daily GUI, and a correct report is generated as expected. However, when I try to replicate the procedure by creating a .bat file for SkylineDailyRunner, it throws the error mentioned below. I have attached the .bat file as well.

Code for the bat file

C:/MSIC_Master/SkylineDailyRunner.exe --in=C:/MSIC_Master/ --import-process-count=4 --import-transition-list=C:\MSIC_Master\Data\Ruwan_SHRSP_MPfeatures_13_TL.csv --import-all-files=C:\MSIC_Master\Data --report-name=PythonTemplate --report-file=C:\MSIC_Master\Data\Ruwan_SHRSP_MPfeatures_13.tsv --report-format=TSV --out=C:\MSIC_Master\Data\

It throws a similar error for all 143 files.

Error: Failed importing the results file SHRSP_001.d.
Message: System.IO.IOException: Error: Failed importing the results file C:\MSIC_Master\Data\SHRSP_001.d.
Message: pwiz.Skyline.Model.Results.MissingDataException: No scans in C:\MSIC_Master\Data\SHRSP_001.d match the current filter settings. ---> pwiz.Skyline.Model.Results.NoFullScanDataException: No scans in C:\MSIC_Master\Data\SHRSP_001.d match the current filter settings.
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatogramsLocked() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 362
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatograms() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 215
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.SetRequestOrder(IList`1 chromatogramRequestOrder) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 548
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 403
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 263
   --- End of inner exception stack trace ---

   at pwiz.Skyline.Model.Results.ChromatogramCache.RunProcess(ProcessStartInfo psi, String importProgressPipe, IProgressStatus status, ILoadMonitor loader) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromatogramCache.cs:line 683
   at pwiz.Skyline.Model.Results.ChromatogramCache.Run(MsDataFileUri msDataFileUri, String documentFilePath, String cachePath, IProgressStatus status, ILoadMonitor loader) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromatogramCache.cs:line 644
   at pwiz.Skyline.Model.Results.ChromatogramCache.Build(SrmDocument document, String documentFilePath, ChromatogramCache cacheRecalc, String cachePath, MsDataFileUri msDataFileUri, IProgressStatus status, ILoadMonitor loader, Action`2 complete) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromatogramCache.cs:line 607

Would be really thankful if someone could provide a possible solution/ cause of error.

Veerupaksh  Ruwan_SHRSP_MPfeatures_13_TL.csv  PythonTemplate.skyr  CreateReport.bat 
view request
centroided or orbitrap?
(2 responses) qiyingziamms 2019-05-29

We used Thermo Fusion Lumos to get DIA data, and the datatype selected "centroid",detector type selected "orbitrap".
In Skyline software, configure full-scan setting Interface, precursor mass analyzer and product mass analyzer have multiple choices, include centroided and orbitrap,so i don't know which should i choice?

view request
Peptide library building from MaxQuant.
(6 responses) luzjpaulo 2019-05-20

I created a library from MaxQuant using the latest Skyline-daily update but I found out an issue. Skyline-daily gives me very bad peptide spectrum matches while Skyline does not have this problem.

 msms.txt  modifications.xml  mqpar.xml  SugarCane_histones.fasta 
view request
Thermo Altis Collision Energy Opt
(24 responses) avizrosenberg 2019-05-21

Ran several peptides with collision energy optimization on Thermo Altis. When imported to skyline I am only seeing peaks for a single transition per peptide rather than all the transitions that Ive optimized (I can see the peptides and transitions in Xclaiber).

view request
Correct peaks not picked by Skyline
(2 responses) wen ding 2019-05-27

Hi Nick,

The peak at 32.4 min was matched to the library spectrum while the correct peak at 38.4 min was not assigned by Skyline although less intensive, but having a dopt0.93 when manually assigned.
How do I make Skyline pick up the peak at 38.4 min instead of at 32.4 min to be matched to the library spectrum?

view request
Questions on baseline intensity and peak calling
(2 responses) lparsons 2019-05-07

I am using a method similar to these two papers ( that combines MS1-only scanning with directed feature ID.

Briefly, our process starts by running two samples in triplicate MS1-only scans. We now use OpenMS for feature detection and consensus mapping to produce a list of peaks of interest that are selected as a result of group t-tests (3 runs of sample A vs 3 runs sample B). These features are then used to produce an inclusion list for the instrument to then do a directed DDA where features are pre-selected for MSMS. The MSMS data then is run through MaxQuant, and the MaxQuant data is fed into Skyline. In Skyline we then omit the raw MSMS data, instead using the raw MS1-only data in its place so that the peak intensities from the MS1-only data will be used for the MSMS features.

The results of this generally look pretty good. However, there are some features that come out of this with vastly different intensities from Skyline than what they had in OpenMS. In particular, there are some features where we know that sample B should not show any intensity while sample A should. On some of these samples, Skyline reports very low intensity (exported as "Total Area MS1" in Peptide Quantification report) where we expect none and yet on others Skyline reports very high intensities (up to 10^6 or more) when we expect none.

This leads me to a couple questions

  1. Is there a way to find out what Skyline sees for baseline intensity at a given time in a given file? I would expect this could be helpful for trying to figure out if the lower intensity peaks are just baseline AUC.
  2. Does Skyline do any kind of baseline correction for Total Area MS1? I didn't see a parameter for it though I may have missed one along the way.
  3. Is there a parameter I can change that will set the tolerances (RT, M/Z) for peak calling? I am wondering if the peaks might be called too generously here and the intensity is coming from neighboring unrelated peaks.

I also looked at the dot products of these features to see if that could lend some insight. When I plot median dot product vs median B intensity - for the peaks where I expect to see zero intensity from B but am getting positive "Total Area MS1" for B - I don't see a strong correlation though there is a wide distribution of median Total Area MS1 in the range of median dot products > .085 and < 1.

thank you

view request
Add optimized CE's to peptides in the Skyline document
(2 responses) Astrid Guldbrandsen 2019-05-27

Dear Skyline team,

Is there a way to manually add optimized CE values to the individual peptides in the Skyline document? So that these will be in the exported isolation list instead of the default 27. I am doing scheduled PRM on a QE-HF instrument and I have optimized CE's for all the peptides, but currently I need to manually change it for all peptides in the isolation list every time I export a new one.

Best regards,

view request
Message "missed 1"
(1 response) plemignot 2019-05-28

Attached a capture of my screen.
What is the meaning of "missed 1"?

Best regards,

view request
Failed Importing result
(4 responses) nr412 2019-05-28

While importing a raw data file from Waters instrument, I got the following error on Skyline. can someone please help ;

At 14:48:
Failed importing results file 'O:\\LCMS1_002_HPMB_BA10_30_01.raw\LCMS1_002_HPMB_BA10_30_01.raw'.
[pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Generic Error

Inner exceptions:
Exception type: System.Exception
Error message: [pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Generic Error
[pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Generic Error
at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, DetailLevel detailLevel)
at pwiz.ProteowizardWrapper.MsDataFileImpl.get_SpectrumList() in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 507
at pwiz.ProteowizardWrapper.MsDataFileImpl.get_HasSrmSpectra() in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 742
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 194

view request
There is no dotted line for peak selection
(1 response) daisy0025 2019-05-27
Dear Skyline team:     I am doing a PRM absolute quantification program, but a problem always comes up again and again, so I wrote to ask for some help.     When I import and analyze the data of repeated analysis, there is always a case that the target peak is not selected in some individual spectra, and the two dotted lines of the target peak are not selected in the whole spectra. In fact, the target peak effect is very well, but it is not selected. What is the reason? Hope to get your help.     Thanks so much!
view request
Another "Chromatogram information unavailable' problem
(2 responses) SanL 2019-05-24


I already saw lots of posts about this issue but I felt like none of them really solved my problem. I apologize if I missed the one that did.

I am new to Skyline like most posters. Some information:

Object: I want to build a spectral library for OpenSWATH use
DDA files: 29 .group files (ProteinPilot), each with Biognosys-11 iRT peptides
Background proteome: Whole SwissProt+iRT fasta

Afterwards, as "results" I loaded in the wiff files of these 29 DDA runs in again. The import went well, I can see TIC and BPC of each. However, for transitions I only get "chromatogram information unavailable" message.

I feel like this issue has a really easy solution and I am just missing something very basic. I sadly cannot figure it out at the moment though. Do you have any idea?

I will add some settings I used below and uploaded the file as well as a zipped .wiff file in case those are of any help. [EDIT: Files apparently too big to upload]

Thank you so much in advance!


(Some additional relevant information if necessary:

Peptide settings

  • Prediction: Is set, time window at 10 mins

Filter transitions:

  • Precursor charge: 2,3,4
  • Ion charge: 2,3
  • Ion types: y,b
  • Product ion selection from ion 3 to last ion-1
  • ion match tolerance 0.5
  • Pick 6 product ions
  • 3 minimum product ions
  • from filtered product ions
  • Full-scan set to TOF, 30.000 resolution, use only scans within 5 mins of MS/MS IDs )
view request
Integration threshold
(2 responses) lzhangok 2019-05-22

I load 200 MRMs into Skyline and do the process, cannot find threshold parameters, so in the end, some of the metabolites have green dot indicating positive detection, where many others have not green dots indicating no detection at all. But I can confirm with the vendor Quant tool that many "not detected" metabolites can have good peaks and be detected. Hope to see your suggestions.

view request
Colour coding of Peptides and transitions
(2 responses) nr412 2019-05-22

Dear All,

I am new to skyline and want to check if there is any way to export the colour coded peptide and transition information while exporting out the data?

view request
Exporting p, q, or e values in Peptide Quantification Report?
(1 response) lparsons 2019-04-24

I imported MaxQuant results into Skyline, and now I would like to export a Peptide Quantification report. I've found all the fields that I'm looking for except for the peptide score (from MaxQuant) itself. Is there a way to get this?

In this case I have 6 samples, so I would expect there could be 6 p, q, or e values for each of them, which would be fine. Or do I have to do a different report in order to get these?

thank you

view request
Isotopes via Insert Small Molecule Transition List
(9 responses) Fabian 2019-05-16

Dear all,

I have a list of small molecules containing various numbers of Isotopes.
For exporting and re-importing of this list I setup a suited document grid with the correct order for:
Edit - Insert - Transition List - Molecules.
However, trying to reimport the list gives me only the monoisotopic precursor.
The option I have is to tell skyline the product m/z which differs between the Isotopes.
This results in the error attached.

Kind regards


view request
Retention Time Calibration for DIA-data
(1 response) yb77618 2019-05-17

Hi Skyline Team:

I have DIA-MS data and pre-built assay library, so I want to use Skyline-Daily for data analysis.

The assay library was loaded successfully, 11 iRT-peptides were chosen as standard for retention time calibration.

However, I found out the criteria for DIA-MS data loading was so stringent, 80% peptides were needed with a correlation of 0.99 in regression.

Is that possible for us to change the requirements for calibration? Like to calibrate by hands or lower the R-square value?

(I want to remove some iRT-peptides for better correlation in the regression, an alert popped up, see attached image)

Thank you in advance for answering my questions.


view request
Issue with Recognizing User Created Modification
(23 responses) dwwilk02 2016-05-27

We were trying to get Skyline-Daily (on Windows 64-bit) to create a library from a final_fragment.csv file from a Waters Synapt G2-Si MSe experiment (csv file is attached). The file has the modification GEE(Q) (an addition of H6C4O2 to Q residues) that was created in-house for a collaborator. We get the following error when loading csv files with this mod:

System.IO.IOException: ERROR: The modification 'GEE (Q)' on line 489 is not recognized.
ERROR: reading file GEE_Test_Mouapi_Pink01_IA_final_fragment.csv

   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in c:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 109
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status) in c:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 53
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in c:\proj\pwiz_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 171
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 130

I've tried several things to get the mod to work (adding the mod to Skyline, changing a current mod with H6C4O2 addition to include Q, adjusting the addition for one of the default mods at Q), but so far have had no luck. I can load the file if I delete the GEE mods, but then I miss the library matches for modified peptides. Is there anything that we could try to get Skyline to recognize the mod? Thanks for any help you can provide,

view request
Small Molecule Zero Peak
bwidner 2019-05-16

Hello Skyline Team!
I am measuring small molecules, and I am looking for a way to get "zero" in the Transition Results table for peaks that are non-existent. At present, I am not quantifying and don't have a good sense of LOD. If I had a sense of LOD, I would handle this in post-processing. Without quantification, I am trying to look at presence/absence or fold change. Often when there is no real peak, but there is some noise, the noise is selected. I was getting around this problem by manually drawing a baseline somewhere else, hoping it would put zero in the table. This worked, but sometimes I draw a manual baseline on a peak that looks small and without zooming in I can't tell if it is a small peak or just a single scan.

Is there a way to screen out peaks that are only 1 or 2 scans? I saw the "Points Across Peak" parameter, but when I manually draw the baseline there are often quite a few scans, so that will not work. It would have to account for the number of scans above the baseline, somehow. Does such a parameter exist? I saw elsewhere on the forums the suggestion to use the FWHM, but this is also driven by how wide the base of the peak is, which for manual integration varies randomly.

Thanks in advance!

view request
Export method to MassLynx 4.2 version
(3 responses) danielacgranato 2018-11-27


I was wondering if the new version of Skyline (v.4.2) allows to export SRM methods to MassLynx version 4.2. Last year I was unable to do so and I received a response that Waters was working on making it possible. As a solution to the problem I have been exporting the method to a computer not connected to the equipment (Xevo TQ-XS) and to an older version of MassLynx (v.4.1). Do you know if it is possible now to export directly to MassLynx 4.2?

Thank you very much. Best, Daniela

view request
creating peptide library from uniprot database human
(4 responses) sponce1 2019-05-13

i downloaded all of the file formats (fasta, XML, ts, etc.) available from uniprot human reviewed:yes

and couldn't get them to load in the Peptide settings -> Building Library -> Input Files -> error
"fasta.gz is not a valid library input file.

I've attached my skyline file, a dataset and the fasta file. please make it work!

view request
MS1 Quantification of Precursor Ions
brown459 2019-05-15


The problem: We have generated a database of glycopeptide identifications using a DDA analysis. Each sample/acquisition (i.e. run 1 through run 26) is a high pH reverse phase chromatography fraction. Therefore, not every acquisition contains each of the glycopeptides identified in the database. In fact each glycopeptide should only be observed in a few runs. We want to quantify the abundance of individual glycopeptides in the identified fraction (run) and the surrounding runs.

Is there a way to use Skyline to very simply, export XIC's for a series (we define) of amino acids modified with variable glycoforms (~300). We have the m/z, charge, RT, fraction, elemental composition. We also have a .blib that is composed of EThcD spectra.

I have approached this problem from both the proteomics and the molecule interfaces and have encountered similar erroneous peak matchings. Because we do not identify every species in each fraction, we are finding that in fractions where a glycopeptide is not observed, skyline ends up quantifying an incorrect precursor MS1 that is near it in RT.

Molecules doesn't seem to restrict IDs based on the explicit retention time window. Proteomics may be the only way to go, using MS1 quant and really reducing the RT window? This however, seems to take a long time as there are many possible combinations of modifications.

Are there any other work arounds that I am not thinking of?



view request
combined MS1 PRM no product ion chromatograms
(8 responses) mohammad abukhalaf 2019-05-10

Hi guys

We are running an alternating MS1 PRM scan strategy on a QEPlus. We built a library from previous DDA and PRM measurements of our targets and also imported the FASTA of our target proteins. That all looks fine. However there is no import of product ion chromatograms. We tried adjusting many of the transition settings, i.e. Transition settings->Full scan->MSMS Filtering->Acquisition Method->Target or none, various settings on Transition setting->Filter->Product ions->From To and so forth but no use. We also widened all of the m/z windows. We are using the newest version 4.2.0. Everything to no avail. Any ideas?

Thanks a lot
Mohammad and Wolfgang

view request
RT prediction not shown for all peptides/samples
(2 responses) ssaleh 2019-05-13

Hi Skyline team,

I recently run around 20 samples in quadruplicate on TSQ vantage (to monitor 12 peptides using MRM mode) and I am currently analyzing the data. When trying to upload the results into skyline I had the following error "unable to finish importing chromatograms because RT predictor linear regression failed". To solve this problem I had to switch off the RT predictor, this way the import was successful.
I then switched on the RT predictor , however the RT prediction was missing in a lot of peptides and samples in a random way (please check attached ppt ). Any idea what could be the reason?
Is there a way to share the in a private mode?

Many thanks in advance!

 RT prediction.pptx 
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Try to set different collision energy for different daughter transitions of a compound
(1 response) yufeng li18 2019-05-13

Hello there,

We were trying to export a method to MassLynx, which each compound has 3 to 4 daughter transitions with different collision energy. When we inserted a transition list in the Skyline, the format would look like in the picture 1 and the exported method in the MassLynx had the same collision energy (always be the collision energy for the first daughter transition in the list) for every daughter transition. We noticed the collision energy can only be modified in the precursor tab (662.1018 in the picture). In order to have correct collision energy in the MassLynx, we had to manually edited the in Skyline (shown in picture 2 ) and changed the collision energy for each precursor accordingly.

Do you have an ultimate way that can export a method from the Skyline with each daughter transitions having the correct collision in the MassLynx? I really appreciate if you could help

Many thanks

 1.jpg  2.jpg 
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Webinars Visualization
(2 responses) GioMag 2019-05-09

Dear all,

I wish to watch some webinars (#17 in particular), unfortunately videos automatically stop the playback after few minutes.
This happens with all browsers I have tested (Chrome, Edge, Safari..) and on different computers.
How can I solve this issue?


 Issue Webinar #17.bmp 
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Library build error (dat file)
(15 responses) Jason Held 2019-02-14

I'm trying to build a library from a large mascot .dat file (2.2 GB) and getting: ERROR unable to read filesize of (my filename).dat.

More info pasted below. Error screenshot attached.


System.IO.IOException: ERROR: Unable to read filesize of F011061_Cys_NewRat_PlusRED_0p0232.dat.

Command-line: C:\Users\lab_held.pcl-held1\AppData\Local\Apps\2.0\NRM38HKN.2B5\OM37C28G.N7K\skyl..tion_e4141a2a22107248_0004.0002_ed14b445d33623de\BlibBuild -s -A -H -o -c 0.9768 -i GloCys055_Cys_PlusRED_Mascot_0p023 "C:\Skyline\SpectralLibraries\GloCys055_Cys_PlusRED_Mascot_0p023.redundant.blib"
Working directory: S:\Users\Jason\GloCys055_131231\Cys
Standard input:

at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 60
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in C:\proj\pwiz_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 178
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 144

 Screen Shot 2019-02-14 at 9.55.01 AM.png 
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Dimer peptide
(2 responses) chanchalmrt 2019-05-09

I want to make a dimer peptide of two monomers each with 9 aa (eg. LVCIALRKK-NH2) linked by a single disulphide bond between the third cys of each monomer. Is that possible with skylink?

view request
small molecules: normalized retention time (iRT)
(4 responses) alessio maiolica15118 2018-07-02

Dear Skyline support team,
I am interested in using the iRT functionality for small molecule experiments.
It seems that this is not yet implemented in the current version of the software.

Are you planning some work on this? if yes would you please let me know when would you expect to have it released?
thank you all for your great work.
best regads,

view request
Include fragment ion annotation when "Copy Data" from Library Match tab
jonasbecker 2019-05-08

Dear Skyline Team,

I'm trying to compare spectra from peptides recorded with different fragmentation methods as well as measured from synthetic pools and out of samples.

Is there any possibility to add fragment ion information displayed in the graph to the data which I get by using the "Copy Data" interaction within the Library Match tab? Unfortunately I only get m/z values and intensities, which I than would have to annotate manually.

Or is there any possibility to get this information from a Custom Report for a single spectrum? This one could combine subsequently with the data copied from the Library Match tab to also include unmatched peaks.

I'm using PEAKS Studio X for my search, export data from there and import into Skyline 4.2.0 (64-bit).

Thanks for your help in advance. Best,

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Viewing Products from DDA
bwidner 2019-05-07

Hello Skyline team!

I am trying to develop a quantitative method using DDA data, and I would like to use the product ions to confirm the identity of the parent. I will quantify from the parent. I find the way skyline draws the product "chromatogram" by connecting scans that are really far apart to be sort of confusing, since I can't tell the retention time of the MS/MS scan without clicking on it. Is there a way to view the MS/MS scans as individual sticks or dots or something, instead of as a chromatogram with the scans connected?


view request
Targeted MS/MS importing - cutting off ID'd peaks
(4 responses) kvancott2 2019-02-12

I successfully built a spectral library from DDA data and then imported a list of proteins for an MRM-HR method using a Sciex 6600 QTOF.

I imported another DDA data file into my Skyline file. In the Transition Settings, I used "include all matching scans" option for Retention Time FIltering.

After importing the data file, the scan extraction width seems to be all over the place. Some peptides it is only a minute wide, some are 10 minutes wide, some are 20 minutes, and some even higher.

What is most frustrating, though, is that quite often the matching/ID'd peaks are cut-off in Skyline. I'm attaching a powerpoint file that shows an example of Skyline cutting off the scan of a yeast ADH peptide (YYVDTSK) that was spiked into the samples.

I've tried re-setting the Retention Time filtering, re-saving the file, re-importing the raw data file, but it doesn't change. I've tried adjusting the Retention Time filtering to 5, 10, 15, or 20 minutes - it doesn't seem to make any difference what the setting is.

I even tried importing one of the raw data files that was used to make the spectral library - same thing. Multiple peptides that are ID'd are cut off by Skyline. In your Targeted MSMS tutorial, it says on page 31 that the entire gradient should be covered in the chromatograms. But I'm not seeing that. Not sure what I'm missing.

 KVC-Feb-12-2019 - MS1 filtering problem.pptx 
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Failure opening Skyline Session
(3 responses) aline doerrbaum 2019-05-06

Dear Skyline Team,
I cannot load my Skyline session anymore.
The following error occurs: "Failure opening "C:[...]. Exception has been thrown by the target of an invocation." (also see screenshot attached).
We put a lot of work into checking and integrating the peaks already, so it would be great if there was a chance to rescue it.
An older version of the session, that I saved under a different name before curating the data, can be loaded without problems.
I'm using Skyline and Windows10.
Do you have an idea how to fix it?
Many thanks in advance!

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No precursor ion chromatograms found
(15 responses) bwidner 2019-04-30


I am new to Skyline and trying to do something very simple, but I cannot get it to work. When I import the transition list with just the precursor information, everything works. I see the peaks as expected. However, when I add the product information, I get the "No Precursor ion chromatogram found" message. I double checked the product m/z and looked through the tutorials, and still I am quite befuddled!

The data is from a Thermo Fisher Fusion Lumos orbitrap. I have attached the skyline files and the raw file I am working with.


 test4.skyd  test4.skyl 
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Import of report template
(1 response) sstoychev 2019-05-06


Would like to import the attached report template, how is this done?

Also, is it possible to export a Skyline library in .csv format?



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Spectral library essential in Skyline for the generation of optimum MRM method
(3 responses) shobhabr20 2019-05-03


Is spectral library essential in Skyline (Peptide Setting-> Library) for the generation of optimum MRM method for a peptide.

Please clarify.


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Calculate the confidence score and q-value for searched peptides
(7 responses) sunbergsoon 2019-04-30


I am doing SWATH analysis using Skyline. We filtered the searched peptides based on dot-product which above 0.8.
Recently we also tried the mProphet model and using the second best peak to calculate the q-value to do more confident
filtering. However, the results are not so good and the Q-q plot shows a bad shape. So how can I use Skyline to do more
confident filtering on SWATH analysis? Can I try other method like using openSWATH model?


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Getting The operation has timed out.
(3 responses) kartik kundgol 2019-05-05

Hi Brendan,

I am using SkylineTool.dll in my application. Unfortunately not able to establish the connection between Skyline and my application.

Getting "The operation has timed out exception" in the SkylineToolClient constructor.
toolClient = new SkylineToolClient("$(SkylineConnection)","Test Interface");

Can you please guide the solution.

Thanks & regards,
Kartik K

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Modified Sequence Full Names - apparent bug
(1 response) Nathan Manes 2019-04-29

There is an apparent bug in the current version of Skyline 64bit (v4.2.0.19072). When exporting a report that includes precursor "Modified Sequence Full Names", sometimes a mod will be missing. Out of 321 peptides that I imported (Edit --> Insert --> Peptides) into a new document, two had this error (in both, cys carbam was missing in the heavy form; strangely it is present in the light form). Inside the attached ZIP (Modified Sequence Full Names - apparent is the Skyline file with the 2 peptides and the resulting exported report (transition list) with the incorrect "Modified Sequence Full Names" values (heavy rows are incorrect; light rows are correct). I didn't check if anything else is incorrect.

 Modified Sequence Full Names - apparent 
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Small molecule method export
(8 responses) mcarey17 2018-12-03

Hi all,

Relatively new to Skyline but could use a great deal the method export feature. That's the one where based on a transition list/retention time and an existing *.exp file (which is the MAsslynx MS method details file) Skyline creates an MS method file ( *.exp file), which would be otherwise really clumsy with MAsslynx when more than 100 compounds are involved.

Re-create the error:
Open Skyline; Import transition list; Change setting to XEVO-TQS; Export MEthod

Operating system: Windows 7; Skyline version: latest; MAsslynx version: 4.2

Error message, see it on 'error support 3.PNG'
System.IO.IOException: Error no known instrument type found.
ERROR: Error no known instrument type found.
---> System.IO.IOException: Error no known instrument type found.
ERROR: Error no known instrument type found.

System.IO.IOException: Error no known instrument type found.
ERROR: Error no known instrument type found.
---> System.IO.IOException: Error no known instrument type found.
ERROR: Error no known instrument type found.

at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in c:\proj\skyline_4_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 59
at pwiz.Skyline.Util.Extensions.UtilProcess.RunProcess(ProcessStartInfo psi, String stdin, String messagePrefix, IProgressMonitor progress, IProgressStatus& status) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Util\Extensions\UtilProcess.cs:line 45
at pwiz.Skyline.Model.MethodExporter.ExportMethod(String exeName, List1 argv, String fileName, String templateName, Dictionary2 dictTranLists, IProgressMonitor progressMonitor) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 3772
at pwiz.Skyline.Model.WatersMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Export.cs:line 3571
at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action1 performWork) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 228 --- End of inner exception stack trace --- at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1835 at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 176
at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 131 at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action1 performExport) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 1885

 error support 1.PNG  error support 2.PNG  error support 3.PNG 
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Failure imorting mzXML
(3 responses) cfarnsworth 2019-05-02

I get the following message when trying to import mzXML files. Note only 6 of 24 files in total failed.
Thanks, Charles

Failed importing results file 'D:\IMAC Keezer\180418_28574_CF_x.mzXML'.
Object reference not set to an instance of an object.

Inner exceptions:
Exception type: System.NullReferenceException
Error message: Object reference not set to an instance of an object.
Object reference not set to an instance of an object.
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.get_PercentComplete() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 806
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.UpdatePercentComplete() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 599
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.GetChromatogram(Int32 id, Target modifiedSequence, Color peptideColor, ChromExtra& extra, TimeIntensities& timeIntensities) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 587
at pwiz.Skyline.Model.Results.ChromData.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\ChromData.cs:line 77
at pwiz.Skyline.Model.Results.ChromDataSet.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 233
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Load(ChromDataProvider provider) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 144
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 452
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 263

view request
Failure to import MS1 filtered data
(1 response) sstoychev 2019-05-02


I'm trying to import precursor ion data from DDA runs but Skyline freezes during the import. Tried importing .wiff or .mzml as well as one-at-a-time vs individually but issue persists. Attached is the Skyline document as well as LCMS data so can see if you can reproduce the issue on your side.



view request
Shimadzu Triple Q data in Skyline
(5 responses) t j f m voermans 2019-04-29


In the article: "The Skyline ecosystem: Informatics for quantitative mass spectrometry proteomics" it is stated that skyline supports SRM files from Shimadzu (.gpd). However, the only files I have are .lcd files (see attachment). This file is of a scan+ measurement done on a Shimadzu LCMS-8045.

Any way to import these in Skyline as well? (When I use the normal way of importing data via File - Import - Results, I get a message that there is no usable data found.

Thank you.


 Test file.lcd 
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Unable to generate calibration curves
(3 responses) o mannion5 2019-05-01

I followed the tutorial for generating a calibration curve but after following all the steps I get a blank calibration curve with the message "Error: All of the external standards are missing one or more peaks. The selected replicate has missing or truncated transitions." I don't understand this error message as all of my standards have clear well-defined peaks. Has anyone else encountered this problem ? Am I missing something?

view request
Collision Energy on QExactive
(3 responses) lisa.j.zimmerman 2013-10-23
Hi Brendan
Has something changed in the way Skyline calculates the collision energy for an inclusion list for the Q Exactive? Previously the values were different depending upon the peptide but now the value is 27 for all peptides?
Also, how is the collision energy calculated for the QEXactive since there is no drop-down option for this….it simply defaults to the Thermo TSQ Vantage?
view request
Issues with downloading Skyline
(1 response) ddoza 2019-04-30


I am currently having trouble downloading Skyline 4.2 64 bit on my PC that is running Windows 7 Professional 64 bit. I get an error message "An error occurred trying to download ''." whenever I attempt to download skyline. Restarting the PC or attempting to download Skyline under a different user does not help. I have attached the setup log file. Can you please help?

Many thanks,

view request
System.IO.IOException: ERROR: Illegal character p found in sequence .....
(5 responses) jason a macgurn 2019-04-15


I am using skyline with MS1 full scan filtering on data acquired from a Q Exactive and searched on MaxQuant. I am using Skyline to inspect peptides (especially phosphopeptides) and validate quantification from SILAC labeling.

I had been using a very nice workflow (based on the MS1 full scan filtering tutorial) that was perfect for our application. But recently, I had some computer problems which forced me to install the latest versions of both MaxQuant and Skyline, and now whenever I try to set up the filtering on my data I receive the following error:

"ERROR: Illegal character p found in sequence AAGpSGESpTPER (line 13)"
(detailed error info in the attached text file)

I only get this error when I attempt to filter data generated with my new version of MaxQuant - if I filter analysis from the old version, there are no errors.

My interpretation is that Skyline doesn't like something about the way MaxQuant is formatting the msms.txt output file (phosphopeptides specifically?), so it chokes on it. I figure I either need to re-format the msms.txt output file or adjust some setting for the input on Skyline. Has anyone else encountered this problem? Is there a fix or workaround?
Thanks in advance for any help / suggestions - I am eager to get back to using Skyline for analysis of some of my data!

view request
save pivot in Report template .skyr
(2 responses) sarah lennon 2019-04-25

Hi Skyline team,

I have got an additional question. I would like to share a report template as a .skyr with collaborators. Is there a way to keep the pivot applied to the report in the skyr?
I was able to save it on my own Skyline installation using : 'Remember current layout' but this information seems to be lost each time I export the .skyr and import it back.

Thank you very much for your help,



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Any plans for a Linux version of Skyline (even with limited capabilities)?
(4 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!
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some peptides show incomplete peaks
(6 responses) lvyayao90 2019-04-23

Hi Skyline team
When I imported my results into the Skyline, some peptides show incomplete peaks, I pasted one in the word file below. Can you please explain it for me and is there anything I can do to avoid this?
Thank you very much!


 an example.docx 
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Retention time difference in library when using PDresult vs. msf file
(3 responses) roberthardt 2019-04-23

Dear Skyline-Team,

I today created a spectral library from ProteomeDiscoverer 2.3 results (Mascot search engine) once using the .PDresult and once using the .msf file(s). I got quite similar results but interestingly, there is a clear retention time shift/offset when using the PDresult file. This becomes immediately apparent when looking at MS1-chromatograms, where the blue vertical peptide-ID lines are clearly "missing" the correct MS1-peak in the case of the PDresults-library.



 ID-times_from_PDresult.PNG  ID-times_from_msf.PNG  example_spectra_from_pdresult.PNG  example_spectra_from_msf.PNG 
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Error importing Bruker xml
(4 responses) fcsigloch 2019-04-23


I tried to import a Bruker XML file of a BSA test run and got the following error message. Conversion of the same file with msconvert BSAAnalysisResults.xml gave the same error.

At 10:53:
Failed importing results file 'BSAAnalysisResults.xml'.
[SpectrumList_BTDX::HandlerCompound] Unexpected element name: fullscan

Inner exceptions:
Exception type: System.Exception
Error message: [SpectrumList_BTDX::HandlerCompound] Unexpected element name: fullscan
[SpectrumList_BTDX::HandlerCompound] Unexpected element name: fullscan
   bei pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, Boolean getBinaryData)
   bei pwiz.ProteowizardWrapper.MsDataFileImpl.HasSrmSpectraInList(SpectrumList spectrumList) in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:Zeile 778.
   bei pwiz.ProteowizardWrapper.MsDataFileImpl.get_SpectrumList() in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:Zeile 475.
   bei pwiz.ProteowizardWrapper.MsDataFileImpl.get_HasSrmSpectra() in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:Zeile 742.
   bei pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:Zeile 194.

Bruker also exports mgf file format. This file could be successfully converted by msconvert BSAAnalysisResult.mgf and also searched with
java -Xmx12000M -jar r:\MSGFPlus\MSGFPlus.jar -s BSAAnalysisResult.mgf -d BSA.fasta -t 0.4Da -inst 0 -addFeatures 1 -tda 1 -e 1 -mod Mods_unlabelled.txt.
I can read in the resulting mzid file as "Peptide Search" into Skyline. When I try to read the mzML as "Results", I get another error:

At 11:04:
No scans in BSAAnalysisResults.mzML match the current filter settings.

Inner exceptions:
Exception type: pwiz.Skyline.Model.Results.NoFullScanDataException
Error message: No scans in R:\BRAIN\Testdateien_AnkeBachert\ProteinAnalysisResults_17.53-39.99.mzML match the current filter settings.
No scans in R:\BRAIN\Testdateien_AnkeBachert\ProteinAnalysisResults_17.53-39.99.mzML match the current filter settings.
   bei pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatogramsLocked() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:Zeile 363.
   bei pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatograms() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:Zeile 215.
   bei pwiz.Skyline.Model.Results.SpectraChromDataProvider.<SetRequestOrder>b__45_0() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:Zeile 556.

 BSAAnalysisResults.mzML  BSAAnalysisResults.mgf  BSAAnalysisResults.xml 
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SkylineDailyRunner.exe error via Symphony
(2 responses) sarah lennon 2019-04-24

Hi Skyline team,

I am trying to use the SkylineDailyRunner.exe via Symphony (a Waters server application allowing the automation of tasks). This used to work with SkylineDailyRunner.exe downloaded the 7th of Sept 2017. It does not work anymore with today's version. I got this error back :

"Unhandled Exception: System.IO.IOException: The handle is invalid.
at System.IO.__Error.WinIOError(Int32 errorCode, String maybeFullPath)
at System.Console.GetBufferInfo(Boolean throwOnNoConsole, Boolean& succeeded)
at System.Console.get_BufferWidth()
at`1 args)
at SkylineRunner.Program.Main(String[] args)"

Do you know what it means ?
Worth mentioning as well that the exact same commands from a terminal works fine.

Thank you very much for your help !



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SD Rat Plasma
(3 responses) Richard Lam 2019-04-23


I would like to predict the peptides of SD rat plasma after trypsin digestion. Can Skyline help? If yes, may I know how to do it? If not what do you recommend? Thanks!



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MS1 filtering - library peptide
(5 responses) mmr6q 2016-02-17
Sorry for the continuing questions.
I generated a spectral library using a Mascot .dat file, which has a high-score match to an 8-residue peptide that contains residue numbers 2-9 in from the protein n-terminus (methionine). When I import the corresponding .raw file, that yielded the .dat file, that peptide is not displayed below the parent protein, despite the peptide settings of 6 AA minimum peptide length and exclude 1 n-terminal AA. This peptide is found in the spectral library explorer, but when I add it from the explorer it appears as a "library" peptide and is not associated with the parent protein (and no RIC is plotted). In addition, this peptide is not included in the long list of possible tryptic peptides shown upon right clicking on the protein in the Targets list. Thank you for any advice you can provide to have that peptide included with the other peptides of that same protein.
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Do we need to add the iRT standards to each fractions when building the library?
(4 responses) zzhang9 2019-01-21

Hi there,

I am new to DIA and I have a question about the iRT values. When we building the library, we usually fractionate the sample and run each fraction individually in DDA mode. However, when we do database search using PD or MaxQuant, we only get one file. How could they use the iRT peptides' retention times in each fraction to calculate the iRT values? Thank you!


view request
peptide count from document grid plus pivot not counted correctly
(2 responses) sas28 2019-04-18

Hi skyline team,
I am exporting total sum area for each protein using your document grid with the pivot function. As an addition I want to know how many peptides per protein were used for the summed area. I have noticed that there are several options for total area in the drop down menu and I went for Proteins -> Peptides -> Precursors -> Precursor results -> total area which gave the same result as protein -> peptides -> peptide results -> quantification -> normalized area. However when adding peptides as count in the pivot function, it doesn't accurately reflect the peptides that are actually in the Skyline document. For example for iRT peptides which i have got 10 of, it reports 18 in the final exported summed area document. Other examples are only 1 off and sometimes the peptide count is correct. What am i doing wrong? Many thanks.

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Retention Time of the Unknown
(1 response) qaporter 2019-04-18

Hi, I have a question about the tutorial.
What does the tutorial Skyline "iRT Retention Time Prediction" mean when it come to predicted retention time, if the time is already unknown? But, if the retention time is not known, then what material is needed to predicted retention time, if the retention time is unknown? Because I don't understand how it predicted retention time, if the retention time was to be known, but if it unknown, then what material do I need and this is for small molecule.

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Retention Time of the Unknown
(9 responses) qaporter 2019-03-15

Hi, everyone. Does skyline allow you to enter chromatography and can skyline predict unknown retention time based on line with no standard for small molecules, because I read the tutorial on IRT Retention Time but, that was based on peptide, so I want to know if you can do that with small molecules?

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(3 responses) jmr 2019-04-18

I am trying to set up skyline for small molecules and starting by going through the tutorial. On tutorial it shows skyline adapted to small molecule user interface, but on my version of skyline it does not have the button to select small molecules. Do I have the correct version of Skyline daily loaded? see attached.

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Vertical blue ID lines without any library match
(4 responses) roberthardt 2019-04-17

Dear Skyline-team,

for some transitions in my SRM-experiment I see dark-blue vertical peptide ID lines while there is no library spectrum in my spectral library. Also the marking "ID" is missing from those lines. I selected to show only matching peptide ID times.

The library was created from MIDAS runs on a QTrap6500+ searched against customized database with Mascot.

Any ideas?



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