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

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

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

If your question is about an External Tool, please contact that tool's developers directly. Contact information can usually be found on Skyline's Tools | Tool Store... menu.  

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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
Adding library peptides to document giving impossibly large number of proteins
(3 responses) Liyan 2022-05-24

Hi Skyline developers,

We run DIA on plasma and serum samples in our lab. We have an in-house library generated from fractionated DDA data that gets updated several times a year when we have a new cohort. This has been done on Skyline 19.1 for past two years. The library searches were done on Proteome Discover 2.3 or 2.4.

We are now setting up Skyline on a server and we also have a new cohort to add on to the library. We know that the library PD search on each of our cohorts has around mid 1000+ proteins, and the concatenated library would have 2000+ proteins at most. However one of the library files is giving me an additional 5000 proteins when I add it to the document. This happens whether I append .blib files or select multiple libraries under peptide settings.

Could I send you the problematic library file to take a look?

view request
Waters Synapt G2-Si data
(6 responses) diaz-galiano 2022-05-23

Dear Skyline developers,

We are currently working on a Waters Synapt G2-Si instrument in IM-MS mode. We have been searching for a way to analyze the samples using Skyline, including CCS/drift time values, but we haven't found a tutorial on that (perhaps we haven't looked well enough?)

Could you please direct us to a tutorial on converting the Waters .raw files with MS and Ion Mobility information for their use on the Skyline software?

Thank you in advance for your time.

Best regards,

F. J. Díaz-Galiano

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Deconvoluted Spectra - Intact Proteins - timsTOF
Diego Assis 2022-05-24

Dear Skyline Developers,

I would like to quantify relative abundance of intact proteins isoforms from data acquired on timsTOF (mobility ON and a deconvoluted spectrum). Is possible to import this deconvoluted mass spectrum (or some file extension) and perform quantification on skyline? If yes, which Bruker's file format would be compatible with this workflow?

Thank you so much

Diego Assis

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DIA Analysis through command line tool.
(11 responses) jingyangzhang0222 2022-05-17

Hi Skyline Team,

I am trying to implement a Skyline workflow through the command line, referring to this document( Command-Line Interface-4_2.pdf?renderAs=DEFAULT&_docid=dav%3A%2F_webdav%2Fc90e7bac-8854-102e-9e41-e08d31b9c205%2F%40files%2Fdocs%2FSkyline Command-Line Interface-4_2.pdf).

It is very useful to open, modify and save the existing Skyline files through the command line, but there seems to be no "new Skyline Project" in the command-line options, and how can I set the work mode to "DIA"? If there is a general DIA workflow for Command Line Tool, it would be very helpful, thank you!

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DIA raw files vs human .fasta file comparison
(1 response) amey shirolkar 2022-05-22
hello Team Skyline,

I have uploaded a human .fasta with decoys onto Skyline and now I wish to analyze my DIA data (.raw or .mzML files) against this.

Please brief few steps, since uploading .fasta onto Skyline it has appeared this way it was stuck in between.
Regards and thank you.
 Skyline_humanFASTA_DIA comparison.JPG 
view request
SureQuant Method export
(3 responses) klemens froehlich 2022-05-19
Dear Skyline Developer Team,
I am currently trying to export a SureQuant method from Skyline for an Exploris system.

I get the following error message:

System.IO.IOException: Thermo instrument software may not be installed correctly. The library System\Programs\dependencies\tng\Thermo.TNG.MethodXMLFactory.dll could not be found. ---> System.IO.IOException: Thermo instrument software may not be installed correctly. The library System\Programs\dependencies\tng\Thermo.TNG.MethodXMLFactory.dll could not be found.
   at pwiz.Skyline.Model.ThermoMassListExporter.EnsureLibraries() in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Export.cs:line 1065
   at pwiz.Skyline.Model.ThermoSureQuantMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Export.cs:line 1992
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1939
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 202
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2232

I checked the Thermo.TNG.MethodXMLFactory.dll and found it here:

Is Skyline looking for this dll somewhere else (where I could copy it to)? I have tried to copy the dll into older Exploris software version eg:
but it still gives me the same error.

Any help would be much appreciated, as the method export for sureQuant would be extremely convenient!!!

Best, Klemens
view request
Can I set up Skyline to exclude quantification results when scan numbers are too low?
(1 response) wangn2 2022-05-20
Hello there!
I got a question about the scan numbers (or also called "points across peak"?) in Skyline.
When the concentration of a target peptide is too low in a sample, usually the scan numbers of the chromatograph peak would be very small, say 3-6.
Can I exclude such peaks for quantification, meaning, ask Skyline to report intensity as N/A or 0, for peaks that have scan numbers less than a specific number, e.g. six?

Thanks a lot!
view request
Relative quantification with surrogate standards
(3 responses) clay davis 2022-05-20

I am trying to quantify different lipid classes using several isotopically labeled surrogate standards. Once the labeled lipid internal standard is chosen as "surrogate standard" and set the "Normalization Method" to the respective standard for the other lipids, the normalized area reports n/a. I’ve looked at several ratio outputs in the report option but only the “Total Area Normalized” (Molecule list – Molecules – Precursors – Precursor Results and Area Normalize (Molecule list – Molecules – Precursors – Transition Results) seems to output a value as a percent and not necessarily the ratio or ratio x IS concentration.

  • Clay
view request
Collision Energy Optimization Small Molecules
(2 responses) klemens froehlich 2022-05-18

Dear Skyline Developers,
First of all thank you very much for your continued support and an amazing software package!

We are currently starting to use Skyline for small molecule projects.
I know that Skyline can do collision energy optimizations for SRM type experiments using transition list method export as described in a tutorial.

However, we only have high resolution instruments (orbitraps) in our lab and would like to do PRM of small molecules.

Accoding to this thread:
Skyline cannot do collision energy optimization for PRM data.

Now my question is:
Wouldn't it make sense to optimize the collision energy for small molecules even if we are using PRM? For example if I go to a publication where a compound was measured on a machine that uses a different type of fragmentation and I do not know in what range my collision energy would be optimal?
Or if only one transition is reported in a SRM experiment and I want to make sure to get the best sensitivity from my orbitrap machine for this transition?

Of course I can use more transitions in PRM but for small molecules I think that collision energy optimization is more important as you often only use one or two transitions as compared to pptide quantitation where you use more transitions.

Would it be possible to integrate PRM collision energy optimization somehow in Skyline?
Maybe by shifting the transition mass also by 0.0001 instead of 0.01 as for SRM experiments?

Best, Klemens

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Integration of single masses in a DIA experiment on Thermo Scientifi Orbitrap ID-X Tribrid Mass Spectrometer
(7 responses) harald schoeny 2022-05-12

Dear Skyline Team,
I am trying to integrate a single mass (184.07332 Da) on MS2 level of a DIA experiment, in which I selected a specific precursor mass range (670-900) and measured a specific product mass range (180-190). I want to obtain a sum value for a lipid class and I have already applied this method on a Thermo QExective HF (see attached publication- AIF method).
Now I have measured the same on an ID-X instrument and the MS2 was recorded in an ion trap instead of the orbitrap.
Unfortunately, I am not able to get anything in skyline although these settings worked for the HF experiment. I have tried to set the product mass analyzer in the transition settings to QIT (but also every other option) but nothing worked.
Do you have any idea, what I can try?

Thnak you very much! All the best,

 Schoeny 2022 Anayltical Chemistry.pdf  Capture.PNG 
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Can I specify the retention time when exporting quantification results?
(6 responses) wangn2 2022-05-18
I am using skyline
I found that skyline doesn't always pick the right retention time peak for quantification, possibly due to ion interference from the background (I am using cell lysate as background). Since I have already got retention times of my peptide by running neat standards, I was wondering if I could set up skyline so that for a specific peptide, it only export the quantification result at a fixed R.T.
For example, by running neat standard sample of peptide A I found its RT to be 60.28 min, then by running a sample with peptide A in cell lysate, I would like skyline to export the quantification of peptide A only at the R.T. of 60.28 +/- 5 min, is this possible?

Thanks a lot for your help!
view request
Installation Skyline-daily
(1 response) dima kovalerchik 2022-05-17

Hi Skyline team,
I am trying to install Skyline-daily. I enter the link but it is written:

"Oops! An error has occurred.
User does not have permission to perform this operation.
Please contact this server's administrator to gain access."

How can I get the permission?
Thanks Dima

view request
Average Mass Error PPM
(4 responses) sarah.lennon 2013-11-27
Hi Brendan,

I'm currently using skyline Daily vr. to do MS1 quantification using Maxis Impact Bruker datas. In order to apply a second filter, I wanted to export the average mass error ppm. Here, it appears, that the number reporter in the .csv file is not exactly the same than the one on the top the peak indicated in the skyline file.
Do you have an explanation? Maybe I'm not exporting what I'm thinking.

Enclosed, you will find the skyline file as well as the .csv file.

Thanks for your help,


 20131028 6 bandes vr2rs 40000 br.csv  20131028 6 bandes vr2rs 40000 
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Optimization of injection or fill time in Exploris 240
(1 response) danielacgranato 2022-05-16

We are performing PRM in Exploris 240 and we would like to optimize the best injection time/fill time for each peptide, especially in the case of peptides that compete in the same scheduled window. Can this optimization be performed in Skyline? Can Skyline extract information of best injection time or acquisition time? Can Skyline help us evaluate what peptides need to improve injection time?

Thank you very much.
Best, Daniela

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DNA and mRNA oligonucleotide mapping using Skyline
Alok 2022-05-16

Hello Skyline Team,

In addition to protein/peptide therapeutics, there is a quite a lot going on with regards to DNA and mRNA therapeutic development. As far as I am aware, one can analyse a small number of DNA or mRNA using 'molecule interface' of Skyline. However, this is not possible when a long DNA (e.g. full length plasmid DNA) or mRNA need to be studied using mass spectrometry based workflows. Similar to bottom-up proteomics, the long DNA or mRNA needs to be digested by endonuclease(s) followed by either MS1 based mass fingerprinting or MS2 based sequencing analysis. I wonder if these kind of full length DNA or mRNA MS-based analysis features are available on Skyline. If not, then is there any plan to develop such features in future?


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Skyline DIA spec. library - FragPipe DDA data analysis issues
(1 response) amey shirolkar 2022-05-12

hello Team,

After running for a sufficient time our latest runs in FragPipe (DIA Skyline spec library v/s DDA raw data files) it abruptly ends mentioning MSFragger or JAVA errors how to tackle this?
Screenshots are attached.
Please help.


Amey, PhD

 DIAspeclibfile_DDAruns.JPG  FragPipe_Java_error_06.05.2022.JPG  DDA_speclib_DIA_raw_files.JPG 
view request
Bad DIA Result, is something I set was wrong?
(12 responses) jingyangzhang0222 2022-05-03

Hello Skyline Team,

I am a university student trying to reproduce some results from this paper( I have used the same setting(including Isolation Scheme, Instrument, etc.) as the paper did, but the result was not good enough to show the data's validity.

Only a few peaks show reasonable shapes, which can be observed in the official DIA tutorial. Besides, I was trying the acquire quantification data from the software, but it was not reliable, either. I have uploaded the project file(.zip) to the server, you can download it here( sharing/%40files/, is there any advice on how to adjust the settings, or to correct my operation? It would help a lot for my project if any guidance should be given.


view request
13C phenylalanine and second isotope abundance of unlabeled phenylalanine
(1 response) Sam110 2022-05-10

Hello, I have a dataset with phenylalanine and isotopically labeled 13C phenylalanine which is only one mass unit from the unlabeled phenylalanine. I wanted to know if Skyline can subtract the value for the second isotope abundance of the unlabeled phenylalanine from the 13C phenylalanine?

view request
Error message when trying to open Bruderer.bcfg on Skyline batch
(1 response) lutho 2022-05-10

Hi there,

I am trying to open the Bruderer.bcfg files in preparation for the online course currently running at the May Institute. I have installed Skyline batch and downloaded the Bruderer.bcfg file. I cannot open the file on Skyline batch and I have attached a screenshot of the error message that I am getting.

 Screenshot 2022-05-10 161435bruderer error.png 
view request
SureQuant Tutorial for Custom Method
(1 response) comtewal 2022-03-31

Dear Skyline Developers,

Would it be possible to create a generic tutorial to use when acquiring data on a Thermo instrument using a SureQuant custom method (as opposed to using the commercial kits of reagents/peptides) supplied by Thermo.

I have been trying to create a Skyline method to quantify our peptides of interest using Skyline v. but the transitions for my peptides (heavy and light versions) are importing but I am not able to visualize them.

Thank you so much,
Susana Comte-Walters
MS Redox Core Facility
Medical University of SC

view request
Support for SureQuant custom panel
(1 response) edf4n 2022-02-14

Dear Skyline,
I read in your release notes for Skyline 21.2 the announcement:
“New! Improved SureQuant support with "SureQuant" MS/MS acquisition method and method export for Exploris.
o Also added new PRM acquisition method (an improvement over the old "Targeted" method – now deprecated).”
I’m wondering if you can point me to using these features in your tutorials? I have been working my way through the skyline Targeted Method Editing tutorial. Eventually I would like to create a transition settings report for a custom SureQuant panel. I’m not sure that I am doing it correctly, especially for the export settings.
I have an Orbitrap Eclipse instrument and am using version of skyline
Thank you!

view request
Prosit score export
(1 response) nifr 2022-05-04

Dear Skyline team,

is it possible to export the dot products from matching of my spectral library with Prosit via the document grid?


 Screenshot 2022-05-04 171832.png 
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Protein group, razor peptide and issue #861
ho-tak lau 2022-05-06

Hi Skyline Team,

I have been hoping to be able to remove repeated peptides in certain ways in Skyline for a long time. Recently, I saw the discussion in issue #861, and I would like to chime in.
I wonder if Skyline can integrate ProteinProphet or to read the prot.xml from ProteinProphet, and then have an option to remove/export the repeated peptides/empty proteins. Alternatively, Percolator also does protein inference now. Percolator wiki Maybe this can be an option for MSAmanda search.


view request
Which formula to choose for combining peptides intensity into protein intensity ?
(2 responses) nicolas pierre 2022-05-05

Hello Skyline Team,

I have measured endogenous peptides with their heavy counterpart by SRM. I have also spike a protein in my sample for normalisation.
I have some questions for translated results at protein level.
When I divide the intensity of endogenous peptide by their heavy counterpart it become a ratio and in many cases this ratio totally change the ponderation of each peptide for their contribution to the protein intensity. Typically, peptides representing the highest intensity (the one which would contribute the more to protein signal with the addition of peptide intensities) for the protein can, when divided by its heavy counterpart, be the one with the lowest ratio among the other peptide of the protein. In this case this will totally change the ponderation. I am not very confident with this phenomena because usually signal with the lowest intensity are those with the lowest quality (near LOD, lowest level of precision, lowest quality of the peak...). It means that with a division by the heavy peptides I will give more importance (not systematically) to signal with the lowest quality.
In fact their is different solution to combine the signal of peptide into protein signal. For instance:

-Signal of protein A= (Signal of endogenous peptides 1/ signal of heavy peptide 1) + (Signal of endogenous peptides 2/ signal of heavy peptide 2)
-Signal of protein A = (Signal of endogenous peptides 1 + Signal of endogenous peptides 2)/(signal of heavy peptide 1 + signal of heavy peptide 2)

As explained, the first formula can change the ponderation of the peptides contributing to the protein signal, the second one will not. We could imagine other solutions...Also, I have spiked a protein for normalising my signal and I do not know where I can put it in the formula, where would be the best place..
Do you have some advises for this problem ? In there some Skyline solutions ?
Thank you in advance for your help.

Nicolas Pierre

view request
Zeno MRM Wiff 2 file
(1 response) bkr 2022-05-04


I am currently working on Zeno MRM data and I am not able to import wiff2 data into skyline.

I get the following error

"At 13:10:
Failed importing results file 'F:\20220504\GA_Zenooff_q1_open_50ms_MRMHR_01.wiff2'.
[WiffFile2Impl::ctor()] Object reference not set to an instance of an object."

Kindly help me with this.

It would be also great if you can suggest transition settings for this type of data


view request
MS converter issue - Most urgent - need help
(2 responses) amey shirolkar 2022-05-02
Hello Team Skyline,

For my firstever DIA experiment I am attempting to convert my .raw files into .mzML files so as to upload them into Skyline for analysis, but I am getting following error message for past 4 days.

Please help its very crucial for me.

- Amey Shirolkar, PhD
Enveda Biosciences, Visakhapatnam
view request
Collision Energy Optimisation on TimsTOF Bruker
(2 responses) karweens jean 2022-05-03

Dear Skyline Team,
I am using a Bruker TimsTOF for proteomic analysis in PRM-PASEF mode and I would like to use the skyline software to optimize the collision energies of the produced ions. I have created a file taking into account the different collision energies to test per ion.

The acquisition went well and I managed to import the file on skyline.

However, skyline refuses to display the full histogram for the different collision energies tested.

I would like to know if it is possible to optimize collision energies with a TimsTOF file on skyline.

Thank you very much for your help,
Kind regards,


 Collision Energy Optimisation on TimsTOF Bruker.docx 
view request
Generating mass list
(1 response) tdpatsch 2022-05-03

Hi, I am quite new in the field of proteomics and therefore my request might be rather trivial. I have a database containing hypothetical proteins in FASTA format. I would need a program that imports these FASTA sequences, does the tryptic digest in silico and provides me with an MS list that I can use as an inclusion list on a Thermo QExactive Plus to search specifically for these proteins. Somebody told me that skyline can do the job. Would be great if someone could explain how to do it or refer to the proper tutorial.

view request
ERROR: No spectra were found for the new library - since updating to v 21.2
(10 responses) Francis Beaudry 2022-04-28
Since I updated to 21.2, I have a very difficult time with Skyline. Sometimes it works, but most of the time it doesn’t work. Currently, we are trying to process DDA datasets and use the option DDA Raw to search and build the library. Attached, you will find the message we are getting 9 times out of 10 trials using the same raw files and search setup. Weird … Can you help ??
ERROR: No spectra were found for the new library.
Command-line: C:\Program Files\Skyline\BlibBuild -s -A -H -o -c 0.95 -i 07ADT_27avr_std_3 -S "C:\Users\francisb\AppData\Local\Temp\tmp8B1F.tmp" "C:\Users\francisb\Documents\FB-PROJECTS\ADT\07ADT_Validation_tests\27avr22\Skyline\07ADT 27avr std 3.redundant.blib"
Working directory: C:\Users\francisb\Documents\FB-PROJECTS\ADT\07ADT_Validation_tests\27avr22
OK More Info
System.IO.IOException: ERROR: No spectra were found for the new library.
Command-line: C:\Program Files\Skyline\BlibBuild -s -A -H -o -c 0.95 -i 07ADT_27avr_std_3 -S "C:\Users\francisb\AppData\Local\Temp\tmp8B1F.tmp" "C:\Users\francisb\Documents\FB-PROJECTS\ADT\07ADT_Validation_tests\27avr22\Skyline\07ADT 27avr std 3.redundant.blib"
Working directory: C:\Users\francisb\Documents\FB-PROJECTS\ADT\07ADT_Validation_tests\27avr22
   à pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) dans C:\proj\skyline_21_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:ligne 149
   à pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) dans C:\proj\skyline_21_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:ligne 201
   à pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) dans C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:ligne 157
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Normalizing Unknowns?
(5 responses) whale 2022-04-29


I am running a peptide quant experiment and spiked the same amount of ISTD into each unknown. . However, according to the BCA assay, a few of the unknowns had a very small amount of protein (likely due to treatment) so I was unable to inject the same amount of total protein for each sample. Is there a way to normalize these small amounts (i.e. I injected 10x less material for unknown 1 vs. unknown 2)?

Many thanks!

view request
Mass Error Plots
(2 responses) philip remes 2022-04-30

When I attempt to view the Mass Error plots for PRM QIT data, no data are displayed. I don't know if this is because mass error plots are not supported for this type of data, or the errors are larger than what the tools are anticipating, or something else. It would be useful to have this feature working for the fragment ions. Ask Lilian if you need some data or a skyline document. It would let the user know what mass tolerance is feasible. A larger request would be if you could implement Jarrett's Fine Tune algorithm to calibrate the mass scale.


view request
Import DIA peptide search
(1 response) dilip singh 2022-04-28
Dear Team,

I am using tool "Import DIA peptide search" and getting error at last step, which is mentioned below. Kindly suggest the solution for it.

OK More Info
System.ComponentModel.Win32Exception (0x80004005): The system cannot find the file specified
   at System.Diagnostics.Process.StartWithCreateProcess(ProcessStartInfo startInfo)
   at System.Diagnostics.Process.Start(ProcessStartInfo startInfo)
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 73
   at pwiz.Skyline.Model.DiaUmpireDdaConverter.Run(IProgressMonitor progressMonitor, IProgressStatus status) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\DiaUmpireDdaConverter.cs:line 162

Dilip Kumar Singh
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idotp filtering
(2 responses) lisa walrond 2022-04-26

I am trying to filter my data based on idotp threshold (0.8). It appears when I apply this filter that peptides from all samples are deleted if peptides from any of the samples do not meet the idotp threshold. As opposed to just filtering out the specific samples that do not meet the threshold and leaving the good result. I am looking for a faster way to remove peaks from peptides that aren't real. I hope that is clear.
Thank you

view request
"Error getting score type for this file" (.mzid)
(3 responses) anita liu 2022-04-27


I have been encountering errors whenever I try to build a spectral library by importing ".mzid" files generated from Byonic (Protein Metrics). The error message is as follows:

"Error getting score type for this file.
ERROR: Unknown exception
ERROR: reading file test.mzid"

This happens even when I try to import files that have previously been imported in the past (when my skyline version did not have the "score type" and "score threshold" columns). Could you please advise on how we currently import .mzid files to build spectral libraries?

Thank you!

view request
Import Sciex 7600 Zeno TOF data (wiff) - get error
(1 response) heather eastwood 2022-04-26

I am trying to import Sciex 7600 Zeno TOF data (wiff file) but am getting the attached error. Do you know how I can fix this?


view request
the error messages when importing result file into Skyline
(6 responses) mei chen8686 2022-04-07

I have two questions related to the error messages when importing PD result files. I am using Proteome Discover 2.1. The questions are in the attached file.
Thank you,

 Sklyine questions 04062022.docx 
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How to make skyline pick the peak with highest dot product?
(1 response) pavel shliaha 2022-04-22

I am not sure how skyline picks which peak corresponds to the peptide from library (I use extremely high RT tolerance) but it picks a peak which has a low dot product (0.3-0.5), but there are other candidates which have dot product as high as 0.95 (which I think is my actual peptide). Is there a way to specify skyline to select the peak with highest dot product?

NOTE: I am NOT asking about filtering on dot product score. If skyline selects a peak with dot product of 0.4, while there is a candidate which is 0.95 and I specify the threshold of 0.9 in refine > advanced > min dot product skyline simply removes the selected peak, but it does not help identify the peak with 0.95 dot product

view request
Retention Times Score to Run Regression questions
(2 responses) roman sakson 2022-04-22

Hi Skyline Team,

I am exploring the accuracy of various RT predictors wenn applied to my experimental data. Skyline is (as always) extremely useful here! Normally, I am using the residuals plot. I would like to understand the visualization a bit better and have some questions:

When applied within a document with several runs I can additionally get a score to run regression for all replicates or for the "best" one. I assume that these replicates have nothing to do with possible condition replicates annotated in my document and that Skyline just takes all present results as replicates here? Is the empirical retention time for each peptides just being averaged if I choose to plot all replicates or how are those combined? How is the "best" one chosen?

Thanks a lot,

view request
Import MS2 spectra from Waters Xevo TQ-XS (Survey Scan)
(7 responses) gioele visconti 2022-03-07


I am using a Waters Xevo triple quadrupole (TQ-XS) instrument that contains MRM and MS2 scans (Survey Scan, which is the equivalent of the full scan).
I would like to use Skyline to search for specific m/z, so created an import list with the name of the precursor, its m/z and the polarity. When importing the RAW file, I can see the preview of the imported spectra (so the import step was fine) but when i select the precursor I don't have any spectral information. By looking the details with SeeMS, the Survey Scan spectra are settled as MS level "2". How can I extract this information in Skyline? Should I modify something into the transition list?

I also tried to convert the full scan spectra usign ProteoWizard but the issue was still present (and spectra are settled as MS level "2").

Thanks in advance and best regards,


view request
Peptide Negative Ion - Transitions
(2 responses) dawn dufield 2022-04-18

Hi Guys,

Is there a way to use skyline to predict peptides in neg ion mode (precursors and transitions?)


view request
How I can retreive the TIC?
(1 response) karin yanagi 2022-04-18

I am try to retreive the total tic current over time in TOF experiement as a normalization method and take a look to see the machine siganl over time. In the result, there'is an option to select TIC column, but I dont have any numbers in the result. Is there anything did i did wrong? I attached the wiff file that I am working with.

 StdA_30um.wiff  StdA_30um.wiff.scan 
view request
Tutorial on Prosit use within Skyline?
(4 responses) wxxx 2022-03-26

I would like to be able to submit a list of proteins to Skyline and get an:

  • in silico trypsin digest
  • predicted RT of each tryptic peptide
  • predicted ms2 spectra of each peptide

What I would like to be able to do is to identify 'best' transitions from eac protein (ms1 and ms2) - i.e. those with highest intensity.

Via googling I figured out Prosit can do it. But I cannot find any tutorial which would discuss how to use it within Skyline (either by googling or searching this page). I would need a step-by-step guide - I rarely use Skyline and am not very familiar with it!
Or maybe I should do this by downloading a spectral library?

Could you please point me to any document that mentions prosit?

Thank you.

view request
How to extract result from two different product ion experiement from QTOF, same precrusor, same product, different collision energy
(2 responses) karin yanagi 2022-04-16

This is a run to look at free fatty acid and I have two collision energy set for each mass (for example 253.22 m/z for palmitiec acid, i setted CE as -25 and -15) I want to look at the product ion as same mass as the prevcursor in different collision enegy. They should have different intensity, but no matter how i define the collision energy in the transition list. It seems to lead me to same chromtagram.

 StdA_20um.wiff  StdA_20um.wiff.scan 
view request
Library Build from MaxQuant Results Error Message
(4 responses) brian mcdonagh 2022-04-13

Dear Skyline,
Having trouble building a spectral library from MaxQuant results and don't know the best way to solve it. Error message is attached but is an unexpected closing parenthesis found in sequence.......NETVVK(NEM)EK (line 84).
ERROR reading file msms.txt.

Any advice or help would be greatly appreciated.

 Skyline error in library build.PNG 
view request
Synchronized Integration - remove peak issue
(2 responses) halwaseem 2022-04-14

Firstly I have to say I LOVE the synchronized integration feature. It has reduced peak picking time tremendously!!!

I have been having this issue with all of my integrated peaks being removed when I click "remove peak" for a sample that was NOT checked under synchronized integration. I usually select all of my biological samples for synchronized integration and leave out neat standards (QC std) because I only use them for RT reference (peak areas too high in comparison to bio samples so I don't want to integrate them). Because I have explicit RT's in my transition list, the QC stds always end up getting integrated. When I click on remove peak for any QC std, this is applied to all of my samples. This happens when I have all my bio samples selected and NONE of the QC std files selected under the synchronize integration option.

I am able to get around this by unselecting all the samples, selecting the QC std files and then clicking remove peak. This only removes peaks that were selected and so I am able to selectively remove all the QC stds. I can then go back and select all my bio samples and integrate them. This is a quick thing to do with a short list of metabolites but with >100 transitions, it adds up.

Is there a way to selectively remove peaks of samples that are not checked under synchronized integration?

Notes: I am using the latest version of Skyline Daily but have had this "issue" with previous versions. I am using the molecule interface mode. This is not specific to certain raw files or transition lists. I do not have my samples grouped.

Thank you for you help!

view request
Skyline for GC-MS data
(1 response) Furuhashi Takeshi 2022-04-13

I would like to ask if there is a Sklyline software,
which can do quantification of GC-MS data (SRM, SIM and XIC)?

view request
Added capabilities for import/export of notes?
(2 responses) apickard 2022-04-13

Hi all,

Thank you again for all of the work that you're doing on this. We have started using skyline to help annotate our LC-MS methods (we're a large metabolomics core facility), and have found that adding molecule list, molecule, precursor, and transition notes and colors are extremely helpful. I'm not sure if I'm missing this, but it would be very useful if we were able to export the note color with the note so that when we get a very large (~300 compound) data table out of skyline, we could sort by note color to know what is good to go and what we need to double-check or exclude. For now, we are working around this by writing the note color at the beginning of the note, so that it exports in the notes field.

We were also wondering if it is possible to have added import functionality for notes. Right now it seems the only field we can import is "Note", which goes to the transition note, pretty far down in the tree, but we are doing a lot of high-res work, so we are typically using notes on molecules and precursors. Right now I just use the document grid to copy these notes over to those other fields so that we can see the notes easier on the UI, but sometimes we will have different notes for molecules and precursors it would be helpful to import the notes into separate fields. This again would be something great if we could also import note colors as well, but right now we go through the list anyway to check everything.

We plan on using these skyline documents for other projects, so these notes are important to alert us of things to lookout for with certain molecules in many more projects down the road.

Thanks again,

view request
Input data
(1 response) asamaras 2022-04-11


My name is Tasos and I a new user of Skyline. I have a problem with a protein list input. My goal is to insert a protein list with 854 proteins in a fasta file. The goal is to find the predicted peptides of these proteins after trypsin digest. The problem that I have is that when I insert my protein list, I can see predicted peptides only to the first 5 proteins in the list, but if you click to the other proteins you can see them. But if you just see the list you can't see them. This could be a limitation of the software in the number of the proteins?
Thank you in advance for your help.

Best regards,
Samaras Tasos

view request
Import NIST_2020_MSMS_HR
(1 response) abrahampe 2022-04-08


I am trying to import the NIST20 HR MSMS library into Skyline. The most obvious file made available by NIST to upload into Skyline is the .msp file. There are multiple collision energy MS/MS spectra per compound that I'd like to have available, but it seems that after import only one CE version is available. Please advise on how to proceed.


view request
issue with chromatogram information unavailable from DDAsearchMS1
(1 response) diletta piana 2022-04-12

Hi, I am Diletta Piana a PhD student and I did a DDAsearch to find a specific protein and transition.
I don't know why I have identified the protein and it shows me the peptide and precursor but the chromatogram information is unavailable.
Do you know what the answer to this problem is?
How can I see the chromatogram information of this peptide?
Do you suggest me to do other search ?
Looking forward to your kind reply,

Kind regards,

Diletta Piana

 chromatogram information unavailable..jpg 
view request
Spectra became dotted lines but are still marked Quanitative
(1 response) steve drake 2022-04-08

Probably a newbie issue but I can't figure it out or find it in the support section. My spectra were working wonderfully - picking peaks, adjusting peak widths, giving dotp values and retention times (Great Program). I imported a raw data file and everything went to dotted lines and added precursor ions in my transition lists. I read that the dotted lines means they are now marked as non-quantitative but I checked and they are still marked quantitative (see attached). I started a new file, loaded a single peptide and the older libraries (without the raw file) and it is still dotted with no dotp or peak picking. I tried removing the precursor ions from the list and Refine/Reintegrate but no luck. What did I do wrong and how do I fix it?


 skyline dotted lines.jpg 
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No data import when PROSIT generated library is activated
(3 responses) pavel shliaha 2022-04-08

I am trying to load in some data against a library generated in PROSIT. However I get an empty chromatogram with RT from 0-1.2min, as shown in slide 1 (my runs are 0-80 min). If I then Go to peptide settings > library and untick the library the runs load normally, as shown in slide 3, but of course dotp cannot be calculated. Any suggestions why? Note in the library the predicted RT are all over 80 min (slide 4). Could this be a problem?

view request
Cannot download or install Skyline Daily
(2 responses) mattkarasu 2022-04-07

Ive tried to install Daily here at UWMC this morning and it has thrown up this error:

Cannot download the application. The application is missing
required files. Contact application vendor for assistance.


Windows : 10.0.19044.0 (Win32NT)
Common Language Runtime : 4.0.30319.42000
System.Deployment.dll : 4.8.4270.0 built by: NET48REL1LAST_C
clr.dll : 4.8.4470.0 built by: NET48REL1LAST_C
dfdll.dll : 4.8.4270.0 built by: NET48REL1LAST_C
dfshim.dll : 10.0.19041.1 (WinBuild.160101.0800)

Deployment url :
Server : Apache
Deployment Provider url :
Application url : Files/Skyline-daily_21_2_1_455/Skyline-daily.exe.manifest
Server : Apache

Deployment Identity : Skyline-daily.application, Version=, Culture=neutral, PublicKeyToken=e4141a2a22107248, processorArchitecture=msil
Application Identity : Skyline-daily.exe, Version=, Culture=neutral, PublicKeyToken=e4141a2a22107248, processorArchitecture=msil, type=win32

* Installable application.

Below is a summary of the errors, details of these errors are listed later in the log.
* Activation of resulted in exception. Following failure messages were detected:
+ Downloading Files/Skyline-daily_21_2_1_455/BaseCommon.dll did not succeed.
+ The remote server returned an error: (404) Not Found.

No transaction error was detected.

There were no warnings during this operation.

* [4/7/2022 10:10:13 AM] : Activation of has started.
* [4/7/2022 10:10:14 AM] : Processing of deployment manifest has successfully completed.
* [4/7/2022 10:10:14 AM] : Installation of the application has started.
* [4/7/2022 10:10:14 AM] : Processing of application manifest has successfully completed.
* [4/7/2022 10:10:16 AM] : Found compatible runtime version 4.0.30319.
* [4/7/2022 10:10:16 AM] : Request of trust and detection of platform is complete.

Following errors were detected during this operation.
* [4/7/2022 10:10:24 AM] System.Deployment.Application.DeploymentDownloadException (Unknown subtype)
- Downloading Files/Skyline-daily_21_2_1_455/BaseCommon.dll did not succeed.
- Source: System.Deployment
- Stack trace:
at System.Deployment.Application.SystemNetDownloader.DownloadSingleFile(DownloadQueueItem next)
at System.Deployment.Application.SystemNetDownloader.DownloadAllFiles()
at System.Deployment.Application.FileDownloader.Download(SubscriptionState subState, X509Certificate2 clientCertificate)
at System.Deployment.Application.DownloadManager.DownloadDependencies(SubscriptionState subState, AssemblyManifest deployManifest, AssemblyManifest appManifest, Uri sourceUriBase, String targetDirectory, String group, IDownloadNotification notification, DownloadOptions options)
at System.Deployment.Application.ApplicationActivator.DownloadApplication(SubscriptionState subState, ActivationDescription actDesc, Int64 transactionId, TempDirectory& downloadTemp)
at System.Deployment.Application.ApplicationActivator.InstallApplication(SubscriptionState& subState, ActivationDescription actDesc)
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivation(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl, Uri& deploymentUri)
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivationWithRetry(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl)
--- End of stack trace from previous location where exception was thrown ---
at System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivationWithRetry(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl)
at System.Deployment.Application.ApplicationActivator.ActivateDeploymentWorker(Object state)
--- Inner Exception ---
- The remote server returned an error: (404) Not Found.
- Source: System
- Stack trace:
at System.Net.HttpWebRequest.GetResponse()
at System.Deployment.Application.SystemNetDownloader.DownloadSingleFile(DownloadQueueItem next)

No transaction information is available.

view request
Issue with importing Skyline document
(1 response) Juan C. Rojas E. 2022-04-07

Hi Skyline team,

I tried merging the results of two skyline documents by using File -> Import -> Document...

However, this is giving me duplicate replicates that I can't figure out how to remove from the "Manage Results..." window (see attached PowerPoint).

Why are the files duplicated? Any suggestions for leaving only one?

In advance, thanks!

Juan C.

view request
Small Molecules - Possibility of adding specified peak rankings or ratios between transitions within a precursor
(3 responses) Allison Haase 2022-04-05

Hi MacCoss Lab,

My lab is currently using Skyline for GCMS processing.
It would be helpful if there was some way to specify the predicted peak ranking for some of the transitions within a precursor for peak identification/picking, or if there is a way to specify the ratio between two transitions within a precursor. We are dealing with chiral molecules that have very similar ions, but the ratios between the ions differ.

I have attached my Skyline document which has my current method.


view request
signal to noise calculation
(3 responses) xue shi 2022-04-05

Hi Skyline team,
I am trying to calculate signal to noise. Does skyline have signal to noise calculation option?
I also found skyline has background area calculation.
However, I don't know how skyline calculate background area for this case. Please find attachment for the figure.
Could you please let me know which choice skyline to calculate the s/n?
Thank you,

 skyline questions.docx 
view request
Import a large # of peptides with heavy isotope labels on c-terminal residue
(1 response) wangqingok 2022-04-03

Hi Skyline Expert Team,

I am trying to import a large number of peptide sequences. After the import, I then chose the "modifications" to have a heavy K/R, and then all the K/R are highlighted in blue. But I only want to have the C-terminal K or R to be heavy labeled, but leave the internal K/R to be just light. Is there anyway to specify a modification position, not only on a certain residue but also a location of it? Such as "C terminal".

Or is there a way to allow me to modify the peptide sequence that will be imported to have a label such as [C13N15] near by the residue that I am going to specify as heavy and can skyline recognize such "C13N15" string and take is as a dedicated modification on this peptide? Thank you so much!

view request
Import Peptide Result Issue in
(3 responses) philip remes 2022-04-01

Use File - Import - Peptide Results, and choose a PDResult file. Presumably other types of peptide result would also cause an issue. The new layout here apparently allows a Score Type and Score Threshold instead of the old method where there was no Score Type, and the Score Threshold was global for all the files imported. Here there was no ability to change the Score Type or Score Threshold, so it wasn't possible to create the spectral library.

view request
Library download
(1 response) r benoni 2022-04-01

Hello everybody,
I have a question regarding the download of library for the identification of an unknown protein. I have a pulldown of protein from the membrane of human cancer stem cell (CSC), I isolated one band and analysed, after digestion, with Orbitrap with DDA method. I try to download few library from some website in internet recommended in some tutorial but were not well read from skyline when I try to do Import peptide search. Can you recommend some website or if you have some library of human proteome (membranome) suitable for the protein identification.
I hope I was clear enough.
Thanks in advance

view request
Prosit mirror dotp broken
(2 responses) Tobi 2022-03-25

Dear skyline team,

the library match with a direct prosit mirror is a great feature,
it just sadly takes all peaks on both sides and matches them blindly against each other,
incl. precurors and fragments which simply do not exist in the opposite spectrum leading to false dotp.

I cant even disable the dotp so for slides etc. you always would need to cover or hide it somehow.
The option not to display the dotp would be nice, or have it calculated after excluding peaks which are null on at least one side of the mirror.

Already pictured in Issue 831

Best wishes,

view request
Creating a Spectral Library from Existing Data Set
(1 response) madeline colley 2022-03-30


I have a series of DDA-PASEF experiments, but I am confused on how to start making a spectral library for these small molecules (some lipids and metabolites). I can see there is an "export spectral library" function, which I am assuming saves your transition list into a spectral library format.

I have used lipidcreator to give me a few transition ions for some lipids I am interested in, but when I load that, I am not sure how to then look at that with ion mobility or then add the ion mobility from this specific data set into the library.

Is there a tutorial on how to do this?


view request
Export Report Extremely Slow
(5 responses) graham delafield 2022-03-29

Hi there,

My current Skyline project includes signal extraction from a large proteomics spectral library in order to do MS1 and MS2 LFQ. The projects complete successfully, even if it takes time to chug through the library.

However, when I wish to export a report (~10-15 columns),it takes an extremely long time - sometimes 12 hours for only 50,000 rows. I have applied filters to reduce the number of rows, but there is always a significant stall when the report is 75-90% complete, regardless of the number of rows.

My questions are 1) any tips on why this may be happening? and 2) is the skyline data accessible any other way? For example, can I write some custom SQL to access file where the data is stored?

Skyline Version:

System: Windows 10 x64, i9-10940x, 64 GB RAM

Thank you!

view request
Creating spectral libraries from Proteome Discoverer result files
(1 response) johannes voshol 2022-03-25

I am trying to create spectral libraries from PD results, using Skyline's 'Import Peptide Search' functionality. When I import from the .pdResult file, the scores are not recognized and all set to 0. When I use the corresponding .msf file, the scores are imported, even though - obviously- they are exactly the same and have the same column name (PercolatorqValue). Is that intended behavior or is there something wrong?
A second question is about the SpecIDinFile parameter. This ends up being a strange looking float like -1602.53037. In all cases the number before the decimal point (here 1602) corresponds to the WorkflowID from PD, but the rest has no obvious relation to file ID or scan number as one would maybe expect. Do you have any info on how this parameter is calculated and/or if and how you could retrieve the actual scan number from it?
Thanks a lot for providing these great tools to the community!
Hans Voshol

view request
(1 response) carlos penno 2022-03-29

Dear all,

During importing transition list for small molecule work would it be possible to include a column to determine if a transition is qualitative and quantitative. not sure if that is already possible?

Best, Carlos.

view request
Fill-in Transition List Table Gone?
(4 responses) kchiti 2022-03-23


I am new to Skyline and have just been using the PRM Small Molecules mode. I usually just fill in the transition list table after going to edit>insert>transition list but after updating to the 64bit 24.2 when I go to insert my transition list the only option I have is to paste it in. Is there a way to get the table back so I can directly fill in?


view request
Generating optimization method for small molecules
(1 response) William 2022-03-28

I generated a optimization method (small molecule) for Shimadzu QQQ. I remember that Skyline recognize different CEs by varying the product ion in one digital difference. However, when I open the method I find that product ions for different CEs are all the same. How can Skyline recognize different CEs?

 LCMS-8060_CSH_Opt.lcm  LCMS-8060_GSH_CSH_Template.lcm  CSH 
view request
Error during File open
(1 response) dawn dufield 2022-03-28

We have recently seen a bunch of errors while trying to open a saved skyline file. This just started last week. Not sure what is the cause. We create and import data from Sciex and then we can't open it again..

See attached screen shot for error

 Skyline Error.png 
view request
HRMS batch analysis
(2 responses) nicholas clanton 2022-03-24

I'd like to configure the Skyline interface to quickly analyze a batch of compounds for HRMS accuracy. I'm able to get the document grid to display almost all the info I need (formula, adduct, calulated mass, ppm error) but I can't find a way to display the measured m/z value from an associated scan. Moreover, I can only display m/z values on the mass spectrum to one decimal place. Is there a way to increase the decimal places displayed on mass spectra or add a column for measured m/z in the document grid?

Thank you for your help.

view request
cannot find and use modifications present in unimod
(1 response) pavel shliaha 2022-03-24
Unimod has shTMT modification under 2015accession. I cannot find it in the structural modification list and using V(unimod: 2015)LHSGSR returns an error. Is it possible to import this modification into my skyline

Unrecognized Unimod id 2015 in modified peptide sequence V(unimod:2015)LHSGSR (amino acid 1, V(unimod:2015)).
OK More Info
System.FormatException: Unrecognized Unimod id 2015 in modified peptide sequence V(unimod:2015)LHSGSR (amino acid 1, V(unimod:2015)).
   at pwiz.Skyline.Model.ModificationMatcher.ThrowUnimodException(String seq, Int32 uniModId, Int32 indexAA, Int32 indexBracket, Int32 indexClose) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\ModificationMatcher.cs:line 396
   at pwiz.Skyline.Model.ModificationMatcher.SimplifyUnimodSequence(String seq) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\ModificationMatcher.cs:line 364
   at pwiz.Skyline.Model.ModificationMatcher.IsMatch(Target target, PeptideDocNode nodePep, TransitionGroupDocNode& nodeGroup) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\ModificationMatcher.cs:line 454
   at pwiz.Skyline.Model.AbstractModificationMatcher.CreateDocNodeFromSettings(Target target, Peptide peptide, SrmSettingsDiff diff, TransitionGroupDocNode& nodeGroupMatched) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\AbstractModificationMatcher.cs:line 644
   at pwiz.Skyline.Model.ModificationMatcher.MoveNextSequence() in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\ModificationMatcher.cs:line 78
   at pwiz.Skyline.Model.AbstractModificationMatcher.InitMatcherSettings(SrmSettings settings, MappedList`2 defSetStatic, MappedList`2 defSetHeavy) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\AbstractModificationMatcher.cs:line 81
   at pwiz.Skyline.Model.ModificationMatcher.CreateMatches(SrmSettings settings, IEnumerable`1 sequences, MappedList`2 defSetStatic, MappedList`2 defSetHeavy, IProgressMonitor progressMonitor, IProgressStatus status) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\ModificationMatcher.cs:line 61
   at pwiz.Skyline.SkylineWindow.ImportFasta(TextReader reader, Int64 lineCount, Boolean peptideList, String description, ImportFastaInfo importInfo) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1629
view request
Questions about the spectral library build by DIA-NN speclib file.
(6 responses) andyzcq 2022-03-20

I have successfully built the spectral library based on DIA-NN speclib file, and found the peptides with N-terminal acetylation can not be recognized by Skyline.

See attachment.

view request
Any algorithm differences regarding the extraction of SWATH and Scanning SWATH data by skyline.
(3 responses) Winnie 2022-03-15

Hi Skyline team,

Recently, I have extracted targeted peptide precursors from SWATH and Scanning SWATH files by Skyline. I am wondering if there are any algorithm differences about data extraction from SWATH and Scanning SWATH by skyline.

Best regards,


view request
How could I see the mass spectrometry in the right window?
(2 responses) 1240470751 2022-03-19

Dear Skyline Team,
I'm a new user for this sophisticated software. The question I want to inquiry is that I was puzzled on how to see the Ion pair information and mass spectrometry of every peptide to theoretically select the best one for the MRM after I input the protein sequence and acquire a series of selected peptides as well as their ion information.

view request
Prosit and propietary data
(1 response) Niels van den Broek 2022-03-16

We would like to use the Prosit predictor for our proteins, but have some proprietary data.
When we would use this tool, will it share/store our data on a server, or is it safe to use?

Thank you

view request
Transition List Not Working In New Version
(14 responses) kaitlyn b donohoo 2022-02-22


I've been using the same transition list in skyline for a while but when I downloaded the new version the list keeps coming up with an error in line 634, column 3. I'm not sure what to do about this, and have attached the transition list below.

 Transition List.xlsx 
view request
Chromatogram not loading
(10 responses) prajita 2022-03-17

I am trying to do some MS1 filtering to determine my precursor ions. I imported all FASTA sequence from the proteins. I also imported the result which is my raw file. The software is saying TIC not available. I have done this in the past and it has worked. Can you please help me with this?

view request
How to export figure legends and Skyline transitions' color codes
(2 responses) wangqingok 2022-03-16

Dear Skyline Team,

We are in the process of analyzing about 2 thousand peptides, and we exported the picture by just Right Click on the image and then choose "Save Image As...". and when we were checking the images, we have 2000+ of them, today and we found that the image we saved is an ".emf" file and THE FIGURE LEGEND WAS NOT ON EACH PICTURE. This is surprising. When we do just copy and paste the Metafile on each picture, the figure legend is preserved into the picture.

So rather than redo everything, I want to ask about is there a way to export just Figure Legends for all the peptides and fragmented ions from a skyline file in a batch process way? And how does skyline decide which color is assigned to which ion? We have a lot of pictures like these, and we already know which peptide sequence each of such picture is representing, but we do not know the transitions and ions without keeping the figure legend. Please help. We have 2k + pictures and sequences like the one attached.

view request
Add support for DIA-NN
(2 responses) Yishai 2022-03-14

Hi, is it possible for Skyline to support importing search results from DIA-NN? Particularly for DIA-PASEF.


view request
Chromatogram information unavailable - Metabolomics
(7 responses) erin weisenhorn 2022-02-09


I am using Skyline daily (version to analyze PRM data collected on a Waters Q-Tof. I am starting to develop a PRM method for metabolites and exported an isolation list using Skyline. When I import results per the 'Small Molecule Targets' tutorial I am not able to view any of my chromatograms. I am also a new user to Skyline.

Thanks for your help!

view request
CE optimization transition list export does not work
(2 responses) bkr 2022-03-13

Hi Skyline team,

I tried exporting the CE optimization list and it does not export the optimization list. the transition list contains only one CE.

I am attaching the skyline daily file 
view request
SureQuant Transition Settings
(1 response) romeally 2022-03-11

Has anyone used the SureQuant transition settings to export method settings in a custom assay? I'm curious how this works since I have always used the export spreadsheets provided by Thermo for this. I assume for import this allows the template not to have to use DIA settings as Thermo had recommended, but does it work for exporting with thresholds and calculate the isolation offsets? Just curious how this works in the transition settings.
Thanks! Bob

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Difference in intensity between labelled peak and just its precursors
(3 responses) sa825 2022-03-12

I have detected a light and heavy peptide in skyline using its precursor ions.
However, I noticed that when I look at both the light and heavy peptide at the same time in Skyline (Attachment A) the heavy peak intensity is ~36 but when I look at just the heavy precursor ions (Attachment B), the heavy peak intensity is ~17.
Can you please explain why this is the case?

 A.png  B.png 
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Small molecule (non-global) standard concentrations
(7 responses) jrenders 2020-09-01

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

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SureQuant Integration Errors
(1 response) romeally 2022-03-10

I am using SureQuant and I sometimes get errors integrating the peaks with a line being drawn across the entire chromatogram. It seems like it accidentally triggers early and uses that point sometimes 10s of minutes early and extrapolates to the actual peak. I am attaching an image of the full chromatogram. It seems like if the software had a feature that would only integrate if there are at least 3 consecutive scans or something like that this first erroneous point would not be incorporated. Has anyone encountered this error and if so is there a workaround? Thank you for your help!

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ion mobility issue in Bruker prm-PASEF method setup
(6 responses) ruoyu wu 2022-02-10

Dear Support team,

I have two problems when I establish a prm-PASEF method in Skyline.
When I finish targeted peptides selection and export the method, it would show that some targets our of my DDA-PASEF method's ion mobility range.
However, the corresponding target's ion mobility is suitable in the method's ion mobility range. The error seems to be occurred from resolving power's setting would give the target a mobility range (defined ion mobility± the specified number) (for example, a target's ion mobility is 0.9223 and my DDA-PASEF template ion mobility range is 0.75-1.3 with ion mobility resolving power is 60 in Skyline; when I export the method, it would show an error that ion mobility range is not within the limit). All I could do is delete the correlative target in my list but I'm wondering that do you have other suggested solution for this problem because the target (ion mobility 0.9223) I've deleted is in the range (0.75-1.3) actually.

Another question is Skyline would request to add the retention time predictor when export method (the Skyline version I've used is but I don't have the retention time predictor. Could you please provide the retention time predictor or other suggestion that I can avoid this problem?

Thanks a lot!

Best Regards,

 prm error.PNG 
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Problems importing Bruker MRMS data
(2 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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"chromatogram information unavailable" after importing .wiff file
(3 responses) heather eastwood 2022-03-08

I created a transition list and am now trying to import the Sciex data (.wiff) back into Skyline. When I view the data on Sciex OS, it looks OK. But when I import the data into Skyline, I just get the "chromatogram information unavailable" screen (screenshot attached). I also attached the .wiff and .wiff.scan files, as well as the Transition file.

 Transition list Set  Set 1_5 peptides_10 fmol_uL w 5uL inj_sch RT_best MRMs.wiff  Set 1_5 peptides_10 fmol_uL w 5uL inj_sch RT_best MRMs.wiff.scan  Screenshot of Data Import_Chromatogram information unavailable.JPG 
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Decoys not showing up with dotp values from Prosit library
(6 responses) cm748 2022-03-07

I've been trying to create an mProphet peak scoring model to pick my peaks but I'm running into a bit of a problem including the library intensity dot product field... I have generated my document with decoys (with random mass shift) and acquired data for the for the targets and decoys (with RepLiCal iRT std for RT prediction), then built and implemented a Prosit library to get the dotp values. However, when I implement the library the decoys do not have a dotp value (the targets do), even though in the view library match window prosit is predicting spectra for them.
I have included a screenshot, where you can see in the targets window for the peptide LHIS[...] that there is a dotp for the target but not for the decoy, even though prosit is showing a prediction for the decoy in the library match window.
Any ideas what could be causing this?

Colleen Maxwell

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Failure to upgrade from 21.1 to 21.2 or re-install from newly downloaded Setup.exe
(2 responses) gustavo palacios 2022-03-07

Dear Sirs, I need help to troubleshooting my inability to update one PC to the latest Skyline version. I also tried to re-install it using the downloaded setup file, but this operation also fails. I made screenshots of the error messages from numerous failures to upgrade and failures to re-install attempts. I have also included an screenshot of the system information where these issues keep happening. Of note: I also have Skyline in another PC, with much less memory RAM and processor power, and in that one the latest update did happen with no inconvenient.

I hope the attached information is sufficient to help me figure out how to update to the latest Skyline version my PC workstation.

With my kind regards,


 Skyline-UpgradeFail.JPG  Skyline-SetupFail.JPG  SysInfo.JPG 
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A protein sequence cannot contain the character 'z' at line 230
(2 responses) nicrdo 2022-03-07


I am new in the Skyline heaven and I am trying to set up a PRM method.

Unfortunately, in one of the first step i can not load the proteome of Pseudomonas putida.

The error message is the following.

Is there any way to solve this?


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problem with DDA search tutorial
(4 responses) zahra mashhadi 2022-03-02


I’m very interested in using skyline but also new to it. As I went to follow the steps in DDA tutorial, I got an error that it can’t find spectra for the new library (screen shot’s been attached here). No idea how to fix it. I really appreciate your help.

Thank you very much,

 DDA Error.docx 
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Measure Peak USP/EP resolution, tailing factor, Theoretical Plates
(1 response) zhuhongbin321 2022-03-01

Is it possible to add features like to read USP/EP resolution, tailing factor, Theoretical Plates which are very import for small molecular testing.

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A fixed and variable modification on the same amino acid
(18 responses) laura declerck 2016-07-13

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

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

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

Kind regards,
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Peak ranking interpretation.
(1 response) wangqingok 2022-03-01

Dear Skyline Friends,

I have a question about how to understand the ranks of each transition cames with the analysis. For example, in the attached picture, the highlighted ion y2 : 130.466++ has an intensity of the 3rd strongest among all other fragmented ions. How come that it is ranked only "11" by the software. What are these ranking numbers in [ ] based on?

Thank you so much for your great help!

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Batch import separate negative and positive polarity files into same replicate
(2 responses) matthias schittmayer 2022-03-01

Dear Skyline team,

I am looking for a way to automatically import two separate measurements of the same lipidomics sample (positive and negative polarity acquired on a Bruker timsTOF) into the same replicate in Skyline. I can do so by e.g. importing all positive results and then adding one by one negative polarity files using "Add files to an existing replicate" or using the "Add one new replicate" and selecting the two matching positive and negative data files, but I have to do this manually for each sample which would get quite laborious with larger sample sets. Is there a way of organizing the data or script importing it so positive and negative files are automatically added to the same replicate?


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Skyline Can Not Update on Win11
(1 response) wangqingok 2022-02-28

Dear Skyline Team,

I tried to open some of my skyline files created under 21.2 on my Win11 system where only the 21.1 is installed. However the update can not proceed with the attached error message. Could you please help? Thank you so much!

 skyline error.png 
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Trouble importing LCMS .d files
(1 response) asaha76 2022-02-28

Hi Skyline team,
I am not able to import two of my LCMS files while one which was done as a control with glycans from a kit are working. I have uploaded the three files as a zip folder, as well as a screenshot of the two files which aren't uploading. My operating system is Windows and I'm using Skyline  importfailure.PNG 
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Trouble importing LCMS .d files
asaha76 2022-02-28

Hi Skyline team,
I am not able to import two of my LCMS files while one which was done as a control with glycans from a kit are working. I have uploaded the three files as a zip folder, as well as a screenshot of the two files which aren't uploading. My operating system is Windows and I'm using Skyline

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Modifications for this peptide do not match current document settings.
(6 responses) licky lck 2022-02-28

Hi Skyline team,

I am having a hard time when I am trying to add some identified peptides from the spectral library explorer to my peptide list since some of my included modifications were somehow could not be interpreted by Skyline. I am quite certain that I have added these modifications manually (in the peptide setting->modifications) and their masses actually matched the ones that Skyline could not be interpreted. I selected all modifications to be variable, and I tried to play with other settings, such as max m/z, max peptide length and etc. However, the problem still remained. So I think I really need your help, thanks. :)


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Can't upload SRM data into skyline anymore
(1 response) boycea 2022-02-25


I haven't used targeted SRM in a few months, so when I tried uploading some raw data acquired from Thermo TSQ Altis (SRM) to Skyline, I noticed the only options for Transitions Settings>Full Scan>MS/MS filtering are DDA, DIA, PRM, and SureQuant. I opened a Skyline document from the last time I used SRM data, and the option changed to "Targeted (obsolete)." How do I upload targeted SRM data into skyline now? Thanks


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Sciex 7500 CE/DP optimization, which equation to use?
(1 response) Erik 2022-02-24

Hi Skyline team,

I would like to perform some CE (and try some DP) optimizations on phosphopeptides on a Sciex7500.
For the Altis there was a single option. However for Sciex I see multiple options:
For CE:

  • QTRAP 6500
  • ABI 5500 Q trap PO4

For DP:

  • Static_PO4

I'm a bit confused which equation to use in my case; a MS model that is not listed and investigating PO4 peptides. What are the different equations based on ? And which one would you recommend?
Also the CE values can change a lot depending on the equation (see attachment).


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