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

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

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

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

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

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

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Showing: limited to 100 requests
Retention times do not fit with scan times in ID picker
(1 response) staab-weijnitz 2023-01-27

Hi all,

I want to use Skyline for MS1 filtering and quantification of collagen PTMs from crude samples, conceptually similar to what we did here:

Right now, I am trying to analyze crude cell ECM from cells where I knocked down a gene (n=2, four samples in total). This is basically a test run, to see whether we get enough coverage of collagens and other proteins of interest.

We use MyriMatch (2.2.19172) for peptide search of thermo .raw files and obtain results in .pepXML format and check them in ID picker (3.1). After the "fine PTM search" where we restrict the search on proteins present in the sample and include decoys in the fasta file, I loaded a merged session of my four files in IDpicker and exported the spectral library (.sptxt file) from the merged idpDB file.

I imported this .sptxt file into Skyline ("Use existing library") and then loaded the four thermo .raw files after conversion to .mzMXL via MSConvert (3.0). This seemed to work.

However, now, when I crosscheck the IDs and the retention times I get in Skyline, they are most of the time completely off compared to the scan times for the same peptides in ID picker.

Is there anything obvious I have been doing wrong? I would appreciate your help on this. Happy to upload files or give more information, of course, too, if needed.

Many thanks,

view request
Not seen glycopeptides working with data from MSFRagger (DDA MS1 filtering)
(12 responses) cpavan 2023-01-18

Dear Skyline staff,

I’m trying to analyze a dataset by DDA MS1 filter to compare XIC of glycopeptides. The peptide search comes from MSFragger – glycol search –. I can create a library with different peptides from the proteins ID but I can see the glyco peptides identified by MSfragger, which is actually my interest in the analysis.
I have tried to add the glycosylation by peptide setting but Still, I only see unmodified peptides ( only Carbamidomethylation and met ox -.
Any suggestion to solve that?

thank you so much in advance,


view request
Split retention time window
(1 response) dennisjakob 2023-01-27

I was wondering if it's possible to implement a slit retention time window for the explicit retention time.
In some cases we have shoulders or a bit of tailing. Of course this is not ideal but just the case sometimes.
adjusting the retention time window with different values would help in these cases.
Best, Dennis

view request
Finding unique peptides
(10 responses) prajita 2023-01-23

I have 15-20 peptides that I would like to confirm specificity against Human proteome. I took following steps:
Settings->Peptide setting->Digestion->background proteome
and have added human Fasta sequence.
Then I took following steps:
Edit-> Unique peptides
Under this option, I could see some peptides that have blue checks meaning they are not unique. However, if I keep scrolling I see a limited number of proteins in human. I am not sure if this option is covering all the human proteins. Can you please provide an insight to this?

view request
Skyline can't load the interact-*.pep.xml and *_uncalibrated.mgf files from FragPipe 19.1 coupled with MSFragger 3.7
(9 responses) fcyu 2023-01-18

It works for the interact-*.pep.xml and *_uncalibrated.mgf files from FragPipe 18.0 coupled with MSFragger 3.5. But not the files from the latest MSFragger. Could you please help to test, and tell me if Skyline or MSFragger need to be adjusted? All files have been uploaded to the attachments.



view request
the problem of merging skyline documents with one raw data in different spectral libraries
(3 responses) zhao 2023-01-19

Suppose I have a raw data, named N1, which generates 300 sky files in 300 spectral libraries. How should I merge these 300 sky files? I try import-document and set it to merge with existing results by duplicate name, but the new results after import have no peak. I want to ask how to merge the sky files of different spectral libraries.
best wishes.

view request
Skyline not opening
(1 response) henry 2023-01-19


We have user whose skyline app continues to crash. When She attempts to open it, she gets the attached error message telling her to check the log in Event View, also attached. I've already uninstalled and reinstalled the program and uninstalled windows updates.

 Skyline error.rar 
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Defining Isolation Schemes for Bruker timsTOF DIA data
(3 responses) Premy 2023-01-13

Thanks again for all the support with Skyline!

I am developing a DIA-IM Lipidomics pipeline on Skyline and was wondering whether an isolation scheme would need to be defined for Bruker timsTOF data or if the isolation scheme can be read from my raw files using "Results Only"?



view request
Fitting cross-linked peptides to an iRT scale
(1 response) Juan C. Rojas E. 2023-01-16

Hi team,

I am trying to fit some cross-linked peptides to an iRT scale (i.e. calculator) generated from endogenous peptides. Unfortunately the column had to be changed, resulting in different retention times. However, I am trying to compensate for this using the background proteome to define some iRTs.

Skyline allows me to add the peptides within the "Edit iRT Calculator" by clicking "Add Results..." but when I try to click OK I am getting a error message (attached).

You will know best, but to me it seems is that here Skyline is not recognizing cross-link modifications?

Any suggestions for a workaround or is it something that has to be patched?

As always, thanks for your time and help.
Juan C.

view request
Raw CCS values and Overriding with Explicit IM Values
(2 responses) Premy 2023-01-05

Thanks so much for all the developments in Skyline. This tool has been an amazing support for my DDA and diaPASEF data!

I'm currently working with DDA-PASEF data (Bruker timsTOF) to build an ion mobility library for a set of lipid standards. I had a few questions regarding the 1/K0 and CCS values presented in the report.

  1. Is there a way to get the raw CCS values (in case there are slight shifts in CCS between replicates)?
  2. In the case that the IM extraction window needs to be corrected by overriding with "Explicit Ion Mobility Value", is there a way to also recalculate the CCS value in the report? It currently still reports the original CCS value and the newly corrected 1/K0 value.

I hope this makes sense and thank you in advance!


view request
Windows 10 22H2 and Windows 11 22H2 Compatibility
(1 response) taperez 2023-01-11


Im trying to find out if the newest skyline upgrade is compatible with windows 10 22H2 and/or windows 11 22H2?



view request
Import FASTA file
(7 responses) prajita 2022-12-19


Can you please let me know to import FASTA file is this the only way:
Or is there any other way?

view request
Non-Canonical Amino Acid
(12 responses) lulmer 2022-10-24

I have a unique use case working with the non-canonical amino acid, benzoylphenelaylanine, and I'm having trouble importing my results into skyline. I'm trying to import comet/peptideprophet search results that have the benzoylphenelaylanine non-canonical amino acid defined (attached interact.pep.xml). When I import these results there is a suggested modification that makes the peptide with the non-canonical amino acid viewable in skyline (attached jpegs 1 and 2 described the modification). However, when I try to reopen the skyline document with that modification I get the error "Variable modifications must specify amino acid or terminus" (attached error.jpeg). I've attached the skyline document that gives this error as well. Any suggestions on how to make these comet/peptideprophet search results with the non-canonical amino acid compatible with skyline would be greatly appreciated.

Lindsey Ulmer
 suggestedmod_01.JPG  suggestedmod_settings_02.JPG  210727_hspb5_b9_nouv_t_mon_interact.pep.xml  error.JPG 
view request
Skyline compatibility with GC-QTOF
(4 responses) higgi022 2023-01-05

I am curious if Skyline could be used to generate data collected on a GC-QTOF. My instrument (Agilent 7200 GC-QTOF) is not capable of generating data using MRM\PRM acquisition modes - all data represents full product ion scans using EI ionization.

Would I be able to use Skyline to extract select ions from the full scan and compare their ion intensities relative to a spectral library entry to detect compounds and obtain quantitation?

view request
Skyline Batch error if decimal symbol is a comma
(6 responses) roman sakson 2023-01-06

Hello Skyline team,

I would like to report an error issue that might not have been mentioned here yet.

I am working with Skyline Batch, where I shared a bcfg file and tried to import it back into Skyline Batch on a different computer. Both systems were using Skyline Batch While trying to import the bcfg file I got an error complaining that the version of my file is newer than the version of the program, which was, of course, wrong (see screenshot).

Since the version number look weird with a comma in the error message, I checked the windows 10 region settings and realized that the decimal symbol on my German system was set as comma and the digit grouping symbol was set as dot. Reversing this in thw windows settings resolved the issue. I am not sure, whether something can or should be done here but I thought I better report this.

Thank you for this great software,

view request
Selecting Prosit in Library Match window results in unexpected error
(2 responses) tmaile 2023-01-04

Hello Skyline team,

I accidentally clicked on Prosit in the Library Match window which lead to the window blanking out and becoming completely unresponsive, showing the message "Prosit is not properly configured".
Right clicking on the blank window resulted in an unexpected error report (submitted):

Object reference not set to an instance of an object.

Source code location:
System.NullReferenceException: Object reference not set to an instance of an object.
at pwiz.Skyline.Controls.Graphs.GraphSpectrum.get_SpectrumInfo() in C:\proj\pwiz\pwiz_tools\Skyline\Controls\Graphs\GraphSpectrum.cs:line 1466
at pwiz.Skyline.SkylineWindow.pwiz.Skyline.Controls.Graphs.GraphSpectrum.IStateProvider.BuildSpectrumMenu(Boolean isProteomic, ZedGraphControl zedGraphControl, ContextMenuStrip menuStrip) in C:\proj\pwiz\pwiz_tools\Skyline\SkylineGraphs.cs:line 1159
at ZedGraph.ZedGraphControl.contextMenuStrip1_Opening(Object sender, CancelEventArgs e)
at System.Windows.Forms.ToolStripDropDown.OnOpening(CancelEventArgs e)
at System.Windows.Forms.ToolStripDropDown.SetVisibleCore(Boolean visible)
at System.Windows.Forms.ToolStripDropDown.Show(Control control, Point position)
at System.Windows.Forms.ContextMenuStrip.ShowInternal(Control source, Point location, Boolean isKeyboardActivated)
at System.Windows.Forms.Control.WmContextMenu(Message& m, Control sourceControl)
at System.Windows.Forms.Control.WndProc(Message& m)
at System.Windows.Forms.UserControl.WndProc(Message& m)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
Exception caught at:
at System.Windows.Forms.Application.ThreadContext.OnThreadException(Exception t)
at System.Windows.Forms.Control.WndProcException(Exception e)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
at System.Windows.Forms.UnsafeNativeMethods.CallWindowProc(IntPtr wndProc, IntPtr hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
at System.Windows.Forms.UnsafeNativeMethods.CallWindowProc(IntPtr wndProc, IntPtr hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
at System.Windows.Forms.NativeWindow.DefWndProc(Message& m)
at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
at System.Windows.Forms.Control.WndProc(Message& m)
at System.Windows.Forms.UserControl.WndProc(Message& m)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
at pwiz.Skyline.Program.Main(String[] args) in C:\proj\pwiz\pwiz_tools\Skyline\Program.cs:line 312

Reloading the Skyline file did not fix the issue, I can see my spectra fine via View>Spectral_Libraries. I imported my library into the Targets list via the Spectral Library Explorer, but this also did not have any effect on the Library match window staying blank.
It seems like the window is stuck for now. Is there a workaround for this?


view request
Issue in Spectral Library building from Timstof pro data (dda .d files)
(1 response) suresh choudhary 2023-01-05

Dear Skyline team,

I am trying to build spectral library from timsDIA PASEF data. I have raw files (.d folders) that were processed using FragPipe/MSFragger/Philosopher. Which, by the way, works great for these data.

I am not able to build a spectral library using the DDA-MS1 quant workflow and DIA workflow, with Import - Peptide results etc for building the list form the search results. Neither using MSFragger pepXML files nor using interact-.pep.xml files after PeptideProphet work. When using interact-.pep.xml files, I get an error that it cannot find spectral files. When using pepXML, it finds the files to get the spectra (from file_calibrated.mgf files produced by MSFragger) but gives an error at a later stage. Can some one help me to get the solution of it.

 Screenshot 2023-01-05 183838.png  Screenshot 2023-01-05 184104.png 
view request
Skyline for crosslinked peptides MSe Waters data analysis
(1 response) floriana capuano28104 2023-01-05


I have found a previous post suggesting that Apex3D pepxml files can be uploaded in Skyline. I am planning to use skyline to identify two crosslinked peptides and I would like to use the output of UNIFI data search to build an ion library and gain confidence in peak picking. I understand from previous posts that it is possible to use PLGS csv output, however at this stage I would not know how to add additional modifications in PLGS. I can see that UNIFI allows to export results in mzml and mgf format. Is there any chance I could directly use these to built the ion library? UNIFI is doing a targeted analysis to match spectra to peptides from one or two proteins added to the processing method. Would this have an impact on dotp or confidence in peak picking? Many thanks, Floriana

view request
Opening DIA for small molecules ((bbCID (MS/MS) and MS)) from Bruker impact II
(1 response) mohamed kaddah 2023-01-03

I am trying to open bbCID (MS/MS) and MS from Bruker, it is a type of DIA. I applied this method for degradation of pharmaceutical compound (small molecule). What is the procedure, how I can insert the range not a list of masses?

Can I inspect the data manually? is the step seeing the precursor and products overlayed available? can I use the software to extract specific ions during by inspection?

view request
Modifications for MSGF+ search within Skyline; chemical formulas don't seem to be accepted
kvancott2 2023-01-01

I am working with v22.2.0.351 and trying to run a MSGF+ search.

However, when running the searches, I got multiple errors related to my digest and modification settings.

Digestion: I had made a customized trypsin with proline digestion, and that was not recognized. I had to revert back to the default Trypsin digest setting (no cut when Pro follows KR). Once I did that, that error went away. My customized digest setting was "-1", which was out of the expected range of 0-9.

Modifications: I got multiple errors for modifications that were defined by their chemical formula in my Peptide Settings. It did not like "H3N" that was defined for pyroGluQ. It did not like "H2O" defined for pyroGluE. It also did not like "O - HN" that was defined for deamidation. I tried clearing all modifications from my Peptide Settings, and re-entering all the default modifications that had these chemical formula definitions, but that did not help. I ended up deleting all the chemical formulas from the modifications, just leaving the numerical values in there, and now the searches run as expected.

Below is the error information I got for the deamidation and pyroGluQ chemical formula instances:

MS-GF+ Release (v2021.09.06) (06 September 2021)
Java 17.0.1 (Oracle Corporation)
Windows 10 (amd64, version 10.0)
java.lang.NumberFormatException: For input string: "O - HN"
at java.base/jdk.internal.math.FloatingDecimal.readJavaFormatString(
at java.base/jdk.internal.math.FloatingDecimal.parseDouble(
at java.base/java.lang.Double.parseDouble(
at edu.ucsd.msjava.msutil.AminoAcidSet.parseConfigEntry(
at edu.ucsd.msjava.msutil.AminoAcidSet.getAminoAcidSetFromModFile(
at edu.ucsd.msjava.msdbsearch.SearchParams.parse(
at edu.ucsd.msjava.ui.MSGFPlus.runMSGFPlus(
at edu.ucsd.msjava.ui.MSGFPlus.main(
Search canceled.

MS-GF+ Release (v2021.09.06) (06 September 2021)
Java 17.0.1 (Oracle Corporation)
Windows 10 (amd64, version 10.0)
java.lang.NumberFormatException: For input string: "-H3N"
at java.base/jdk.internal.math.FloatingDecimal.readJavaFormatString(
at java.base/jdk.internal.math.FloatingDecimal.parseDouble(
at java.base/java.lang.Double.parseDouble(
at edu.ucsd.msjava.msutil.AminoAcidSet.parseConfigEntry(
at edu.ucsd.msjava.msutil.AminoAcidSet.getAminoAcidSetFromModFile(
at edu.ucsd.msjava.msdbsearch.SearchParams.parse(
at edu.ucsd.msjava.ui.MSGFPlus.runMSGFPlus(
at edu.ucsd.msjava.ui.MSGFPlus.main(
Search canceled.

view request
Using R script in Skyline Batch
(14 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?

Thank you in advance.

 Rscirpt path.jpg  Configuration.png 
view request
Calculation of asymmetry factor and tailing factor of chromatographic peaks
(2 responses) Juraj_Lenco 2019-08-06
Hi, it would be great, and I guess appreciated by many users, if Skyline could determine asymmetry factor and tailing factor of chromatographic peaks. Skyline can determine peak widths at 50% intensity or at baseline so far. Unfortunately, the width at 50% intensity is not sufficient for assessing chromatographic peaks quality. The peak width at baseline is almost never used in LC-based analytical chemistry since it is challenging to determine real peaks boundaries . The peak width at baseline should be thus replaced by asymmetry factor and tailing factor that are very popular methods of measuring peak shape in chromatography.
view request
Feature request: color dotP labels based-on value
(1 response) gabe 2020-04-06

We analyze datasets with many samples (typically 96) and rely critically on the dotP values displayed above each Peak-Area bar. Depending on the monitor and resolution, sometimes the dotP text is so small that it's very difficult to read. It would be helpful if these values could optionally be colored based on the value -- something like >0.9 green, between 0.7 and 0.9 orange, below 0.7 red. Ideally these would be user-configurable.


view request
Document Grid: Molecules missing Predicted Retention Time and/or Average Measured Retention Time when using a Spectral library
(11 responses) jtsorren 2022-12-13


As the titled states I am using skyline-daily in molecule mode. When I add all of my molecules from a spectral library from the Spectral library Explorer I find that if I navigate to View -> Document Grid -> Reports -> Molecules the columns with either Predicted Retention Time or Average Measured Retention Time contain #N/A.

I am wondering if one of the columns should have picked up the molecule retention time given that the spectral library contains scans at explicit retention times. Maybe there is a setting I am not correctly using...

Also I am wondering if this is the cause of some incorrectly selected peaks for precursor that fall outside of the observed retention time seen in my spectral library for some molecules.

Thank you!

view request
Internal standard, relative response factor and quantification
(1 response) naymin saw 2022-12-20

Hi Skyline community,

I am using Skyline for small molecules quantification.
For the quantification of some metabolites, I don't have standard but I have two internal standards in the samples and relative response factors of my interest metabolites.
Is it possible to implement these data in Skyline workflow for the quantification of large-scale metabolites?
I have not found the documents in Skyline and please advise me.

Thanks and regards,
Nay Min

view request
An error occurred trying to download Skyline
(3 responses) amahan 2022-12-19

An error occurred trying to download ''.
See the setup log file located at 'C:\Users\amahan\AppData\Local\Temp\VSDF115.tmp\install.log' for more information.

when I enter "" directly in browser I get xml code

<?xml version="1.0" encoding="utf-8"?>
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<assemblyIdentity name="Skyline.application" version="" publicKeyToken="e4141a2a22107248" language="neutral" processorArchitecture="msil" xmlns="urn:schemas-microsoft-com:asm.v1" />
<description asmv2:publisher="Skyline" asmv2:product="Skyline" asmv2:supportUrl="" xmlns="urn:schemas-microsoft-com:asm.v1" />
<deployment install="true">
<deploymentProvider codebase="" />
<dependentAssembly dependencyType="install" codebase="Application Files\Skyline_22_2_0_312\Skyline.exe.manifest" size="142364">
<assemblyIdentity name="Skyline.exe" version="" publicKeyToken="e4141a2a22107248" language="neutral" processorArchitecture="msil" type="win32" />
<dsig:Transform Algorithm="urn:schemas-microsoft-com:HashTransforms.Identity" />
<dsig:DigestMethod Algorithm="" />
<compatibleFrameworks xmlns="urn:schemas-microsoft-com:clickonce.v2">
<framework targetVersion="4.7.2" profile="Full" supportedRuntime="4.0.30319" />

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An error occurred trying to download ''.
(2 responses) kguehrs 2019-01-22

I tried to install Skyline on my computer running Windows7 64bit. The installation did not start giving the error message you can find in the attached screenshot together with the log file the message is referring to. Can you give me any advice to bypass the error and install the new Skyline version. Is there any possibility that the error is related to settings of the locales or regions because I run the English keybord and locales on the computer my IT installed a German version of Windows 7 on. If not I will keep the actual version 4.1 which is currently working without any problem.

I looked into the support issues list but I did not find any request with the same error description.

Thanks for your assistance.

 190122_installation_error.png  install.log 
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XIC overlay
(2 responses) sstoychev23513 2022-12-16

Hey guys,

Is it possible to overlay the XIC of two samples/runs in Skyline of have to export the data?


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AutoQC-no updating of raw files
(1 response) danielacgranato 2022-12-16

We have just installed AutoQC to monitor our System suitability runs, but although we were able to generate a file with 3 runs in Panorama, it does not update the new runs added to the folder. It does not move forward from "waiting for files". Could you help us troubleshoot this issue? I have attached the skyline file document (.zip) were using and the error log information. Please let us know if you need other files. Thank you very much. Best, Daniela

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Generating GC-MS "transition list" from .msp
(9 responses) brynnsundberg 2022-12-14

Hello Skyline team!

I have looked at the user instructions to view GC-MS data in Skyline, but I'm stuck without an initial transition list. The dream is to import my NIST .msp file and to be able to generate "transitions" from the 1710 small molecule spectra it contains to find them in my GC-MS results (using the strategy of selecting DIA in the MS2 transition settings and using a fake precursor mass). Unfortunately, the library explorer doesn't recognize the spectra in my .msp file, maybe because it's missing precursor mass and charge information. Is there a way to make this work? Do I have to learn how to code so I can pull the information from the .msp file and create the transition list I need? Any help you can offer will be greatly appreciated!


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files with dual polarity
(7 responses) naymin saw 2022-12-09

Hi Skyline community,
I am new to Skyline and I found some issues with dual polarities. My .raw files are positive and negative polarities in one file. When I prepare the standards, QC and blank samples in positive mode, it worked well.
However, when running in negative mode, there is no chromatogram found.
Could you please advise how to work with these dual polarities .raw files? Or please check the transition lists I have prepared and let me know if anything is not correct.

Many thanks,
Nay Min

 Transition list Pos.PNG  Transition list neg.PNG 
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Keyboard Shortcuts
(14 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!


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Spectronaut assay library import failed
(9 responses) Rita 2022-12-08


I tried to import a large Spectronaut Mouse library - MouseRefSWATH_iRT.csv by Krasny, L. et al 2020 ( Skyline was able to read .csv and check the file for errors but the process ended up with an error message (please see the attachment). I have Skyline running on 64-bit Win10 Pro 128 GB RAM. Would you have any suggestions?


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Negative transitions (and precursor) of a peptide
(5 responses) dilip singh 2022-12-12

Hi Skyline Team,

I am working on project in which inactive protein undergoes PTM (carboxylation) and leads to active protein (carboxylation at gamma position of glutamate (Glu) residues). I am not getting good response in positive ionization mode and I want to try in negative ionization mode.

Is it possible to have the negative transitions (and precursor) of a peptide in skyline? if I generate data in negative mode, Is skyline able to read that data?

Kindly suggest.


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Bibliospec not recognize modification from PLGS file
(4 responses) jcaceresvergara 2022-12-02
I'm trying to create a library from a peptide search made using PLGS. I get the error at the end. It does not appear to understand the modification I set, I try to change the final_fragment.csv file or add the modification to my skyline file but nothing seems to work. can you help me with this?

The modification should be a loss of' -2HS' variable on cysteine. I'm attaching my final_fragment.csv file.

ERROR: The modification 'Dehydroalanine+C(4)' on line 1429 is not recognized. Supported modifications include: "12C d0", "13C", "13C N15", "13C d9", "1H d0", "2H d8", "Acetyl", "Amidation", "Biotin", "C-Mannosyl", "Carbamidomethyl", "Carbamyl", "Carboxymethyl", "DUPLEX_TANDEM_MASS_TAG126", "DUPLEX_TANDEM_MASS_TAG127", "Deamidation", "Dehydration", "Dimethyl", "Farnesyl", "Flavin-adenine", "Formyl", "Gamma-carboxyglutamic", "Geranyl-geranyl", "Glycation", "Hydroxyl", "ICAT-C", "ICAT-C13C(9)", "ICAT-D", "ICAT-D 2H(8)", "ICAT-G", "ICAT-G 2H(8)", "ICAT-H", "ICAT-H13(6)", "Isobaric 114", "Isobaric 115", "Isobaric 116", "Isobaric 117", "Isobaric 8plex 113", "Isobaric 8plex 114", "Isobaric 8plex 115", "Isobaric 8plex 116", "Isobaric 8plex 117", "Isobaric 8plex 118", "Isobaric 8plex 119", "Isobaric 8plex 121", "Lipoyl", "Methyl", "Myristoyl", "N-Glycosylation", "NATIVE_TANDEM_MASS_TAG126", "NATIVE_TANDEM_MASS_TAG127", "NIPCAM", "O-GlcNAc", "O-Glycosylation", "O18", "O18 label at both C-terminal oxygens", "Oxidation", "Palmitoyl", "Phosphopantetheine", "Phosphoryl", "Propionamide", "Pyridoxal", "Pyrrolidone", "S-pyridylethyl", "SILAC 13C(1) 2H3", "SILAC 13C(3)", "SILAC 13C(3) 15N(1)", "SILAC 13C(4) 15N(1)", "SILAC 13C(5)", "SILAC 13C(6)", "SILAC 13C(6) 15N(2)", "SILAC 13C(6) 15N(4)", "SILAC 13C(8) 15N(2)", "SILAC 13C(9)", "SILAC 13C(9) 15N(1)", "SILAC 15N(2) 2H(9)", "SILAC 15N(4)", "SIXPLEX_TANDEM_MASS_TAG126", "SIXPLEX_TANDEM_MASS_TAG127", "SIXPLEX_TANDEM_MASS_TAG128", "SIXPLEX_TANDEM_MASS_TAG129", "SIXPLEX_TANDEM_MASS_TAG130", "SIXPLEX_TANDEM_MASS_TAG131", "SMA", "Sulfo", "Trimethyl".
ERROR: reading file 20221122 C418U_iRT MSE JC_LC-05_IA_final_fragment.csv

Command-line: C:\Users\jcaceresvergara\AppData\Local\Apps\2.0\T0VY925W.HAY\GJ5BNRYZ.4Y7\skyl..tion_e4141a2a22107248_0016.0002_c984fd7cba69179e\BlibBuild -s -A -H -o -i lib -S "C:\Users\jcaceresvergara\AppData\Local\Temp\tmp6856.tmp" "D:\lib.redundant.blib"
Working directory: C:\Users\jcaceresvergara\Downloads\PLGS Search Results\PLGS Search Results\Trypsin\20221122 C418U_iRT MSE JC_LC-05_20221202133552
OK More Info
System.IO.IOException: ERROR: The modification 'Dehydroalanine+C(4)' on line 1429 is not recognized. Supported modifications include: "12C d0", "13C", "13C N15", "13C d9", "1H d0", "2H d8", "Acetyl", "Amidation", "Biotin", "C-Mannosyl", "Carbamidomethyl", "Carbamyl", "Carboxymethyl", "DUPLEX_TANDEM_MASS_TAG126", "DUPLEX_TANDEM_MASS_TAG127", "Deamidation", "Dehydration", "Dimethyl", "Farnesyl", "Flavin-adenine", "Formyl", "Gamma-carboxyglutamic", "Geranyl-geranyl", "Glycation", "Hydroxyl", "ICAT-C", "ICAT-C13C(9)", "ICAT-D", "ICAT-D 2H(8)", "ICAT-G", "ICAT-G 2H(8)", "ICAT-H", "ICAT-H13(6)", "Isobaric 114", "Isobaric 115", "Isobaric 116", "Isobaric 117", "Isobaric 8plex 113", "Isobaric 8plex 114", "Isobaric 8plex 115", "Isobaric 8plex 116", "Isobaric 8plex 117", "Isobaric 8plex 118", "Isobaric 8plex 119", "Isobaric 8plex 121", "Lipoyl", "Methyl", "Myristoyl", "N-Glycosylation", "NATIVE_TANDEM_MASS_TAG126", "NATIVE_TANDEM_MASS_TAG127", "NIPCAM", "O-GlcNAc", "O-Glycosylation", "O18", "O18 label at both C-terminal oxygens", "Oxidation", "Palmitoyl", "Phosphopantetheine", "Phosphoryl", "Propionamide", "Pyridoxal", "Pyrrolidone", "S-pyridylethyl", "SILAC 13C(1) 2H3", "SILAC 13C(3)", "SILAC 13C(3) 15N(1)", "SILAC 13C(4) 15N(1)", "SILAC 13C(5)", "SILAC 13C(6)", "SILAC 13C(6) 15N(2)", "SILAC 13C(6) 15N(4)", "SILAC 13C(8) 15N(2)", "SILAC 13C(9)", "SILAC 13C(9) 15N(1)", "SILAC 15N(2) 2H(9)", "SILAC 15N(4)", "SIXPLEX_TANDEM_MASS_TAG126", "SIXPLEX_TANDEM_MASS_TAG127", "SIXPLEX_TANDEM_MASS_TAG128", "SIXPLEX_TANDEM_MASS_TAG129", "SIXPLEX_TANDEM_MASS_TAG130", "SIXPLEX_TANDEM_MASS_TAG131", "SMA", "Sulfo", "Trimethyl".
ERROR: reading file 20221122 C418U_iRT MSE JC_LC-05_IA_final_fragment.csv

Command-line: C:\Users\jcaceresvergara\AppData\Local\Apps\2.0\T0VY925W.HAY\GJ5BNRYZ.4Y7\skyl..tion_e4141a2a22107248_0016.0002_c984fd7cba69179e\BlibBuild -s -A -H -o -i lib -S "C:\Users\jcaceresvergara\AppData\Local\Temp\tmp6856.tmp" "D:\lib.redundant.blib"
Working directory: C:\Users\jcaceresvergara\Downloads\PLGS Search Results\PLGS Search Results\Trypsin\20221122 C418U_iRT MSE JC_LC-05_20221202133552
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\pwiz\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 161
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\pwiz\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 412
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 161
 20221122 C418U_iRT MSE JC_LC-05_IA_final_fragment.csv 
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DDA search hangs on "Running percolater..." with a specific FASTA file
(8 responses) Remco van Soest 2022-09-20

Skyline (64 bit) (c99206313)

When using the "Import DDA Peptide Search" workflow Skyline hangs on "Running percolator..." after the search has successfully finished. I have to manually end the "percolator.exe" task in order to make the software respond again.

The issue seems related to the FASTA file (attached; downloaded from NCBI; sequence is correct). If I manually remove line 11 and 12 everything works just fine. It is not the length, as everything works fine as well if I replace line 11 and 12 with A's.

Why does the percolator task hang when using a specific FASTA file? I don't see anything wrong with the file, and obviously want to be able to use the full sequence for my search.


 sequence (2).fasta 
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Skyline on apple IOS
New to Skyline 2022-12-08


As i cannot use the Prosit server on the Skyline software on Trust PC, i thought i could access it through my Laptop, however i am unable to do this on my laptop, is this due it being an Apple IOS and not on Windows? is there a way to overcome this?

Kind regards.

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MGF files with product ion charges are ignored during spectral library build
(14 responses) jtsorren 2022-12-01


I am using skyline-daily in molecule mode and using the built in feature to build a spectral library from a .ssl file and mgf files.

In mgf files you may now explicitly denote the product ion charge for measured peaks in individual scans. I am wondering why when I build the spectral library and add the molecules with transitions to the target space that the product ions are all assumed to have 1+ charge.

Thank you 
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Error message: Failure opening - "the file contains an error on line 13438 at column 9"
(6 responses) katherine wongtrakul-kish 2022-12-06

Hoping you can help me get back into my skyline file please... I added a new molecule to my transition list recently ( (HexNAc)2 (Deoxyhexose)1 (NeuAc)1 + (Man)3(GlcNAc)2 with m/z 876.8) by copying and modifying an existing one.

Usually this works alright if I'm copying and creating multiple entries for isomers, but on this occasion I modified the mass as well to make an entirely new glycan molecule -- which was perhaps where my mistake was?!

I managed to integrate peaks for the new glycan, but then at some point hit a clipboard error. When I closed and tried to re-open the file, I couldn't (error message in the attached screen shot).

I've also attached the SKYL file. Thanks for any advice you can give
 Total Ascites_EpCAM+_CD45_NG.skyl  skyline error 5Dec.PNG 
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SureQuant with two FAIMS CV voltages on Orbitrap Fusion Lumos - ZigZag shaped peaks on Skyline
(16 responses) aheinone 2022-08-11

Dear Skyline Team,

We are starting to do SureQuant on Orbitrap Fusion Lumos with FAIMS by using two CV voltages (-50 and -70). We are dealing in total with about 2000 peptides, but < 500 peptides at a time in one Skyline document. We have imported the raw files to Skyline for further processing. We have built Ion Mobility Libraries by using the results. Some peaks are drawn correctly, but in many cases product ion peaks are jagged (zigzag shaped in MS2 level). But still precursor peaks (MS1 level) look ok. It seems that this happens with CV -50 peaks. Probably Skyline takes data points from the both CV values for some peaks? We have tried many different settings for MS/MS filtering, but we haven't found any solution. We tested to change the CV values manually in the Ion Mobility library, but it doesn't solve the problem. And the CV values seem to be correct in the library. If we split the original raw files to two separate files according to the CV voltage, and then import them to Skyline, the peaks are fine. But if we build the Ion Mobility Library by using the two separately imported files, it didn't solve the problem. Is there any other way to handle multiple CV values than split the files according to the CV voltage. We would appreciate for any help with this, thanks.


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Waters Synapt G2-Si - multiple issues to access CCS values
(9 responses) nicolas macorps 2022-08-31
Dear Skyline support,

I have been trying to use Skyline to analyse data obtained with a Water Synapt G2-Si with ion mobility (IM) experiments. Experiments are performed with the HDMS mode (ion mobility, elution time and full scan MS only). The ion mobility technique is TWIMS that use a calibration curve (mob_cal.csv file in the sample root) that the proprietary software uses to correlate measured drift times to CCS values.
I want to use to have a solid workflow with this software to quantify PFAS (small targeted molecules) in various samples. To confirm that I am indeed looking at a PFAS, I need to access CCS values (which include the need to create an IM library) so I can then have it when exporting a "report'.

I encountered some issues when using Skyline features which is why I am opening this support feed. You can find uploaded in the "File Sharing Folder for Skyline Support" my folder under the name "". This file contains *.raw data from the Water Synapt, *.sky file (with the trageted list molecules in a *.csv in case it is needed) and snapshots of some error message I got along my tests.

1) Skyline crashing when importing *.raw files :
When trying to import Synapt *.raw files (220621008.raw and 220621009.raw in the *.zip folder), skyline crashes at the importation step (software closes without error message). This issue was encountered and reported in another support feed. The proposed solution in this feed was to convert *.raw into *.mzML files, but it seems that this approach doesn't allow for CCS values when creating IM library.

I went looking into the *.raw folders and found that when changing _FUNC002.DAT into _FUNC002.temp (in other words I "remove" this function from the *.raw file) I was now able to import without crashing the *.raw file and was able to obtain chromatograms with full scan spectrum and drift time (ms) distribution.
I initially thought that this "Function 2" contained "lockmass" data which can cause some issues when converting into *.mzML so I thought that removing this function could fix the problem. However I cannot be sure that this function is indeed the "lockmass" when looking in the "_extern.inf" file nor in the "_HEADER.TXT". The only information I have on these files are for the function 1 : "MOBILITY MS FUNCTION". So function 2 may also contain IM data from the HDMS experiments but I cannot be sure.

In any case, I kept going with modified *.raw files with the change of _FUNC002.temp (220621008_modify.raw and 220621009_modify.raw) to try to obtain CCS values

2) Negatives CCS values when creating IM library with "use results"

Now having something to work with, I tried creating the IM library for my molecules. I was happy to see that I could obtain some CCS values by doing so. Unfortunatly they are all with a negative sign and as such completely incorrect. I had two thought on that :
- Conversion of drift time into CCS values using the mob_cal.csv file (as done by the proprietary software) is not performed in Skyline in this manner or Skyline needs another type of file to do so.
- We need the function 2 to be active to have CCS values

For the latter, I tried to reactivate the function 2 in the *.raw file and reimport them. Doing so led to no crash so I followed with the creation of an IM library. I ended up with an error message "Failed using results to populate ion mobility library: [SpectrumList_Waters:spectrum()] Bad index: 18446744073709551615" (you can find the snapshot in the *.zip file and the extended error message in a *.TXT file in the *.zip).
Further trying with reimportation led to a crash of the software as mentionned in my first point.

I hope you have enough elements with this description to help me ?
- Do you know why Waters HDMS experiment *.raw files would make the software crash ?
- Do you know why I have negative CCS values when creating the IM library with the "use results" feature?

Let me know if you need anything else from me.

Best regards,
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Gas-phase-fractionation and iRT
(16 responses) Joerg 2022-11-18

I have a problem with iRT alignment with my gas-phase fractionation-SWATH-runs. As I am doing GPF for the SWATH-analyses some of the iRT peptides (Pierce C18) are fragmented only in fraction while others are fragmented in fraction 2 and so on, but none of the peptides are fragmented in the different gas-phase-fractions. I have full-MS-spectra for the entire precursor region in every run, though. Is there a possibility to align the runs according to the precursor signals or do I have to fragment each peptide in each run?
Many thanks!

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New to SkyLine- In silico
(11 responses) New to Skyline 2022-11-29

Hi All,

I am new to skyline. I am doing a research project in measuring a particular hormone. Is there a 101 document on how to use the Software, I have been through the tutorial (targeted method editing) but unable to replicate it for my project. We have the water Xevo. I wanted to do an in-silico for my peptide but dont know how to conduct it. Thank you for any support.

Kind regards,

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Results found 2 samples instead of 34 samples from the msms file
(5 responses) yael hirschberg 2022-12-02

Hello Skyline Team,

I ran 34 samples on Maxquant and tried to import the msms file on Skyline. However, Skyline only continued with 2 samples and 17 replicates of each. I attach a screenshort of the import and the msms file. What did I do wrong?

Thank you,

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timsTOF Scheduled PRM Data Import
(11 responses) robwsprung 2020-02-19

Dear Skyline Team,

Thank you for your continued development of an excellent software package.

I am having trouble importing scheduled PRM data from a timsTOF Pro instrument. When I look at the results in Compass DataAnalysis, it appears that the method worked as expected - MS1 signals for the expected isotope labeled peptides are observed and MS/MS spectra are acquired at the retention times of interest. However, when importing into Skyline Daily ( no precursor and no product ions are observed.

I've successfully imported other PRM runs from the instrument. Only this scheduled method is causing problems. Any insight you could provide would be appreciated. Thanks.


 timsTOF MHC  timsTOF MHC settings.pptx 
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Prosit Server Unavailable with Skyline Latest Version
(1 response) cm748 2022-12-01

I have just updated Skyline to the most recent version and am getting the "Server Unavailable" error with Prosit (attached image), as are my colleagues so I don't think it's a problem my end with connection etc. Could you advise?

Best wishes,

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Enhancement Requests: Filter a Range of Fold Changes and Multi-Proteome Homologous Peptide Filtering
philip remes 2022-11-30


I had an idea for an enhancement request. There is a workaround involving highlighting potentially 100's of lines and deleting them, it just isn't as nice. I created a targeted assay in a two-proteome mixture, and before performing a dilution curve that would take a few days, wanted to confirm with a quick experiment that the peptides chosen were from one proteome and not the other. Two replicates were injected and imported at each of two concentrations, and a Group comparison performed between the concentrations. The sorted Bar plot does a good job to show which peptides do not exhibit the expected fold change. Using Refine / Advanced / Group Comparison and entering a Fold change value appears to be useful in the case where one is interested in peptides that change by more than some ratio, up or down. But in this case, I am interested in accepting peptides with a range of acceptable fold change values, for example between 0.5 and 1.5, and rejecting the others. Does that make sense? Something you could add to your queue of new features?

A related feature request for these types of two-proteome experiments is the ability to filter out peptides that are homologous between organisms. This comes up also when using a carrier proteome: one wants to make sure not to pick peptides that could possibly exist in the carrier proteome. One could imagine a filter tool that allows to enter a path to another fasta file, and to filter out any peptides in common between the current document fasta and that one.


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BS3: how to insert in two peptides, which are probably cross-linked, two different site of BS3 modification?
(1 response) francesca grassiscalvini 2022-11-30


I would like to verify an option of cross linking between two peptides:

ATAQDNPKSATEQSGTGI (with BS3 linked on K) and SYCR (with BS3 linked on S which is the amino-term)

Is it possible in the panel modify, clicking on the sequence of one of the two peptide, insert two different site of modification for the two peptides?

or is it possible to insert my modification with K as a position and also C term, in the same modification? I know that is possible but then the program search for K only in the position C term and in the peptide ATAQDNPKSATEQSGTGI it's not on the c term

many thanks

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Orbitrap library
(2 responses) MS-SZAOmics 2022-11-28
Hi I'm Merve and I'm very very new in Skyline. We have an Orbitrap (Exploris240). I wanted to study with my data on Skyline but I couldn't find a library for Orbitrap. As a beginning I started with BSA analysis. In library site like NIST, I guess there isn't a BSA library for Orbitrap. I'll be glad if you can help me about this issue.

Many thanks,
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Removing Redundant Transitions
(4 responses) eajarett 2022-11-28


I am trying to exclude all redundant transitions in a DIA assay library from quantitation. I was able to export all transitions and manually determine which ones were identical with others based on their molecule list name, precursor m/z, precursor charge, product m/z, and fragment ion.

I tried manually deleting all transitions in my DIA assay library and reimporting the "mixed transition list" containing only the non-redundant transitions, but this didn't work. Is there a better way to exclude the redundant transitions from quantitation?

Thank you so much!

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Molecule mode - Spectral Library Build
(6 responses) jtsorren 2022-11-27


I am working with skyline-daily in molecule mode and trying to understand an error in my spectral library build.

I have used Molecule settings -> Library -> Build... to attempt a spectral library build from empirical data from an .ssl file. I have constructed multiple ms2 data files from multiple DDA experiments and referenced each in the .ssl file it by the "file" and "scan" columns.

After inspection in the Spectral Library Explorer, for each molecule only the first spectra from the first file for each will display in the viewer. When I change file to another scan I am met with the error "Failure loading spectrum. Library may be corrupted." When I explore Library Details many of the Files have 0 matching Spectra even though I have found the molecules' scans in the .ms2 file.

Is there a setting I am not using correctly?

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Past Releases?
(2 responses) nate 2022-11-23

The docker image of Skyline is a bit behind - can anyone help me find previous releases for windows? See below.

Thanks! Nathaniel

The document format version 22.2 is newer than the version 22.13 supported by Skyline-daily (64-bit : automated build) (804ca65).

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Problem with Import or Search of TMT Labeled Peptides
(6 responses) philip remes 2022-11-22


I'm trying to build a library of Yeast peptides labeled with 11-Plex TMT, using Eclipse DDA MS2. I've tried doing this in several ways:

  1. Search the data in Proteome Discoverer, import the .pdResult files. There are 17k peptides with q-value < 0.01, however I'm not able to successfully import the results. We get the old, "Importing the FASTA did not create any protein targets".

  2. Search the data in Skyline. I tried using MSAmanda, and MS-GF+. There appears to be an error parsing the TMT modification string. I'm pretty sure that this is the string that Skyline supplied for that modification.

Based on this thread I made sure that there were a number of good targets in PD. I've had no trouble importing standard, unlabeled HeLa results from PD or searching them in Skyline, so it seems like the TMT modifications must be the issue.

I've uploaded the pdResult files, raw files, and Skyline files to the file sharing server with the name Could you have a look and see if the problem is on my end, or the Skyline end?


view request
MS2 Quantitation with Orbitrap ddMS2 Data
(1 response) johnny perez28236 2022-11-21

Good Morning, I hope this email finds you well.

I am using Skyline to interpret ThermoFisher Exploris data. My method performs SIM analysis followed with MS2 analysis if about a certain ion threshold. What I have noticed is that Skyline extrapolates data for the entire LC run time. Therefore, if the ddMS2 data was only collected for 30 seconds and the run is 5 minutes, it will extrapolate the baseline from 0 - 5 minutes. See attached picture. This causes a problem when attempting integration. Is there any way to prevent skyline from doing this?

I've also attached what ThermoFisher's qualitative analysis software shows for the same MS2 experiments.

Thanks again!


 MS2 Data - Skyline - Orbitrap Exploris.png  MS2 Data - FreeStyle - Orbitrap Exploris.png 
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Issue with loading transition list with deuterium as H'1 in formula for newer Skyline version
(4 responses) j3 menzel 2022-11-12

Hi Skyline team,

I'd like to ask, whether the notation for heavy atoms has changed recently?
I used to enter deuterium into transition lists as H' (one deuterium being H'1), which worked fine for Skyline (64-bit)
I can still use this older Skyline version without problems, but the newest one shows an error when trying to load such transition lists.
The error message I get is:

Failed reading the file C:... .csv. The given key was not present in the dictionary.

The same transition list without any H' is loading fine in the latest Skyline version.
Is there a way I can generate transition lists with deuterium containing precursors and products that will work in all versions of Skyline?



 D problem snip.PNG 
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Cannot read Bruker .d without TIMS enable
(4 responses) ho-tak lau 2022-11-15

Hello Skyline Team,

I have a set of TimsTOF pro2 files (.d) that Skyline sees them as normal folders rather than spectra files. These data are recorded as MS1 or MRM mode without TIMS enabled. Is there a way to force Skyline to read them?

MSConvert also see them as normal folder, so I cannot convert them manually.

I can upload a few of them if that will help.


view request
Unaligned retention time issue
(3 responses) dominik kopczynski28488 2022-11-14

We have an issue with Skyline which looks like a potential bug. Maybe you could have a look at it, here is the description:

  • We set up a scheduled targeted method for our lipidomics measurements with (among others) a retention time window from 13.9 to 15.9 min.
  • An example is shown in the attached Image Analyst-correct-retention-time.png. You can see that both chromatograms are aligned.
  • When importing the data (minimal example in:, we get a shift in retention times as visible in Image Skyline-incorrect-retention-time.png.
  • However, this shift should not be there, it should be aligned.

Thank you for your support.

 Analyst-correct-retention-time.png  Skyline-incorrect-retention-time.png 
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Error during import DIANN .speclib as library
(1 response) ziyue wang 2022-11-14
I was trying to import the .speclib file generated by DIANN to skyline for building library, however, it failed several times.
My skyline is up to date and I tried .speclib with both .wiff and .wiff2.dia as suffix of File.Name column, both networking.
please find the error and my .speclib file in attachment.
 skyline error.JPG  report-lib.tsv.speclib 
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Exploris PRM data import issue
(4 responses) wal mair 2022-11-10

Dear Skyline team,

I am trying to setup a PRM method on our new Exploris 240 and to analyze the data using Skyline. There is no "PRM" mode per se on the Exploris and the engineers have told me to use "product ion scan" mode which should result in equivalent data. The resulting raw file, opened in FreeStyle, contains no ms1 spectra, but ms2 spectra continuously acquired for the precursor mass added in the inclusion list during the time window specified. I am new to PRM so I have no comparison but it's exactly what I expected. I imported the file into my Skyline project and it says "Chromatogram information unavailable". I figured my Skyline document might have wrong settings, so I manually added a BSA peptide and fragment masses from the Skyline PRM tutorial and imported one of the associated Agilent QTOF data files (3-BSA-1fmol). The file imports without problem and the precursor and fragment ions are displayed.

Another user had submitted a PRM&Exploris question in 2020 so I assumed it should be possible to analyze data from that instrument with Skyline, generally. However, not sure if relevant, I noticed that user used a different "MS/MS filtering" - "Acquisition method" --> "Targeted (obsolete)" which is not available in my Skyline (see his post).

Some info that might be relevant:
I am working with small molecules, in particular I am targeting derivatized fatty acids. I am transferring info from a MRM method which is why I only have 2 product ions at this point. Data was acquired in positive mode. I'm using Skyline 64-bit, Windows 10. I will upload the skyline document with the name "".

+Is there an issue with the way I acquired the data?
+Is the data ok but Skyline is confused by the derivatization adduct?
+Is there a mistake in the way I setup the transitions?

Any help is appreciated! Thanks!

 MethodProductIonScan.PNG  RawFilePrecursorPlusProducts.PNG  Skyline.PNG 
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Partial missing fragment ion exracted from FAIMS-DIA file by skyline
(5 responses) Winnie 2022-11-08

Hi skyline team,
I used skyline to extract the interested peptide precursors from the FAIMS-DIA files. The interested peptide precursors were almost extracted from files acquired from Q Exactive HF instruments or TripleTOF instruments. But there are some weird things about those peptide precursors extraction from FAIMS-DIA files. 1. Some of the peptide precursors show good MS1 extraction but without fragment ions. 2. Some of the peptide precursors could extract fragment ions from A files but could not extract from B files. The A and B files are technical replicates.
I do not know whether there are some issues with skyline settings, so I upload the skyline documents if you can check it would be better. Many thanks.

I have uploaded the file to the link.
Best regards,

view request
Library Build for SILAC labelled PTM spectra from MetaMorephus mzID and mzML files ERROR No spectra were found for the new library.
(5 responses) gwitek 2022-09-21


I am trying to build a library from my spectra files that contain peptides digested with Pepsin, grown in SILAC media, and that have phosphorylation on S/T/Y (PTM search).
I get mzID and mzML files from MetaMorpheus but I get an error that No spectra were found for the new library.
I tried changing files to lowercase letters mzid and mzml both or each and it does not work.

I did read another request that said PTM searches can cause library build issues and wanted to reach out to you if this might be another issues with PTM or SILAC.

Thank You,

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Issue with Protter Tool
(1 response) dilip singh 2022-11-10

Hi, I want to use Protter tool to show the unique peptide selected in skyline in the protein structure (Skyline--Tools--Protter)

But I am getting error, attached herewith.

My operation system is windows 10 enterprise with 11th Gen Intel ® Core (TM) i7-1165G7 @2.80GHz 1.69 GHz; RAM 16 GB, 64-bit operating system
Skyline (64-bit) (c99206313)

Could you take a look at this problem? Thank you very much!


 Protter Error.docx 
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ERROR: Failed to find required column named 'file'.
(28 responses) zzhu248 2022-05-26

Dear Developers and all researchers,

When I was trying to build the library via peptide setting, I upload my .ssl file and it pops up warning as title.

For the specific workflow, I am following a paper from nature protocol:
I was working on the crosslinking setting and stuck on the step 39.

I would be very appreciated for your help!

Best Regards,

 Screenshot 2022-05-26 143119.png 
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(6 responses) ebru9679 2022-11-08

Dear Skyline time,

I am trying to download encyclopedia-1.12.31 but I get an error "SSL_ERROR_RX_RECORD_TOO_LONG". could you please check your ssl record?
Thank you

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Unable to use 'External Tool' feature
(2 responses) dilip singh 2022-11-07

Dear Skyline Team,

I am trying to use Protter from tool store. To do so, I need to add "Protter" in external tool option.

I am getting error, which is attached for your reference.

Kindly suggest solution.


 External Tool_Error.docx 
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export protein-peptide list from skyline
(1 response) kmsd 2022-11-07

Hello, I am new to skyline so I am still learning new things. I have followed MS1filtering tutorial to import my short listed protein target into skyline along with 8 orbitrap pdresult files. After cleaning them now I need to export out the protein-peptide list from skyline to hard drive for future use. Is there any supporting tutorial available?


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Latest CIRT library
(2 responses) Brett Phinney 2022-11-03

Hi All, does anyone have the latest list of CIRT peptides skyline looks for? Preferably in a bilb library with predicted ion mobilities ? Just curios , I looked and couldn't find it



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Exporting scheduled method for collision energy optimisation
(13 responses) matthew daly 2022-10-17


I am having difficulty exporting a scheduled method for collision energy optimisation.

My colleagues ran a panel of peptides and I have the RT for these peptides, however I don't have the .raw files. I have pasted the results into the iRT calculator.

I am trying to run these same peptides using the same chromatography on a different platform, so I need to optimise the CE and as I am sample limited would like to do it in as few injections as possible. When I try to export a scheduled method optimising CE I get the error "Retention time predictor is unable to autocalculate a regression". Is it possible to export a method for peptides that you've used to train the iRT calculator?

Many thanks

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export dot p result between MIDAS vs Library
heyang 2022-11-02

Hi Support team,

Could I export dot p value of MS2 spectra comparison between MIDAS vs library?


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Error retrieving chromatogram "UV 1"
(1 response) licky lck 2022-11-01

Dear Skyline team,

The samples run from Our Q Eaxctive had a hard time recording the UV data, so we could not retrieve the UV data after the sequence run. So when I am trying to import the raw file into skyline, this error message pops out "Failed importing results file 'XXXX.raw'.
[ChromatogramList_Thermo::chromatogram()] Error retrieving chromatogram "UV 1": [RawFileImpl::getChromatogramData()] Unknown UV/PDA packet type". (The full error message can be found below)

Do you have any suggestions to recover the data from my raw file, or can skyline ignores this UV signal but import my MS data?

Full error message:
Failed importing results file 'XXXX.raw'.
[ChromatogramList_Thermo::chromatogram()] Error retrieving chromatogram "UV 1": [RawFileImpl::getChromatogramData()] Unknown UV/PDA packet type
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'XXXX.raw'.
[ChromatogramList_Thermo::chromatogram()] Error retrieving chromatogram "UV 1": [RawFileImpl::getChromatogramData()] Unknown UV/PDA packet type ---> System.Exception: [ChromatogramList_Thermo::chromatogram()] Error retrieving chromatogram "UV 1": [RawFileImpl::getChromatogramData()] Unknown UV/PDA packet type
at pwiz.XX.msdata.ChromatogramList.chromatogram(Int32 index, DetailLevel detailLevel)
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetQcTraces() in C:\proj\skyline_22_2\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 846
at pwiz.Skyline.Model.Results.GlobalChromatogramExtractor..ctor(MsDataFileImpl dataFile) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\ChromDataProvider.cs:line 129
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_22_2\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 87
at pwiz.Skyline.Model.Results.ChromCacheBuilder.CreateSpectraChromProvider(MsDataFileImpl dataFile, ChromFileInfo fileInfo) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1306
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 244

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Rugged extracted ion chromogram of targeted peptide precursors from FAIMS-DIA file by Skyline.
(5 responses) Winnie 2022-10-30

Hi, skyline,

I extracted chromotogram of targeted peptide precursors from FAIMS-DIA files by skyline, the xic show rugged profiling. The screenshot is attached. I was wondering there are issue on ion moblity library because i did not set ion mobility sepctral library. The sepctral library was imported by the following strategy. File>import>assay library. I found the column named precursor ion mobility in this spectral library is NA. The spectral library was build by Fragpipe with files acquired by FAIMS-DDA. I first communicated with Fragpipe team, they told me it is normal the column named precursor ion mobility is NA, because they this the FAIMS is low resolution device for ion moblity. So they set NA of precursor ion mobility for the library genetated by FAIMS-DDA.

So, did you have suggestion on the rugged xic and how to spectral library generated by FAIMS-DDA

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Can't connect to prosit server
(7 responses) sp807 2022-10-27

Everytime I try to conncet to the prosit server, it says "server unavailable".
I have updated my software to Skyline (64-bit) (c99206313) but the same things happens.

Any way to fix this?

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Skyine-Daily failing due to application error thrown on Windows 2016
(7 responses) hchandler 2022-10-22

Dear Skyline support team:

When launching Skyline-Daily or even trying to run the program, we are getting the same error that we have attached to this trouble ticket. We first thought that RAM was problematic as another product had taken so much, but even when the other program is not running and the server is rebooted to clear resident memory and caches, the error occurs. It states that the system did not have enough resources to complete the operation. The server is pretty beefy with 128GB of RAM, plus 2 Xeon 3.2GHz CPUs in an enterprise level Dell server running Windows 2016. The drives all have plenty of disk space (500GB or more) available, plus the NAS drives not only have plenty of free space, but the Network load does not reach above 10% utilization on the 1TB network interface. So not sure of the resources needed, other than we thought that the browser level was still using the old deprecated IE 11 ( we just installed Edge, but IE 11 is still on the system) and maybe the .NET needs to be updated (it is v4.8). So rather than guess at the issue, we are asking if you could read the error and determine the possible issue at hand.

Thank you for any help or assistance on this matter, in advance.


Henry Chandler

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Using an earlier version of MSstats
(2 responses) susannah hallal 2022-10-27


I hope you're well.

Is it possible to install and use an earlier version of MSstats in Skyline? I currently have the most recent version installed, but would like to use an earlier version to compare my data to an analysis I had done a couple of years ago

Kind regards,

view request
Determination of LOD and LOQ using the height of the peak and the background signal amplitude
(1 response) gomesrja 2022-10-25

Hi, good afternoon

Is there a way of getting the peak intensity and background signal amplitude to determine the LOD and LOQ (being LOD 3x and LOQ 10x)? And is this the best way to determine these parameters using DIA MRM-like data?

Many thanks,

Ricardo Gomes

view request
High Energy DT Offset
(5 responses) sxu374 2022-10-20

Hi Skyline support team,

I have some data acquired from Agilent 6560 IM-QTOF. I noticed that for some analytes, the IM drift times of the fragments are lower than their precursors, leading to unsuccessful alignment. In a literature, I found a function called "HighEnergyDTOffset", which could set in skyline transition list. This function indicates how much the fragment drift times could be lower than their precursor. I added the "HighEnergyDTOffset" column to my transition lists of different batches of data. Something interesting is that this function only works for certain transition lists. In other transition lists I loaded, the IM drift time window applied in MS/MS spectra was the same as the precursor, not having any drift time offset at all. I wonder if there is a guidance on how to set "HighEnergyDTOffset" correctly, or if there is any restriction on how the data is acquired by MS (AIF, PRM, etc.) in order to enable this "HighEnergyDTOffset" function.


view request
Sciex SWATH data we see precursor response but not fragments
(1 response) jlaycock 2022-10-21

Dear Skyline Team:

We have 5 replicates of predigested BSA injected with fixed window SWATH on our Sciex a 7600 ZenoTOF. The BSA Fasta was imported and the 5 .wiff data files were imported. I have tried a several different settings in the Transitions form but still do not see the fragments.

I have confirmed by viewing the data in OS Explorer that there is response for at least some of the fragments.


Data files available to upload if helpful.

 SkyLine SWATH no fragments.docx  BSA Digested Standard 
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Using software for MacBook user
mhossai9 2022-10-21

I am using MacBook. I can’t able to use the software from my MacBook. Is there any solution?

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Target peptide monitoring using DDA Orbi-Lumos LFQ data
(1 response) kmsd 2022-10-19

Hello, I have a list of peptides that I want to monitor against lumos LFQ (DDA) raw data file. I know there are lot of good articles and Ques/Answer available in skyline but I am new in this platform and need some guidance. Can someone direct me how to do this using skyline weblinks/tutorials etc?


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I can't choose my quantification ion
(10 responses) antonin padioleau 2022-10-11

Hi all,

I have a problem with my transitions. I work with GC-(EI+)-HRMS and LC-(ESI+/-)-HRMS data acquired in full-scan mode and I use the small molecule part of Skyline.

I created my excel sheat with 1 quantifier ion and 1 or 2 confirmation ions, the quantifier in the upper line of my sheat. When I imported this sheat with copy/paste, I selected all ions as precursor (it didn't work with precursor and product ions, no data in the chromatograms). Next, in the left side, for each molecule, my precursors were rearranged from the smaller mass to the highter.
My problem is that for all the molecules, the quantifier ion is automatically the first precursor ion, the smaller one, even if it's not the best choice. I tried to select the "Quantitative" choice only for my quantifier ion but, when it's not the smaller ion, the message "Error: All of the external standards are missing one or more peaks" appears in the calibration curve window. I can only quantify with the smaller ion...

I tried to change the monoisotopic and average masses of my molecules, which are always the mass of the smallest ion, to my quantifier ion mass but Skyline automatically adds the difference between the former masses...

How can I choose my quantification ion and confirmation ion in another way?


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Problems importing Bruker MRMS data
(3 responses) dennisjakob 2022-03-07

I've been running samples on a Bruker Scimax MRMS. When I import the data I get this error message:

At 3:47 PM:
Failed importing results file 'D:\Users\Data\2020-11-24-Aminoacid mix\Amino acid_1_2_1_123.d'.
unknown compressor id: 6bb2e64a-27a0-4575-a66a-4e312c8b9ad7
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'D:\Users\Data\2020-11-24-Aminoacid mix\Amino acid_1_2_1_123.d'.
unknown compressor id: 6bb2e64a-27a0-4575-a66a-4e312c8b9ad7 ---> System.Exception: unknown compressor id: 6bb2e64a-27a0-4575-a66a-4e312c8b9ad7
at filename, MSData result, Int32 runIndex, ReaderConfig config)
at pwiz.ProteowizardWrapper.MsDataFileImpl..ctor(String path, Int32 sampleIndex, LockMassParameters lockmassParameters, Boolean simAsSpectra, Boolean srmAsSpectra, Boolean acceptZeroLengthSpectra, Boolean requireVendorCentroidedMS1, Boolean requireVendorCentroidedMS2, Boolean ignoreZeroIntensityPoints, Int32 preferOnlyMsLevel, Boolean combineIonMobilitySpectra, Boolean trimNativeId) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 194
at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 291
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 188
--- End of inner exception stack trace ---

Do you have an idea what the issue could be here?


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Peptide Identification using DIA SWATH Data
(1 response) gomesrja 2022-10-17

Good morning

I have a question...How to I know each peptide and proteins are considered identified and present in a sample using DIA SWATH data?

I am doing an untargeted global analysis of all possible proteins in the library. Is the library match peptide score?

Many Thanks,

Ricardo Gomes

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Q Exactive method export
(1 response) ad6628 2022-10-14


I was at the training on October 10 and 11 this week.Now I am trying to use a Skyline for the lab's needs. I was wondering if you could, please, give me some directions on a couple of problems. I was able to create a transition list for the peptide we are studying. However I would like to export a method for the Q Exactive mass spectrometer. When I go to file-export-method, I do not see Q Exactive in the list "instrument type". Also, when I tried to export one of them, the program is asking for a template. I would appreciate if you could direct me how the template should be for the Q Ex method and how to find the Q Ex in the drop down list. I tried to export the transition list method from a Fusion mass spectrometer since it is showing in the list of instrument just to practice, but I get an error message that I am attaching to this request.

Thank you very much

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Mobility filtering and data importing issues
(3 responses) mmgadz 2022-10-11

I am working on developing PRM-PASEF methods. I have encountered some issues with importing data and have some questions about the mobility filtering/method exporting.

Importing: Importing the same raw file with the same peptide/transition settings into separate skyline files produces A) peak area, RT, and spectral data and confident identifications or B) no peak areas or RT data displayed, unconfident identifications. I cannot figure out why this is happening.

Mobility filtering: The filtering window is frequently offset from observed maximum - is there a way to mediate this in skyline? When exporting a bruker method, how does skyline incorporate the mobility filtering? Is it necessary to refilter the data with the same library used in the method/data acquisition or should I use "Use Data" and refilter for a given dataset?

Attached is a powerpoint with screen grabs demonstrating the above challenges, as well as descriptions. The slides are segmented into "Issue 1" and "Issue 2".

Any help will be greatly appreciated - thank you!!

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Custom fragment ion for ECD
(2 responses) ankittjain89 2022-10-12


I was trying to look for a custom fragment ion from ECD and ETDhcd spectra for the isoAspartate residue of the peptide that gives off either c+57 or z-57 ions. I am able to add c and z ions in transition settings but unable to add any other loss/ gain to it. One workaround I had to this problem was to use a peptide modification of 57 gain/ loss and then select the c ions with a 57 loss or z ions with 57 gain. So is there any other simpler way to do this custom fragment ion search?


 SkylineECD settings.pptx 
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Q executive method
ad6628 2022-10-13


I was wondering if you could, please, give me some directions on a couple of problems. I was able to create a transition list for the peptide we are studying. However I would like to export a method for the Q Executive mass spectrometer. When I go to file-export-method, I do not see Q executive in the list "instrument type". Also, when I tried to export one of them, the program is asking for a template. I would appreciate if you could direct me where to get a template for the Q Ex method and how to find the Q Ex in the drop down list.

Thank you

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Collision Energy for Thermo Altis
(4 responses) Gao 2022-10-12

I noticed the collision energy calculated by Skyline Daily for Thermo Altis were dramatically changed in recent updates. They are much higher than the previous versions. Would it be possible to have the conversion of CE between current version and previous version? The collision energy of previous versions work better for me on my Altis. Thank you!

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MRM assay development
(3 responses) ad6628 2022-10-12


I have attended the introductory training on October 10 and 11 but I am not sure how to apply the training to my immediate problem. My boss wants me to establish MRM conditions for a QExecutive mass spectrometer to check on a synthetic peptide. I will attach the slides I have. The peptide has modifications as oxidation, acetylation and has an amide attached to the n-terminal. It can be analyzed as heavy versus light. I was wondering which tutorial I should follow to create the assay. Any advice would be greatly appreciated.

Thank you

 MRM assay development.pdf 
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Command line to set the peptide as a global standard
(3 responses) jianqiaoshen 2022-10-08

Dear Skyline team, I am working on the automation for Skyline. I am wondering is there any command lines or ways to set one peptide as the global standard?


Jianqiao Shen

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Multiple precursors library match
(1 response) Juan C. Rojas E. 2022-10-11

Hi all,

I could have sworn that a recent version of Skyline-daily was displaying at least one of the PSMs for one of the charge states if the selection was focused on the peptide. But on Skyline-daily this useful feature seems to be gone (attached pic).

Would it be possible to get it back? :)
Juan C.

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Ghost peak in Skylin
(3 responses) ychiu2 2022-10-07

Hello Skyline team,
I have been using Skyline to do the quantification for small molecule and recently we found a ghost peak integration issue. In Skyline, you can see that the software integrate a peak at m/z 1055.5057, but when I plot m/z 1055 in Freestyle (Thermo software), no peak was found. I really want to know if there is a way to fix this as we use Skyline heavily for quantification. If the peak integration was not good, then the data validation will be very difficult. I have attached the skyline file and two graphs demonstrating the peak
Thank you

 221007_Skyline_GhostPeak.PNG  221007_GhostPeak_Thermo.PNG 
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unify raw file can't be imported to sklyline.
(1 response) juan xu918 2022-10-09

Dear Skyline team,

I am trying to import 2 Waters Xevo G2 raw files into skyline. The skyline document has only 1 protein and the raw files being imported were collected using MSe method. I began importing the data and didn't see any chromatograms exported. After a few mins, the program skyline closed down by itself.

I am using Sklyine (64 bit) 22.0.255 (C99206313) on Windows 11 Pro. The processor is an 12th Gen Intel(R) Core(TM) i7-12700H 2.30 GHz and 40 Gb of Ram.

Many thanks,


 unify raw data import issue.pdf 
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Concentration Multiplier in Molecules
(1 response) franzone 2022-10-07

I am doing quantification on amino acids so I am using the Molecules layout for my analysis. I read previous posts about using Concentration Multiplier in the Peptides layout. Is this function available in Molecules and, if not, will it be added in the near future?

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Trouble in export method to MassLynx
(3 responses) guioliveirareis 2022-10-05
Dear Brandon and Skyline Team,

I've downloaded the new Skyline version to run a SRM experiment in Waters Xevo TQS-micro, however when I try to export the method this message pop-up:

An error occurred attempting to export.
ERROR: Saving the method was unsuccessful.

Command-line: Method\Waters\BuildWatersMethod -w 5 -m "C:\MassLynx\\Acqudb\template_skyline.exp" "C:\MassLynx\\Acqudb\t5oo4e1q.bxc\transitions.txt"
Working directory: C:\MassLynx\\Acqudb
OK More Info
System.IO.IOException: ERROR: Saving the method was unsuccessful.

Command-line: Method\Waters\BuildWatersMethod -w 5 -m "C:\MassLynx\\Acqudb\template_skyline.exp" "C:\MassLynx\\Acqudb\t5oo4e1q.bxc\transitions.txt"
Working directory: C:\MassLynx\\Acqudb ---> System.IO.IOException: ERROR: Saving the method was unsuccessful.

Command-line: Method\Waters\BuildWatersMethod -w 5 -m "C:\MassLynx\\Acqudb\template_skyline.exp" "C:\MassLynx\\Acqudb\t5oo4e1q.bxc\transitions.txt"
Working directory: C:\MassLynx\\Acqudb
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_22_2\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 161
   at pwiz.Skyline.Util.Extensions.UtilProcess.RunProcess(ProcessStartInfo psi, String stdin, String messagePrefix, IProgressMonitor progress, IProgressStatus& status) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Util\Extensions\UtilProcess.cs:line 45
   at pwiz.Skyline.Model.MethodExporter.ExportMethod(String exeName, List`1 argv, String fileName, String templateName, Dictionary`2 dictTranLists, IProgressMonitor progressMonitor) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Export.cs:line 4674
   at pwiz.Skyline.Model.WatersMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Export.cs:line 4466
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_22_2\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_22_2\pwiz_tools\Skyline\Util\Util.cs:line 1963
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 204
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\skyline_22_2\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2240

Here, we use MassLynx 4.2 SCN 977 (with TargetLynx installed).

I've tried reinstall and uninstall several times, with several ways. I have no ideia what is happening, and then I was wondering if you have seen this message before in Waters equipaments?

Thank you very much for your help,

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peptide absolute quantification from PRM data sanity check
(2 responses) manguy jean 2022-09-23


I learned how to use Skyline on the job for a peptide absolute quantification project. I read a lot of the tutorials, some of the posts I found here from different google searches, and looked at the methods sections of diverse papers. I think what I did makes sense I just want to make sure I didn't make a mistake because of some blindspots, so any help or advice is welcome.

We want to quantify some peptides from a hydrolysate (not for protein quantification but just for peptide quantification so we didn't have the choice of which peptides would be best). We have heavy variants of each of them. We built an internal calibration curve for each peptide using varying concentrations of heavy peptides. The first series of runs (PRM on a QE) uses a wide scale of concentrations to find the approximate concentration of light peptide, then another series of runs to adjust the calibration curve for each peptide to quantify and then replicated that again.

I used Skyline to analyze these data. I used the retention time of these peptides in previous DDA runs using the same LC parameters and the presence of peaks for the heavy and light products to select the correct peaks. Removed the product ions interfering with the peak. I then extracted the ratio of heavy/light for each analytical replicate and for each concentration. I used these ratios to make a calibration curve (linear regression) and I looked for the ratio heavy / light == 1 to find out the concentration of light. I had to go outside Skyline for this step as all my samples are marked as Standard, no sample being just Unknown, but being both at once, and as far as I know I cannot ask Skyline to look for a ratio of 1.

I hope someone can tell me if I am completely off the tracks or not.

Thank you


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Analysis of uncharacterized peptide-modifications
laura boffel 2022-10-07
Dear Skyline team,

I am currently investigating drug-protein adduct formation of reactive chemicals at Cys34 of human serum albumin. More specifically, I am investigating whether a specific drug becomes reactive after in vitro metabolization and whether this reactive metabolite is captured by the thiol group of Cys34 and consequently, a covalent bond is formed between the reactive metabolite and amino acid.

So my question is if I can use Skyline to detect such protein/peptide modifications, by comparing IDA data obtained from a blank sample and a sample incubated with the drug, to be able to detect differences in the fragmentation pattern of the peptide? Since I don't know the chemical composition of the reactive metabolite on beforehand, an untargeted modification approach should be used.

Thank you in advance!

Kindest regards,
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Feature request: Maximum pressure value for reports
(2 responses) cashwood27088 2022-10-06

Dear Skyline team,

I hope you're doing well. I'm loving the new Skyline-daily update featuring the pressure traces, but currently the chromatogram visual alone makes automatic QC difficult. Could you please add a parameter in the report section that provides the maximum height observed in the pressure trace? I imagine it would be similar to the "Total Ion Current Area" parameter except only returning the maximum pressure. It would also need to ignore the automatic peak picking of the pressure trace since the maximum height would be the maximum y-axis value for the entire retention time range.

I've attached a mock-up of what I think it should look like.


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Signal to noise in Skyline
(1 response) kh 2022-10-06

Dear Brandon and Skyline Team,

I would have a question regarding S/N, is it somehow possible to access it in Skyline?
We currently run an instrument benchmarking experiment (small molecules, neg and pos mode, high resolution) and would like to compare also the signal-to-noise values for several metabolites in a fast and easy way (if possible). Would you have any recommendations for us?

Thank you for your help!

All the best,

view request
exporting Prosit dotp scores
cabarnescabarnes 2022-10-04

Hi all,

I've seen this requested a few times, but is there any update on the ability to export the Prosit dotp scores for each peptide in a mirrored database match-based spectral library?



view request
highlighting peptides that map to multiple proteins
(2 responses) cabarnescabarnes 2022-09-30

Hi all,

Simple question, but is there a way to highlight which peptides are matching to multiple proteins? I have a proteogenomics type of effort going on and there is utiility in me finding protein evidence for multiple isoforms that might all individually have the same tryptic peptide, but that tryptic peptide may only map to that novel isoform group and nothing else, which would be helpful in and of itself for me as I want to keep all of the isoforms in there.



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