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.

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Showing: limited to 100 requests
Need help finding a tutorial
(2 responses) sean davidson 2019-02-21

About a year ago, I worked through a tutorial that I thought was from you guys. It was about using DDA data to compare plasma samples that had been spiked with known amounts of a few proteins. The tutorial took you through how to analyze the data and make comparisons. Trouble is that I can't seem to find that tutorial again. Am I imagining this?! Any help appreciated. Thanks.

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Add modifications to Skyline
(7 responses) francescomattia mancuso 2013-11-24

Dear Brendan,

I would like to know if there is an automatic way to add modifications that are not present in Skyline.

I saw the possibility to add one by one, but the best would be importing them directly from unimod xml, all in one step.


Many thanks for your help,

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An error occurred attempting to export
(3 responses) kpathak 2019-02-19
Hi I am trying to export peptide MRM method for Waters TQ-S and I get below error.

Please provide solution.

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

OK More Info
System.IO.IOException: ERROR: Saving the method was unsuccessful.
 ---> System.IO.IOException: ERROR: Saving the method was unsuccessful.

   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 59
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 54
   at pwiz.Skyline.Util.Extensions.UtilProcess.RunProcess(ProcessStartInfo psi, String stdin, String messagePrefix, IProgressMonitor progress, IProgressStatus& status) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Util\Extensions\UtilProcess.cs:line 44
   at pwiz.Skyline.Model.MethodExporter.ExportMethod(String exeName, List`1 argv, String fileName, String templateName, Dictionary`2 dictTranLists, IProgressMonitor progressMonitor) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Model\Export.cs:line 3817
   at pwiz.Skyline.Model.WatersMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Model\Export.cs:line 3616
   at pwiz.Skyline.Model.ExportProperties.<>c__DisplayClass138_0.<ExportWatersMethod>b__0(IProgressMonitor m) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Model\Export.cs:line 770
   at pwiz.Skyline.Util.ProgressWaitBroker.PerformWork(ILongWaitBroker broker) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Util\UtilUI.cs:line 123
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 232
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Util\Util.cs:line 1852
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 180
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 132
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 1967
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Building and using the existing spectral libraries in skyline
(9 responses) shobhabr20 2019-02-18

I am new to Skyline.

Need guidance on **building and using the existing libraries**. I have gone through the tutorials available in Skyline website. Have many queries.

My experiment is on **'Infliximab'** as a case study in **buffer, rat, and human matrices**.

1. I need to **download files from the online available libraries** i.e Peptide Atlas, GPM, NIST. But am not sure which files to be downloaded and also the file formats supported by Skyline for my experiment on 'Infliximab' Light Chain.

2. I want to build a library by making use of BiblioSpec spectral library. Again I need a input file for the same.

3. I need a FASTA file for Creating a Background Proteome File.

4. Require peptide sequence file

5. Require Protein sequence file

**Kindly respond asap.**

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Precursor intensity
(5 responses) smanda 2019-02-18

Hi Team,

I am trying to export spectral library as a report with the OpenSwath template. I have the fragment intensity figured out but I would like to get the precursor intensity as well as one of my columns, where can I find that?

Any pointers appreciated. The spectral is built from a mzid input.


view request
(1 response) jzaia 2019-02-20

Can skyline be used to quantify TMT-labeled glycans? The concept would be similar to quantification of TMT-peptdies, using the reporter ions to produce abundances for multiplexed samples. Thanks. Joe

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mProphet Modeling for MS1 XIC Analysis
(8 responses) aaron robinson 2018-09-17


I was wondering if you can do mProphet modeling for MS1 XIC analysis of DDA data in Skyline.

Is this something that you guys have tried? I don't see why it wouldn't work and it would make MS1 quant a bit better/more accurate.

I've tried it by adding decoy peptides and training a model and it seems like it works ok...have you guys tried this at all/can you give me any feedback on if it is a reasonable way of filtering MS1 quant data...It seems like it would be the preferred way of analysis for MS1 data, especially since you can set a q-value filter for group comparison analysis.


Aaron Robinson

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Problems in "Peptide Setting→Library"
(1 response) Yan_LM 2019-02-19

When I build a new library, the window pops up, leading to no spectrals were added. Before today, I have never encountered this kind of problems.

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Getting started, developing MRM's for the 1st time.
(1 response) neil dodsworth 2019-02-19

Hi All
I'm new to all this and don't have a proteomics background. I'm trying to rapidly develop MRM methods for some known proteins to start with, with a view to quantitating them in mixtures. I have some digests ready to go on. I don't need databases to search for unknowns at this point. I've pasted my protein sequence into the box, and created an in silico digest of my protein with all the tryptic peptides listed, with their resulting theoretical fragment ions, displayed below as per tutorials, home page etc.
However, ..... that's as far as I've got and attempts via Edit and Settings etc. in the toolbar to create Transition lists and MRM's have failed. I've tried editing some of the fields such as instrument type, m/z ranges etc but nothing happens. I don't generate an MS/MS spectrum or any MRM's. So what am I doing wrong and what steps do I need to take to create my methods?

many thanks


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Full-scan settings not enabled-
(5 responses) JCPrice 2015-02-04
Hi Brendan,
I am new to Skyline and am attempting to use the MS1 Full-Scan Filtering to quantify some peptides. I started the import peptide search wizard. I have successfully built the spectral library, by bringing in .bibliospec files from Protein Prospector. Then the wizard stops. I am trying to import the results to extract chromatograms, but I get the message that I have incorrect full scan settings (see attached). I did go through the transition settings before beginning and check the full scan settings were at 'none' as recommended in the tutorial.

I would appreciate any input,
 MS1 scan error.JPG 
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Unsupported score in search output file generated from Peptideshaker and several different search engine output
(6 responses) weixiandeng 2019-02-11
Hi Skyline team,

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

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

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

Can you please help me figure out the problem?


ERROR: .mzid file contains an unsupported score type

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

   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 59
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in C:\proj\skyline_4_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 171
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 137
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DAT file to XML converter
(9 responses) t j f m voermans 2019-02-14


I have done a full scan on a sample with digestion products from BSA. I am following the MS1 Full-Scan Filtering tutorial, but page 4 you need to upload a few .xml files. I only have .dat files. So I need to find a way to convert these files. However, no such converter seems to exists. Does anyone know another way around this problem?


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does skyline use MS manufacturer's proprietary centroiding algorithm ?
(6 responses) Antoine 2018-03-21

I tried to extract some Thermo QExactive data acquired in profile mode (DIA) with Skyline using the centroid mode.
It seems that skyline convert the profile data into centroid data before extraction. Is it correct?
In fact, MS data looks centroided when open with the "full scan" window.

If it is correct, does skyline use the Thermo's algorithm for centroiding the profile data?
That would be very convenient.

Thank you

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Exporting peak areas to compare multiple runs
(1 response) lparsons 2019-02-15

I'm having a hard time doing this in as I used to in previous versions of skyline; in particular I'm not getting exported tables to come out in the format I'm expecting.
For example, if I have 6 MS files (A1, A2, A3, B1, B2, B3) that were read into Skyline against a list of transitions, and I'm trying to compare the peak areas of the same peaks across the 6 samples, I'm not getting the best table formatting for this. I would like to see a table that is roughly:

But what I'm getting instead (by way of File -> Export -> Report -> Peptide Quantification, or File -> Export -> Report -> Peptide Ratio Results) looks more like

Where each peak is repeated on six consecutive lines. I would like to have each peak on one line with all of the observed areas on that line with it.

I looked at "Skyline Custom Reports" ( but it still generated a report where each peak was represented by multiple lines. In previous versions of skyline I was able to generate reports where each peak had just one line.

Any suggestions would be much appreciated.

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Do we need to add the iRT standards to each fractions when building the library?
(3 responses) zzhang9 2019-01-21

Hi there,

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


view request
Protein Molecular Weight
(1 response) ekuhn 2019-02-06

Is there a way to list (and subsequently export) protein molecular weight for the entered Uniprot protein as a column in the Skyline report form?

view request
Problem in importing assay library
(1 response) ankit1517.niper 2019-02-07

Hi Skyline Team,

I am trying to import the spectral assay library which I built using the SpectraST on DDA data. It is in .tsv format, I am trying to follow your tutorial, but when I Click "the Create button to create a pseudo spectral library for the document to store the transition intensities" I got the message source spectrum not present. Although I kept the raw files as well as the iProphet output file in the same folder.
I also want to tell that my library has only Biognosys iRT standard petide detected, so I am using the iRT standard peptide's Assay library provided by you in the tutorial material. So can I do this or it is a wrong approach? If it is wrong could you please suggest the possible way out for this.
For your information, I used CiRT peptides for RT normalization of peptides in the spectral library during the library generation step.


view request
DIA data upload and quantitative transitions
(8 responses) Tomas Vaisar 2019-02-14

I am uploading some DIA data into a document populated with proteins/peptides/transitions from another run (with spectra/chromatogram library generated from separate runs on different day/column). When I upload the new data some peptides/precursors/transitions show up as "non-quantitative" even though there is excellent match to the library spectrum (dotp 0.98, and accuracy <2ppm.
I have iRT peptides and have made a calibrator from them prior to the upload, but I am uploading whole chromatogram (not only 5 min window around predicted RT.
What controls this "non-quantitative" assignment during the upload? Any way to prevent it?


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Multiplex PRM data
(7 responses) Andrea 2019-02-11

I plan to process PRM data in skyline produced by multiplexing the endogenous and heavy labeled peptide targets. The MS2 spectra will be a mix of heavy labeled and non labeled fragment ions. Is this possible to do?, if yes, are there any special processing parameters I would need to change

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Library build error (dat file)
(5 responses) Jason Held 2019-02-14

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

More info pasted below. Error screenshot attached.


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

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

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

 Screen Shot 2019-02-14 at 9.55.01 AM.png 
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Skyline 4.2 upgrade issue
(1 response) zhangqing 2019-02-13

Dear all,

I am installing Skyline 4.2 in a windows 7 PC, but it occured an error, the error message is attached.
I also tried to upgrade from 4.1 to 4.2 in my laptop, it also occured the same error.

Best regards,

view request
Custom Reporting -Small Molecule
(5 responses) davis 2019-02-13

Hi Skyline Community!

I would like to report my small mole data in the following reporting format (see excel for an example of preferred reporting format). I have reviewed Skylines custom reporting tutorial but it is not clear if this format is achievable? Has anyone generated a report like this using Skyline or would I have to copy and paste each species into excel to achieve this reporting format?

Thank you! Sonnet Davis

 20190206_Denali_LIPID_POS transitions.xlsx 
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Accept peptide function could not match modified sequences
(1 response) schen19 2019-02-13

Hi Skyline team,

I had a problem after importing DDA peptide search data into Skyline. I was trying to focus on only a list of peptides using the "Accept peptide" function in the Edit>Refine menu. However, even if I select "match modified sequence", it doesn't seem to keep the modified sequences. Only the unmodified ones would stay.

When I try to accept the peptide that only has modified version (I can clearly see the peptide in the Skyline file), there would be an error message saying the peptide does not exist in the file.

Am I doing anything wrong or is it a bug in the software?

Thanks in advance,

view request
short request non-quantitative transitions
(2 responses) Tobi 2019-02-08

Dear Skyline team,

with the change of the M-1 chromatograms to non-quantitative, I noticed that all non-quantitative transitions appear always as unsmoothed chromatograms no matter which transformation is applied. Is there a reason that non-quantitative transitions are always displayed by untransformed chromatograms? Just from my side I would prefer having the same, selected transformation applied to all chromatograms.

Second small question: Is it possible to add the M-1 chromatogram to all peptides or to build a peptide list where all targets include showing the M-1, at least in the document grid?

Thank you very much for your support

Best regards,

view request
Version differences
smanda 2019-02-12

Hi Brendan and Team,

I have mzid from three search engine results (X!Tandem, Mascot and MSGF+) which I am importing in skyline for spectral library. I can import successfully if I use Version But when I use the same file with the updated version it throw an error "ERROR: .mzid file contains an unsupported score type".

Are there any changes the way the scores are read now?


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

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

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

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

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

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

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

 KVC-Feb-12-2019 - MS1 filtering problem.pptx 
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Tips for a long small molecule transition list?
(5 responses) lparsons 2019-02-11

I am trying to insert a long (12,500) transition list of small molecule information into Skyline ( I have four columns in the list; m/z, rt, charge, rt window (in that order). If I insert a small portion of the list at a time (say 2,000) everything is fine. However if I try to import the entire list at once, it invariably crashes. The program itself gets killed off by Windows and never reports an error (windows simply says "Skyline has stopped working", and gives me no options but to close it).

If anyone has any suggestions on how to troubleshoot this, I would really appreciate it.

thank you!

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Retention Time Window for Insert Transition List
(1 response) lparsons 2019-02-08

I am using Skyline to produce AUC values for specific ions observed in MS1-only acquisitions. Previously I would produce a two-column table (M/Z, charge - where charge is set to "1" for all lines) that I would import into Skyline via Edit -> Insert -> Transition List. This works really well though the chromatograms produced for each ion from the list goes across all time (and I presume therefore that the AUC does as well).

Therefore I wanted to try adding a time window in the interest of getting only AUC for a defined time window. I added "Explicit Retention Time" and "Explicit Retention Time Window" back to the list of columns in the Transition List. Then I imported a new list with those four columns through there (assuming that the two new columns are both in units of minutes).

This works fine, however the chromatograms that are drawn for each ion go across the full time of the MS file regardless of how wide or narrow the time window is. Does this mean that the AUC numbers are also produced for the full curve (rather than just the RT +/- the Time Window)? If so, is there something I can do so that only the window is considered?

thank you!

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jittery speak shape from SWATH data with overlapping windows
(2 responses) blattmann 2019-02-07

Dear Skyline Team,
I get jittery peak shape probably due to import of SWATH data with overlapping window (see example picture). How can I prevent this?
Thanks for your help and advice.
Best regards

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Could not open the chromatogram file.
(13 responses) Vineel 2019-02-07

Hi, I could not open the skyline chromatogram files. Showing error 3429 at column 7 for 1 file; error 10689 at column 7 for the second file and error 8379 at column 7 for the third file.
Please help.
Thank you.

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Error Line 705 at column 9
(3 responses) Kendra Adams 2019-02-06

Hi Skyline Team,
I am having an issue with a document I just created in Skyline Daily. Seemed to work fine while I had it open, and then fails after I close and try to open it again.
The error message is attached.

 Skyline Error 020619.PNG 
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Failed importing results file
(7 responses) sandberg 2019-01-22

Dear Skyline Team,

I am trying to import 4 wiff2 files into a Skyline peptide search but one of the files keep giving me the “Failed importing results file” message, picture attached (Pic1). All wiff2 files are fine when I open them in BioPharmaView so I don’t think that the wiff2 file is corrupt. It does not help to just import a FASTA file as my library and then import the wiff2 file directly.

Could you please help me to figure out what the problem is?

I also attached the problematic wiff2 file.

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Missing retention time when importing MSP file
(5 responses) bart van puyvelde 2019-02-04

Dear Skyline team,

Is it possible to include retention time in an .msp file? In Progenesis QI for proteomics, we have the possibility to export our spectral library as an .msp file (with RT in mins included), after importing mascot search results. However, when I try to add an .msp library into Skyline, RT's are missing. Is it possible for Skyline to read the RT's as well?

At last, I would like to thank you for the amazing work on the new features (Audit log, Waters lock mass correction).

Kind regards,
Bart Van Puyvelde

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Heavy labeled peptides (standard type in MSstats input)
(3 responses) danielacgranato 2019-01-31

I have exported from Skyline the excel file in MSstats input format (SET1.csv). I would like to use it for sample size calculation in MSstats, using R. My experimental design contains heavy synthetic labeled peptides as reference peptides plus iRT spiked in to the protein digest. I would like to use this data that was obtained by SRM in order to predict the number of experimental replicates for the next experiment.

Unfortunantely, our results using MSstats in Skyline and also in R indicate a large number of samples even with a low power size (80%) and a FC of 2. I am starting to believe it is because we are not indicating properly the heavy peptides and the iRT in the Syline document. How can we indicate in the document grid that the heavy peptides are our normalizers of the endogenous counterpart and that the iRT is our control for RT drifts. Should I set iRT as global standard ? How can I set the heavy peptides as normalizers in Skyline?

Thank you very much in advace. Best, Daniela

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TMT11? (131H)
(1 response) jeffrey culver 2019-02-06


I'm working with TMT-labeled peptides and have a TMT11 experiment (using the 'heavy' 131 tag), and I don't see this available as a modification. Is it available and I've just missed it somewhere? Or am I able to introduce it myself?

Thanks for your help!


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Error while exporting methods on Thermo TSQ Altis
(1 response) hober 2019-02-06

I was really happy to see that you included support for exporting methods for the Altis in the latest version of Skyline. However, I have an issue while exporting. When I try to save the method I get the following error message:

ERROR: TNG Method is not valid

I am currently running Skyline on the instrument computer, which runs Windows 7.
The system also has version of Xcalibur and I have tried the template method with both a particular dwell time and cycle time, but neither works.

Any help would be much appreciated!

Andreas Hober

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(1 response) Frederique van Holthoon 2019-02-06

I'm just starting to do a little bit more with skyline. I succeeded in importing raw data files. Everything looks pretty good, however some integrations/IDs look strange (see attached file). What is going wrong, how do I fix it?
With kind regards

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How are "second best peaks" determined during mProphet modeling?
(2 responses) a. schroeder 2019-02-02

Dear Skyline Team,

during mProphet modeling "second best peaks" can be used instead of (or in parallel to) decoy peptides.
As far as I have gotten into the subject, Reiter et al. only mention decoy peptides in their mProphet publication.

Is the use of second best peaks Skyline-specific?
And how are they determined during modeling?
Basically: What are second best peaks?

Thanks a lot for your great work!

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Report file
(2 responses) zainab noor 2018-11-15

Dear Skyline team,

I have to export the report file from skyline with the peptide and peptide intensity column. Which column should I use to extract the total intensity of each peptide in each replicate? Is it Max Height under Precursor Results?

Kind regards.

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Phosphopeptides identified from MaxQuant
(5 responses) without1102 2019-01-15

Dear MacCoss Lab,

I would like to import identified phosphopeptides search from MaxQuant to Skyline (version I put modifications.xml , mqpar.xml, and msms.txt files in the same folder. When I uploaded msms.txt to build spectral library, it showed "illegal character p found in sequence xxx". I attached the error message below. Thank you.


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Error in standard curve
(2 responses) mvillal1 2019-02-05

Hello Skyline team,

I am trying to plot the standard curve of 28 AQUA standards. I removed several transitions without signals from the PRM data that I collected (n=3 technical replicates per point in curve) but even for the best peptides (i.e. IALAG...) I keep getting "All of the external standards are missing one or more peaks - The selected replicate has missing or truncated transitions".

Can you please point me to something that I may be missing?

Attached is my skyline file.

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BlibBuild Command
(1 response) smanda 2019-02-03

Hi Brendan,

I am wondering what are the exact arguments I need to give to command line BlibBuild/BlibFilter to get exactly same results If I import using the Skyline interface the same mzid file and create a library. How do I specify the modifications in command line.


view request
Batch uploading Skyline files to PanoramaWeb
(2 responses) Gao 2019-02-01

I am just wondering whether there is a way (maybe command line tool) that I can back upload hundred of skyline files to PanoramaWeb. Thank you so much!

view request
Import notes in skyline
(3 responses) Fabian 2019-02-01

Dear Skyline team,

I have a transition list containing small molecules.
I can export this list in a way that it contain notes:
Protein Note:
Peptide Note:
Precursor Note:
Transition Note:

However I do not see a possibility to import these notes into skyline.
This is also interesting for me since some notes I made are not added in skyline.
Btw. is this possible for peptides.

Any advices are very wellcome!



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Peak picking MS1-only data
(1 response) lparsons 2019-01-31

Is there a way that I can import MS1-only data to skyline for peak picking? Previously I have used other software to do the peak picking and then taken the results from that in as a list for Skyline, is there a way that Skyline can pick peaks for me? I have ~12 files in a current data set, I would like for Skyline to pick peaks from them and then produce a consensus list of peaks and intensities across the files (functions I would normally do in OpenMS). I was looking at the "Skyline MS1 Full-Scan Filtering" tutorial though it seems to expect that I have a list of peaks ready to go to give to Skyline.

thank you

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How to include CiRT peptides
(1 response) ankit1517.niper 2019-01-30


I am trying to build a spectral library based on the DDA data, for this I am using the comet and X!Tandem as sequence database to be searched into. Then I am using PeptideProphet for the statistical validation and Mayu for FDR estimation. I am using Biognosys iRT-C18 peptide kit for the RT normalization. But the problem I am facing is that the out of 11 iRT standard peptides, only 2 are present in the output pep.xml files of Comet, none in X!Tandem. So, due to which I am not able to normalize the RT properly. So is there any way out that I can perform RT normalization?

If no, then I want to use CiRTs for the RT normalization, but I am confused from where to start, do I need to follow the same protocol as explained for SiRT (Biognosys) explained in your tutorial or is there any way of doing this in Skyline?

Thanks in advance.

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NON stable isotope internal standards for absolute quantification.
(7 responses) MP 2018-04-29
Dear Skyline team,

I have got a collection of small molecules which I am quantifying using a targeted approach. One part of those molecules has got a 13C internal standard in the sample (so all works as usual, peak rations are automatically determined). However, the other half does not have a stable isotope labelled standard, but only a chemically very close internal standard.

The question is: Can I assign the non-labelled internal standards to certain analytes, so that peak ratios are automatically calculate, just as this is done for the 12C/13C pairs? And if yes, does this work in the same transition file?

Many thanks for helping.

Best Regards,
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Importing results of MS1-only data "raw full-scan settings must be enabled"
(2 responses) lparsons 2019-01-29


I've been away from Skyline for a while (now on version, 64bit windows; not sure what version I last used before). My goal is to take in a transition list of "parent" ions (which have not been selected for fragmentation) from MS1-only data and get AUC for every ion on the list.

I started with Edit -> Insert -> Transition List, where I pasted in a two-column list of small molecules where the first column is "Precursor m/z" and the second "Precursor Charge". In my case, all the ions in the list have a charge assumed to be +1 for now. The list imports as 1,703 molecules without error.

Then I try to import my MS1 data (Thermo raw - I've tried importing as mzML and the result is the same). File -> Import -> Results; I leave the radio button to "Add single-injection replicates in files; Optimizing "none"". I select a file and a couple seconds later I get an error from "Importing Results...":
"To extract chromatograms from (file) full-scan settings must be enabled"

I tried also going to Settings -> Transition Settings -> Filter -> Small Molecules. In there I set "Ion types" to "p" (for precursor) or "f,p" (full, precursor) but neither of those settings make a difference from the default of "f". Precursor adducts is at "[M+H]", fragment adducts "[M+]".

Any suggestions on this would be appreciated. I'm not sure what I need to change to resolve the "full-scan settings must be enabled" error that is preventing my files from being imported. My raw files are MS1-only acquisitions, my goal is to get AUC for the defined list of features across a number of MS1-only files. Right now I have 6 files though any one or any combination of the six will give the same error.

thank you!

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Offline installer trying the access internet
(2 responses) CD 2019-01-16

Dear Skyline team

I am trying to install the latest version of Skyline on a PC without internet access using the offline installer. During the process, the installer tries to access the Skyline webpage and then fails. How should I overcome this?

Thank you

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Skyline analysis of LTQ XL Targetted Metabolomics
(12 responses) sawhelan 2019-01-24

Are files from Thermo LTQ XL compatible with Skyline analysis? Trying to upload targeted analyses of small molecules and it appears the data is loading but I am unable to see any peaks, RT, etc. All uploaded raw files are blank.
Thank you for any insight or directions provided.

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Absolute concentration question
(2 responses) jung165 2019-01-28

Hello Skyline team.

I have a quick question about absolute concentration.
I followed the absolute concentration procedures with Absolute Quantification.pdf&_docid=attachment%3A%2Faded0ebd-d12e-1034-84af-a631c495ef40%2FSkyline%20Absolute%20Quantification.pdf, that Skyline provided.

So, my question is that light peptides are required for absolute concentration?? Is there any other way to measure absolute concentration with only heavy labeled SIS peptides and those endogenous samples??

I appreciate for your help.

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MS1 extraction issue - varying imported results on 2 different computers/windows versions
(6 responses) thomas gossenreiter 2019-01-22

Dear Skyline-Team,

I just observed that I get different peak areas and mass deviations when importing the same data (MS1 filtering of PRTC peptides) on 2 different computers (1 running windows 7, the other one windows 10).

  • running same version on both computers: Skyline-daily 64-bit, version:
  • I have used the same settings for MS1 extraction on both computers

It seems like the peak area extracted on the windows 10 computer is the correct one, because it corresponds to the intensity observed in the raw file when using Xcalibur Qual Browser.

I attached the used settings and results of one peptide in a ppt.

It is quite irritating for me, as I have no idea where this is coming from. Did you ever observe this before or have an idea what the reason could be? Of course I can provide more information if necessary.

Best regards,

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PRM FDR control
(9 responses) jfoe 2018-12-13

Dear skyline team,

I have been experimenting with the advanced peak picking model based on mProphet.
I have an assay with heavy internal reference and I would like to have q values for each peptide.

Now due to our quite tight scheduling for the PRM acquisition I can't imagine for scoring based on second best peaks to be of use.
When generating decoys however, they get +10 precursor mass so the would need another window during acquisition.

Do you think a workflow is possible, where one uses decoy transitions with the PRM data as is?
I think FDR in PRM is an important topic and I would like to put some effort into making this work.


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Differences between Skyline and ProteinPilot microapp Results
(1 response) kosmatchev 2019-01-25

Hello Skyline team,

we have a SWATH Project with multiple DDA runs. Doing the data analysis with the ABsciex Software (ProteinPilot) we get 1000fold smaller amount of proteins as after processing the same data set with the Skyline Software. Do have an idea why threre is such a differnce?

Thank you and best regards,

Olesea Kosmatchev.

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Dotp vs Rdotp confusion
(1 response) jfoe 2019-01-25

Dear skyline team,

I have read up on dotp vs rdotp.
For some reason though, the rdotp is almost always much higher in my assays.

I have attached a picture of my issue.
The top row is for the analyte peptide.
The bottom row for the heavy reference.
The Library entries in both rows should be the same.

Why is the rdotp so much better than the dotp for the analyte?
This doesn't make sense to me because the reference seems to be basically identical to the library.

This is my understanding so far:

dotp - between product peak areas and the corresponding intensities in a library spectrum
rdotp - between analyte peak areas and reference standard peak areas (e.g. light to heavy)

What I would conclude then is that if for any given peptide and its heavy reference peptide:
Both peptides use the same library entry for the dotp comparison.
If the dotp of heavy standard to library is 1, then dotp = rdotp.

Your help would be much appreciated!

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Lipidomics and MS1 filtering
(4 responses) alejandro.cohen 2019-01-17

Hi Skyline people!

I'm setting up Skyline to analyze lipidomics samples. Running LC-MS on a QExactive, I'll be using Avanti's Splashmix of QC purposes. I've set up Skyline in MS1 Filtering mode, and successfully imported the Splashmix components and adducts. Data is so far, so good (expected adducts are there!), however, I'm not seeing the M+1 and M+2 isotope signals on each individual adduct as I typically did when I used MS1 filtering for precursors in proteomics studies. Is this correct for small molecules applications or am I doing something wrong?

Attached the .zip


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Peptide/Protein intensity
sbhosale 2019-01-25

Dear Skyline team,

Regarding the summation of intensities to determine protein and peptide values for statistical testing with Skyline:
If I compare two or more conditions at the protein level and I have several peptides per protein that all reflect the difference, although are at different intensities, can this be used to influence the statistical significance of the summed protein intensity, i.e. on account of having three measures, or is the information lost by summation (particularly when the base intensities of the different peptides differ by an order of magnitude)?

Thanks in advance,

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Filtering Dominant Charge State
(2 responses) meyermr 2015-11-25
Hi Skyline Team,

I have a peptide tree that is filtered to contain only sequences and charge states that are present in an imported spectral library. If there are multiple charge states observed for a peptide, is Skyline capable of further filtering a peptide tree by dominant charge state?

Thank you,
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Modifications XML Error
miguelramirez2020 2019-01-24


I am having an issue exporting a method file for an orbitrap fusion. I have been attempting to set up a PRM method using the Skyline Targeted MS/MS (PRM) worksheet on the website. However, when I attempt to export the method file, I select Thermo Fusion and a method file template but before the export is completed I receive a Modifications XML Error.

Could someone guide me in the right direction, please. 
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Chromatograms from SIM scans
(1 response) michael plank 2019-01-13

Hi Skyline team,

apologies in case the answer to this is somewhere obvious that I missed.
I set up a PRM method where, in addition to the targeted MS2 scans, I`m recording an MS1 full-scan and for some of the precursors also (MS1) SIM scans (not for all precursors due to cycle-time considerations).
I was wondering: will Skyline automatically extract the MS1 chromatogram from the SIM scans where available or will it use the MS1 full-scan or both? Is there a way I can set how this should be handled?

Thanks a lot,

view request
Building library for searching MS1 data using Spectrum Mill
(2 responses) carol_ball 2019-01-22

Skyline version 64bit 4.2.0
Spectrum Mill version B05.00
I created a pep.xml file of the valid peptide hits in Spectrum Mill
I then Opened Import Peptide DDA search results and put that pep.xml file in place
The software then gives the attached error - looking for a mzXML results file. If I pull the mzXML file from within the .d file Skyline will continue on but fails when it tries to use the mzXML as the result file. I can get it to work if I choose File-import result file. Why doesn't the wizard offer the .d files as the results file choice since the mzXML that I can pull out of Spectrum Mill isn't the file it actually needs?
In addition, the valid hits are all human as I used that as a filter in Spectrum Mill. When I import the database (swissprot.FASTA all species) that I used for searching instead of getting just the list of peptides that are in the pepXML I get LOTS of other peptides. It was my understanding that the pepXML file contains the sequences used to build the target list I'm not understanding how I can get many peptides in my list which are not in the pepXML?

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Major neutral loss peaks not extracted
(7 responses) michael plank 2019-01-20

Hi Skyline-team,
can you please help me with this: I performed a PRM experiment for some phosphopeptides, searched them in MaxQuant and generated a library from the MSMS.txt. I loaded phosphopeptides of interest from the library explorer into the target list. These contain positional isomers. Then I imported the data.
When I select e.g. peptide ‘K.INRTRTMSVFDNVSPFKK.T [52, 69] (missed 1)’ the library spectrum is dominated by b-ions with phosphate loss (see sc2.png attached), but the EICs are of the much less abundant y-ions (sc4.png). In ‘Transition Settings – Filter – Ion types’ ‘b’ is included (sc3.png). Is there anything I need to specify that the neutral loss is considered?
(This is also the case when I look a library spectrum of the same sample as the EICs and the RTs match, so its not simply because Im looking at the wrong peak.)
The peak areas view shows the discrepancy between library spectrum and integrated peaks (sc5.png).
The MS2 spectra in QualBrowser reflect the relative ion-intensities as in the library spectrum, apart that the library spectrum is charge deconvoluted.
On an unrelated note: When I change transition settings and then try to re-import data via ‘manage results’ I get the error in sc1.png even though the data have all been imported at this point. Am I doing anything wrong?

Thanks for your help.

 sc2.PNG  sc3.PNG  sc1.PNG  sc4.PNG  sc5.PNG 
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Incomplete MS1 XIC in case of additional ms/ms filtering
(6 responses) Fabian 2018-05-08

Dear all,

I,ve encountered an issue when trying to load MS1 and MS2 XICs into skyline.
The method is dd with an inclusion list on a Q-Exactive HF.
I would like to see the complete MS1 XIC, therefore I set "Include all matching scans" in "Transittion settings" in the "Full Scan" tab.
This works well in case no MS/MS filtering is included, however setting "MS/MS filtering" to "target" to include the ms2 informations, the first and last ms2 are used as the borders for ms1 and ms2 XICs.
This is rather unpleasant for my purpose and seems to influence ms1-based quantification as well, as one might interprete from the attached figure

In case of only 1 ms2 is present, the ms1 trace is complete, but the ms2 XIC looks pretty odd.

Many thanks in advance


 MS1XIC_DLGEEHFK_487_7325_MS1_only.PNG  MS1_and_MS2_XIC_DLGEEHFK_487_7325_MS1_only.PNG  Only_one_MS2_CCTESLVNR_569_7526.PNG 
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Quantitative measurement peptide
(4 responses) m p j van hoorn 2019-01-10

Dear Skyline,

I have a question about a quantitative measurement of a specific protein.
One of the two screenshots that is attached is giving calculated concentrations as it should (peptide:LFLEPTQ..), but the other one (peptide:GTYSTT..) isn’t giving me a calculated concentration (NaN) and a very weird Ratio to Standard (the 4 samples with the normal ratio to standard I have integrated manually). The same calibration curve is used and also the same settings for both peptides.

Do you know what there could be wrong about my settings or how I could solve this problem?

With kind regards,

Maarten van Hoorn

 Skyline no NaN.png  Skyline NaN.png 
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An error occurred trying to download ''.
(1 response) 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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CE optimize method XML parameter issue with Agilent 6495B qqq
wangqingok 2019-01-18

Dear Skyline Group Experts,

I am using Skyline to optimize a set of over 10k peptides, and would hope to do it in a high-throughput way. I started with a batch of 200 peptides.

After I exported the method with a template method and transitions of these peptides (one method per peptide), and I put them into the worklist of the 6495B qqq, and after I initiate the run, it shows in the MassHunter Acquisition Logbook as "
Error AcqEng MSQQQ_1: Failed to download parameters to the instrument. Invalid XML string. Failed to process the child elements in element [timeSegments]
Failed to process the child elements in element [timeSegment]
Failed to process the child elements in element [scanSegments]
Failed to process the child elements in element [scanSegment]
Failed to process the child elements in element [scanElements]
The Maximum number of [scanElement] elements allowed in this collection is 99".

However, when I just export the transitions as with CE optimizations and then copy-and-paste these transitions into the template method, it runs flawlessly in MassHunter on 6495B. I am wondering if the Skyline is actually not compatible with the parameters required by Agilent 6495B therefore the template method is not properly loaded and outputted through Skyline??? Thanks a lot!

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Spectral library generation
(1 response) smanda 2019-01-16

HI Brendan,

I am using mzid from peptideshaker (3 three search engines results, combined using PeptideShaker) to build a spectral library in Skyline. I export the report on openswath format. This all works fine for me.
What I am concerned is the number of transitions in the resulting library. When I create a library using Peakview, I see the number of transitions and peptides are atleast 5 times more than when I create using Skyline. Is there some setting which I need to be checking before exporting the report in Skyline?
Do you have a tutorial /steps to ONLY create a library in Skyline? The videos I could find are for analyzing the entire data in Skyline and not just library creation and export.


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Redirect issue?
(1 response) llparker7765 2019-01-17

Hi again,

I am also finding another issue on the website. When I click through to an install download page on the website, and have forgotten to sign in first so it gives me the "do you want to sign up or sign in?" message, it successfully brings me to a log in page, but then after I click "submit" on the log in, the redirect doesn't take me back to where I was ans instead ends up with this error:

Not Found
The requested URL //labkey/wiki/home/software/Skyline/page.view?name=SkylineInstall_4-2 was not found on this server.

Apache/2.2.15 (CentOS) Server at Port 443

It does log me in, so once I manually head back to the install page, I'm logged in and able to proceed. It just seems to be the redirect (maybe the URL is old?) that is the problem.

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custom modidfication
(1 response) edi goihberg 2019-01-11

we are trying to modify c terminal lysine on a peptide, the modification is unique in that it adds a permanent charge via quaternary ammonium ion
we tried creating the transition in silico using previous experimental data after adding the chemical formula at the modification setting the transition that we got did not correspond to the published experimental data probably since the double and single charge that the program creates is by adding a proton to the molecule while our peptide is already charged, is there a way to add a charge to the modification setting?
Thank you,

view request
Error with validation of installation
(1 response) llparker7765 2019-01-17


update a few minutes after posting this: the regular non-beta version of Skyline 32-bit DOES work, so it seems to be an issue with the Skyline Daily 32-bit install

I've had a version of Skyline Daily 32-bit working for at least a year on my Windows 7 (with, as far as I know, all current Windows updates) that's installed in Parallels Desktop on my Mac. I'm trying now to update my installation to the newest version of Skyline Daily (32-bit). I've tried both the online installer and the offline installer, and get the same error with each: it says it "Application validation did not succeed. Unable to continue" after installation, so the install doesn't complete. Am I missing something crucial? The detailed error message it gives is this:

Windows : 6.1.7601.65536 (Win32NT)
Common Language Runtime : 4.0.30319.42000
System.Deployment.dll : 4.7.3221.0 built by: NET472REL1LAST_C
clr.dll : 4.7.3324.0 built by: NET472REL1LAST_C
dfdll.dll : 4.7.3221.0 built by: NET472REL1LAST_C
dfshim.dll : 4.0.41209.0 (Main.041209-0000)

Deployment url :
Deployment Provider url :
Application url : Files/Skyline-daily_4_2_1_19004/Skyline-daily.exe.manifest

Deployment Identity : Skyline-daily.application, Version=, Culture=neutral, PublicKeyToken=e4141a2a22107248, processorArchitecture=x86
Application Identity : Skyline-daily.exe, Version=, Culture=neutral, PublicKeyToken=e4141a2a22107248, processorArchitecture=x86, 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:
+ Manifest XML signature is not valid.
+ No signature was present in the subject.

No transaction error was detected.

There were no warnings during this operation.

* [1/17/19 8:51:24 AM] : Activation of has started.
* [1/17/19 8:51:25 AM] : Processing of deployment manifest has successfully completed.
* [1/17/19 8:51:25 AM] : Installation of the application has started.
* [1/17/19 8:51:25 AM] : Processing of application manifest has successfully completed.
* [1/17/19 8:51:29 AM] : Found compatible runtime version 4.0.30319.
* [1/17/19 8:51:29 AM] : Request of trust and detection of platform is complete.

Following errors were detected during this operation.
* [1/17/19 8:53:01 AM] System.Deployment.Application.InvalidDeploymentException (SignatureValidation)
- Manifest XML signature is not valid.
- Source: System.Deployment
- Stack trace:
at System.Deployment.Application.Manifest.AssemblyManifest.ValidateSignature(Stream s)
at System.Deployment.Application.ComponentVerifier.VerifyStrongNameAssembly(String filePath, AssemblyManifest assemblyManifest)
at System.Deployment.Application.ComponentVerifier.StrongNameAssemblyComponent.Verify()
at System.Deployment.Application.ComponentVerifier.VerifyComponents()
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 ---
- No signature was present in the subject.

    - Source: System.Deployment
        - Stack trace:
                at System.Deployment.Internal.CodeSigning.SignedCmiManifest.Verify(CmiManifestVerifyFlags verifyFlags)
                at System.Deployment.Application.Manifest.AssemblyManifest.ValidateSignature(Stream s)

No transaction information is available.

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SWATH Identification
(2 responses) Lehnert 2019-01-10

Dear Brendan,

we have done a SWATH project with a large library consisting of thousandes of proteins and are now trying to analyze our DIA run (HCP-Project). Unfortunately Skyline identifies and quantifies almost any protein present in the library although for most peptides there is only background noise. So far I am unable to remove those false postitives since I have not found a way to define a peak detection threshold. Is there any way to do that (or what am I doing wrong) and if not what can I do to remove those false positives?

Tahnks and best regards,


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Crashed during a save and corrupted my file
(4 responses) Asger 2019-01-15

Dear MacCoss Lab,

I was using Skyline (64-bit) on a Windows 10 Pro on a small molecules data set with about 68 transitions and 200 samples.
Usually I dont encounter any problems even with bigger data sets, but Skyline have crashed a couple of times so I have adopted a habit of saving a lot, however this time Skyline crashed as I was saving and corrupted my files (Document, Chromatogram data, View and SKYL file), resulting in the error:

Failure opening
Exception has been thrown by the target of an invocation.

Is there any hope for recovering my data?


 Skyline crash.PNG  Skyline Log.txt 
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Error loading wiff files
(13 responses) Ryan Hill 2017-06-27

  After updating to the new version, I seem to have issues monitoring wiff files that are currently in progress. I have a weeks worth of samples and normally monitor tech reps in skyline throughout the day. However, when trying to open the batch file that still had samples being added to it, I now get an error:

"System.Reflection.TargetInvocationException: [read_file_header()] Unable to open file F:\Analyst Data\Projects\Hanen lab\201704\Data\201706 Humacyte.wiff (invalid permission or file locked) ---> System.Exception: [read_file_header()] Unable to open file F:\Analyst Data\Projects\Hanen lab\201704\Data\201706 Humacyte.wiff (invalid permission or file locked)
   at pwiz.CLI.msdata.ReaderList.readIds(String filename)
   at pwiz.Skyline.FileUI.ImportResultsDlg.<>c__DisplayClass8.<GetWiffSubPaths>b__4() in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\FileUI\ImportResultsDlg.cs:line 402
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 228
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Util\Util.cs:line 1935
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 179
   at pwiz.Skyline.FileUI.ImportResultsDlg.GetWiffSubPaths(String filePath) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\FileUI\ImportResultsDlg.cs:line 402"

I can get around this by using the sciex scripts to extact subset files. However, even when loading these extracted samples, there seem to be some bugs that don't go away until I either transfer everything to a different computer or restart the system.

If I stop the acquisition, the samples seem to load fine and are not corrupt despite the warning. I'm not sure if this was in line with Enrique's problem, but just thought I'd bring it to your attention.

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Building Library Issue
(7 responses) stephanie zalesak 2019-01-09

Hello, I just updated Skyline and now am having trouble loading my spectral library. I had no issues yesterday when I tried to upload the same files (before I updated Skyline). I attached a screenshot detailing the error message.

 20190109 skyline error.pptx 
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The document format version 4.2 is newer than the version 3.73 supported by Skyline (64-bit) .
(7 responses) voellmy 2018-11-12

Hi Skyline team,

Since upgrading to the latest Skyline version (I now have, I am having trouble using SkylineRunner. I get the following error:
"The document format version 4.2 is newer than the version 3.73 supported by Skyline (64-bit) .". What can I do to resolve this ? I've tried saving an older (and functioning) Skyline document with the newest version and this reproduces the error, so I'm fairly certain the version is the issue.

Thanks for your help,

view request
Spectral Library export
(3 responses) sstoychev 2019-01-11


Is it possible to export a spectral library in format compatible with Excel?

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Support for mixed acquisition modes
(4 responses) Yishai 2017-11-21
We have some data running targeted analysis using the Lumos where we run EThCD PRM in parrallent to other modes (DDA MS3). We also run other mixed modes on the Lumos but are unable to load the data into Skyline. Do you plan on supporting such modes?

view request
Unable to train peak scoring models
(2 responses) mmidha 2019-01-10

Hi Brendan,

I am processing the DIA experiment on Skyline I am unable to reintegrate and train the peak scoring model based on mprophet after the processing is complete. It seems there are not many available features scores to do that, only first two features are enabled but rest are disabled (please see attachments). Kindly request you to look into the issue.

Thank you


 Reintegration_issue_Skyline.PNG  Reintegration_issue1_Skyline.PNG 
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Building library issue
(2 responses) Vera 2019-01-09

Hello Skyline Team,

I just upgraded my skyline to the latest version but I ran into the same problem as Stephanie described. I installed Skyline daily and problem solved. I just want to report this issue and draw your attention. Hope it could be solved soon. Thank you.

Best Regards,

 Skyline building library issue.PNG 
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Skyline Metabolomics Course/ Industry Consultancy
(1 response) davis 2019-01-09


I am a Scientist working at Denali Therapeutics. Wil there be a Metabolomics Skyline Course offered or consultancy services for Skyline metabolomics training?

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Strange decission by the "apply peak to all" function in one case
Fabian 2019-01-08

Dear skyline team,

in one specific case the apply peak to all function makes really strange decission and I do not understand why?

Applied is a powerpoint showing the issue.

Kind regards


 strange apply peak to all decission.pptx 
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Multiple Peptide Area Histograms
(2 responses) dawn dufield 2019-01-04

Is there a way to be able to arrange multiple peptide histograms similar to how you can tile or stack multiple chromatograms. I find the connected dot plots that it automatically changes to when you select multiple analytes very difficult to view for many samples. it would be much better if we could compare multiple histograms and arrange graphs accordingly. Is this something that is possible or could me added?


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short question absolute peptide/protein quantification with internal heavy labeled
(2 responses) Tobi 2019-01-03

Dear all, wish you a good start into to the new year.

Is there a way to average peptide quantification results directly within skyline into protein quantification results? I can imagine this beeing troublesome in cases where peptides belong to several proteins, but when working with preselected representative peptides this might be helpful to quickly get to a complete results report. Do you have plans to implement something in this direction or do you want to stay on the peptide level?

Question on internal standard based absolute quantification.

Is it possible to do internal absolute quantification with different amounts in each replicate/file? So far I only get no or messed up quantitative results based on some regressions which I might not be able to avoid if I want the quantification result at all.

I would like to set to Replicate/Analyte Concentration to values ranging from 115-140 fmol, meaning in replicate 01_1 all heavy labeled peptides are present at an amount of 115fmol, while in replicate 04_01 the value is 140 fmol etc. All light peptides should be quantified against heavy but only within the same file.

As far as I can see I don't get the expected output for Quantification in Peptide Ratio Results. All light BSA peptides should have around 100 fmol for all replicates, while spectrin peptides should have ca. 20 fmol in replicate 02_01 and 02_02 but close to 0 fmol in other replicates.

I know this might not be the usual approach but it is for comparisons and it would be nice to know if it is technically possible or not.

Best regards,
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providing a list of modified peptides
(2 responses) pavels 2019-01-03

I understand that it is possible to paste a peptide together with its transitions (as shown in the videos and tutorials), but can I load modifications as well? E.g. if I have many peptides with different modifications, could those be introduced in a column e.g. Y3P and K4me1 to also make them modified or do I have to go through them manually one by one and introduce the desired modifications?

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quick reloading of data, upon changing peptide and transitions
(2 responses) pavels 2019-01-02

I have tried to search for this feature in the manuals and the videos, but failed so far. Lets say I have specified the list of peptides with modifications, loaded the data, but then decided to change a modification on a peptide. While this change is made the data is not reloaded and the analysis appears blank. Is there a button somewhere that simply reloads all the data with the current transitions (when changes to peptide modifications are made), or even better updates this automatically?

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mProphet missing decoys error
(2 responses) dkueltz 2019-01-02

Hi Brendan, Nat,

Using the newest version of Skyline (4.2) an error pops up from mProphet that no decoy peptides are present in the document even though they had been generated (Refine - Add decoys) and are clearly present. This behavior is different from previous Skyline versions we have used in my lab. I just updated to the latest Skyline daily and the problem is the same. A screenshot with the error message is attached. Thanks for looking into this!


 Skyline decoys error.jpg 
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mzXML import error - Expected Sorted Data - Agilent Spectrum Mill
(2 responses) carol_ball 2019-01-02

Skyline Version: 64 bit version
Spectrum Mill Version: B05.00 SP1
Win 10 OS
I used the DDA import wizard of Skyline to build a library based on the SpecMill results by importing the pepXML export files. I used the human SwissProt FASTA for both SpecMill searching and in Skyline. The library built successfully from my identified SM results.
I then imported some MSMS .d files and the target list (library) extracted the MS and MSMS signals just fine.
I then built a new project and instead used the mzXML files that SpecMill creates during extraction. It is my understanding that this should be an alternative to bringing in the .d file? That did not work and I get the following error:
Failed importing results file 'C:\Users\carhaney\Desktop\SpecMill to Skyline\P182_AJS_8uL.mzXML'.
Expected sorted data
I attach the full error message and one of the Spec Mill mzXML files.

My question is, is the mzXML file created in Spectrum Mill supposed to work in Skyline (Import—Results) for searching against the target list of identified peptides? I have already asked for support within Agilent but they referred me back to you. I'm certain I am doing something incorrectly as I am brand new to Skyline.

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Invalid Link for BlibBuild.exe, BlibFilter.exe and BlibToMs2.exe Download
(1 response) li zhiyu 2019-01-02

Happy New Year!

I tried to build a spectra library from DDA data by using BlibBuild, and BlibFilter.

On webpage: and build
There is a link to precompiled binaries for BlibBuild.exe, BlibFilter.exe and BlibToMs2.exe, which is:

I clinked the link, and got the following information:
This site can’t be reached unexpectedly closed the connection

My computer can get without problem. It seems that the original download page itself ran into some problem.

Would someone please provide another link for the download?

Thank you very much.

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Build spectral libraries using a large number of DDA files
(8 responses) benoit fatou 2018-12-04

Hi all,

I am trying to analyze DIA data and then extract peak areas for peptides/proteins of interest.
I would like to use a spectral library I generated from a large number of DDA files (about 1300 files).
When I was building it into Skyline, I received a error message saying I have reached the maximal number of spectrum files which is 500.

I am contacting you to know if it possible to increase the capacity to Skyline to analyze a larger number of DDA files for spectral library generation.

Thank you very much for your help !

Best regards,

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Error building specra library from PD 2.2 files
(1 response) leah0330 2019-01-01
When I built spectra library using .pdresult (The file size is about 52 GB) created by PD 2.2 version (see attachment Pic 2), I always got the ERROR message (see attachment Pic 1). The .pdresult file is from 12 msf files merge result. I do not know whether my file is too big to load. But when I imported all the 12 msf files or .pdresult files seperately (see attachment Pic 3), it succeeded, I do not know why.
 Pic 1.jpg  Pic 2.jpg  Pic 3.jpg 
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Shimadzu QQQ methods with a large protein can not be predicted by skyline
(2 responses) spklongz 2018-12-31

Happy new year everyone.

Recently, I use skyline to predict LC-MS/MS method of a large protein (the fasta file of this protein is attached), while it failed to export transitions if I chose precursor charges (2,3) and ion type (y, b). If I set precursor charges (2) and ion type (y, b), it success. Do you know how to solve this problem?


 tetanus .fasta  failed to export.PNG 
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pFind spectral library
(4 responses) user4380 2018-12-28


is it possible to import pFind search results into Skyline to obtain extracted ion chromatograms for identified peptides (MS1 XIC filtering)?
(the Open-pFind paper has just been published in Nature Biotechnology, volume 36, pages 1059–1061 (2018) ).

Kind regards,

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How to input the .sptxt file
(1 response) sunrui 2018-12-30

what is the format of the skyline, which column is the necessary for the skyline to recognize. I input the sptxt file, however there is a error that the sptxt file is not a valid file. if someone know the answer, please tell me.

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Error when selecting "log2 fold change"in regulation
(2 responses) sstoychev 2018-12-28


I'm getting the following error when trying to edit the regulation table by adding columns such as "log2 fold change"

Skyline version: (64-bit)
Installation ID: ed56f5a5-04ae-49bc-bfc9-021aa0ce9225
Exception type: NullReferenceException
Error message: Object reference not set to an instance of an object.

Stack trace:

at pwiz.Common.DataBinding.AbstractViewContext.CustomizeView(Control owner, ViewSpec viewSpec, ViewGroup viewPath) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\DataBinding\AbstractViewContext.cs:line 327
at pwiz.Common.DataBinding.Controls.NavBar.CustomizeView() in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\DataBinding\Controls\NavBar.cs:line 303
at System.Windows.Forms.ToolStripItem.RaiseEvent(Object key, EventArgs e)
at System.Windows.Forms.ToolStripMenuItem.OnClick(EventArgs e)
at System.Windows.Forms.ToolStripItem.HandleClick(EventArgs e)
at System.Windows.Forms.ToolStripItem.HandleMouseUp(MouseEventArgs e)
at System.Windows.Forms.ToolStrip.OnMouseUp(MouseEventArgs mea)
at System.Windows.Forms.ToolStripDropDown.OnMouseUp(MouseEventArgs mea)
at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
at System.Windows.Forms.Control.WndProc(Message& m)
at System.Windows.Forms.ToolStrip.WndProc(Message& m)
at System.Windows.Forms.ToolStripDropDown.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.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_x64\pwiz_tools\Skyline\Program.cs:line 304

 Slyline error 20181228.JPG 
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Importing out of memory
(10 responses) lihaikuo 2018-04-17
Hi, I am trying to import results of more than 200 raw MS datas on Skyline, but it will cost RAM more than 30G, especially when the last 40 or 50 raw datas are being imported. And the computational speed is pretty slow. (At the beginning of importing, I can get a .skyd file in 20 minutes, but when the 170th raw data is imported, it will take more than 5 hours.)
Our computer currently has a RAM of 32G, so it can not run well.

I am using the latest version of Skyline, and here I do not want to seperate my files into groups to import.
I want to know how other users solve such problems.

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Overlapping MS1 data
(2 responses) Tobi 2018-10-30

Dear Skyline Team,

even though i looked up the DIA tutorial and other forum posts concerning overlapping data i just dont see the right explanation to a question I have, it would be great getting some hints from you.

The question is, can skyline deal with overlapping MS1 data in a way that chromatograms are built without any overlap causing twice the amount of points across the peak?
For example tSIMs measurements will be done from 399-421, 419-441, 439-461 (overlap of 2 m/z) etc…
Can you built MS1 chromatograms as if 1 m/z (half the overlap) would have been cut away from each side of each scan? (400-420,420-440,440-460…) Or is the Isolation Scheme option limited to MS2 data?

It would be great getting in contact with you, and sorry if I missed something in the tutorials.

Best regards, tobi

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Points Across Peak
(8 responses) Yasin 2018-11-11

Dear Skyline team,

when creating a report template it is possible to add the "points across peak" - column. However, this number only counts the number of data acquisition points. Often it would be nice to filter for peaks with a certain number of points above 0 in between the integration boarders as this would save the time of manually deleting peaks with less then a specific number of (connected) points above 0. Hope that is something you would consider to implement.

Anyway, thank you in advance!


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Extracting precursor intensities from MS2 targeted scan
(2 responses) kentsisresearchgroup 2018-12-20

Dear Skyline community,

we used a Thermo Fusion Orbitrap to collect targeted MS2 of peptides, with an isolation window including two isotopologues. We are now trying to use Skyline to quantify these peptides based on their precursor intensities, extracted from these MS2 scans (the precursors are indeed detected, based on inspection of the spectra). However, for most peptides the light isotopologue is not extracted at all by Skyline, despite being in the targets list.

Can this depend on the scan header? Does the software filter spectra in any way, for example based on the nominal target mass in the header? Is there anyway to force Skyline to use all scans covering a mass range containing both isotopologues?

Thank you very much in advance for your help,

Paolo Cifani
Kentsis Research Group
Memorial Sloan Kettering Cancer Center

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Error while removing peaks and then apply to all function
(5 responses) ssharma02 2018-11-06
So while analyzing, I wanted to remove a peak and then use the apply peak to all function to remove peaks form the different samples. An error keep on showing. A screenshot of the error is attached a powerpoint file.
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How to add RT and Analytes Name to Peak labels
(3 responses) dawn dufield 2018-12-04

When we have a method with multiple peptides or small molecules and I want to look at the overall TIC for whole run I shift select the ions and then it labels them on the chromatogram. 2 questions. If the names are too long it puts a .... and truncates the name. Is there a way to expand that name so I can see the whole name. 2) how do I add the ion name and the RT to the chromatogram? When I click on just 1 ion it shows the RT, but when I select multiple ions it changes to the ion name and no RT.


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