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

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

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

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

  • A detailed description of your problem or question, including instructions for re-creating any problem that you are encountering. Screenshots are often helpful.
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  • 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
peptide count from document grid plus pivot not counted correctly
(2 responses) sas28 2019-04-18

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

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

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

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

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

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(3 responses) jmr 2019-04-18

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

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

Dear Skyline-team,

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

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

Any ideas?



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Cannot Import Top5 Data from a QExactive
(1 response) khader awwad 2019-04-18

Hi all,

I analyzed a antibody digestion with a Top5 method on QExactive. In Skyline I generated the peptide list. I wanted to import the files in skyline, but I get the error message that chromatograms cannot be loaded before full scan data are enabled.

I have attached the error message from skyline.

The questions are:
Is the import of Top5 data not possible in skyline?
Or where can I enable full scan data in skyline?

Thank you in advance for your help.



 Skyline error 2.JPG 
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System.IO.IOException: ERROR: Illegal character p found in sequence .....
(4 responses) jason a macgurn 2019-04-15


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

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

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

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

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

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Chromatogram unavailable issue
(4 responses) Bo An 2019-04-15

Hi everyone,

I encountered a "chromatogram unavailable issue" with Sciex MS2 data, I have two transitions for the targeted peptide, if I only select one transition in Skyline method, the chrom is ok for both; but if I included both transitions in the Skyline method, only one chromatogram will be displayed, the other one will not be recognized, have any body encountered this issue before?

Many thanks,

 1.PNG  2.PNG 
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Isobaric labeling in small molecules
(2 responses) Juho 2019-04-16

Dear all,

I would like to go trough results of isobarically labeled small molecules (TMT/iTRAQ etc) that were combined for smaller number of samples and measured using scheduled PRM. Now the individual reporter ions present individual samples or standards with different concentrations and I would like to set the analyte concentration based on product m/z and replicate. For the quantification I try to set concentrations from the document grid individually, but skyline prefills all same analyte concentrations of the same replicate and same precursor regardless of different product m/z.

Is there a way to fill individual product m/z of the same precursor m/z and same replicate with different concentration for quantification with Skyline or should I just export integrated peaks and do this in a spreadsheet?

Many thanks,

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Export of Scan Indexes
(2 responses) jfoe 2019-04-16

Dear Skyline Team,

I have been exporting skyline chromatograms to perform some diagnostics with scan header information.
For export of the chromatograms I use the Report Objects:
Proteins/Peptides/Precursors/Transitions/Transition Results/Chromatogram/Raw Data/...
.../Raw Times
.../Raw Intensities

When automating this with a lot of files etc, it turns out that matching diagnostic data via scan retention times can get quite messy due to loss of precision on export.
You also store a Vector of ScanIndexes with Chromatograms. Could you maybe add an option to export those?

Best regards,

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

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

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

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

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

thank you

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Unclear Peaks in Skyline for Heavy Labelled PRMs
(5 responses) sa825 2019-03-24

Operating System: Windows


I am looking at the detection of specific proteins using their peptides in heavy labelled (Oxygen 18) plasma samples on the Thermo Q exacitive. I manually generated a peptide list using Uniprot and pasted it into Skyline. Then I exported this list as an isolation list (Non-scheduled) and searched for them on the Thermo. Some of the peptides were detected from this run but at times the detection of the peptide and its heavy labelled counterpart did not match up as expected in Skyline.

To overcome this, I removed the peptides that were not detected and re-exported the Peptide list as a "Scheduled" Isolation list and searched for them in the sample on the Thermo. This produced a second set of data but this second set of data appears very rough with no clarity in the detection of the peptide or its heavy labelled counterpart.

I have attached 2 images of the detection of the same peptide in the Non-scheduled and Scheduled runs and I was hoping you could tell me why this is the case and how to overcome it?

Thank you,


 Non-scheduled.jpeg  Scheduled.jpeg 
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File text/Free text column into document grid?
(1 response) waltteri hosia 2019-04-16

Hello. Is there a way to copy paste file descriptions from instrument sample lists to document grid? In other words is there a free text column to choose somewhere in edit document grid views? I have tried to look but have not found.
We use forward numbering as file name, and actual sample description, i.e. what was injected, is written to "file text" (waters) or "sample ID" (Thermo) fields, and this info does not come into Skyline. Then with larger document grids it gets hard to follow what's what when one only has this file number-name.

BR, Waltteri

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Issue with Recognizing User Created Modification
(22 responses) dwwilk02 2016-05-27

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

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

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

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

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Unexpected Argument
(1 response) kartik kundgol 2019-04-15

Hi Brendan,

I am using SkylineRunner.exe and was exiting because of the below command,


Can you please guide me regarding this.

Please find the attachment.

And what is the major difference between SkyLineRunner and SkyLineCmd

Thanks & regards,
Kartik K

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calibration curve with spiked ISTD for multiple peptides
(8 responses) DeWi 2019-03-29

Hey there,

I'm quite new in working with skyline.
I've got an issue or I didn't get how it works with skyline, however:
I tried to create my calibration curves with spiked ISTD (5nM) of 18 pepiteds ( à 3 Transitions) simultaneously.
So, I already found the option to define the calibration points of only one peptide in the menu View-> document grid-> replicates.
And the other option to define the spiked-in concentration of the ISTD for all peptides in the documentation grid -> Peptide Quantification.
But I have not found a solution where I can definde both (and for all peptides instead of just one, because every peptide has a slightly different concentration in each calibration point).

Is this possible to do both with skyline?
Maybe you can help me.

Thank you!


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Import a precursor isotopes transition list
(10 responses) davidz 2019-04-09

Hi there,

Is there a way for me import a transition list with precursor ion, precursor isotopic ions, retention time, and ranking, here is what I have:

but I keep on getting this error:
"The product m/z 34 is out of range for the instrument settings, in the peptide sequence VSDFGLTKEASSTQDTGKLPVK. Check the instrument tab in the Transition Settings."

I take the format is a little off, but I'm using the format from an exported transition list from previous experiment.


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Shimadzu Library file not loading
(3 responses) Alan Barnes 2019-04-08

Dear Skyline Team,

I am having trouble importing a Shimadzu Library file. I have followed the following path is this correct?
Settings > Peptide Settings > Library > Edit LIst > Add > Browse

I have found mlb file format is not read. 
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NIST mps phosphopeptide library issue
(1 response) anne-christine uldry 2019-04-03

Dear Skyline developers,

We've just had a little issue with one of the NIST msp libraries, the human phosphopeptide spectral library human_hcd_labelfree_phospho_selected_passed.msp, which is deposited for instance here:

When we loaded it into Skyline via Peptide settings/Library, we noted that no phosphorylation (or any other usual modifications) was marked anywhere upon inspection of the library in View/Spectral Library, although the msp file loaded without warnings or error messages.

Upon inspection of the msp file, I noted that the format of the modifications in the phospho peptide msp was different to that, say, of hsa20140422.msp, which I found here
and which seems to exhibit modifications as expected when viewed as spectral library inside Skyline.

In the former the mods are marked thus


while in the latter they are marked as, for instance,


So I managed to solve my problem of reading the NIST phospho msp in Skyline simply by changing all the "(" of Mods into "/" and the ")" to nothing; that seems to work, in the sense that I now do see the relevant modifications in the spectral library, what I see in the file appears on the spectra, and there were no warnings or error messages while loading.

I am not familiar with the msp format, and cannot tell which of the two Mods formats I saw is the standard.

My questions to you however are

  1. Do you see any issues with my simple "sed" solution, or could they be other surprises with this (perhaps not very standard) human_hcd_labelfree_phospho_selected_passed.msp file ?

  2. Will Skyline be able in some future version to read this kind of msp format?

Many thanks in advance for your reply.

Kind regards,
Anne-Christine Uldry

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Paste of peptides with PTMs misses some peptides
(3 responses) Nathan Manes 2019-04-11

A new bug seems to have popped up in v4.2.0.19072. If a text list of peptides with PTMs is copy-pasted into Skyline such as:
IADPEHDHTGFLTEY[Phospho (Y)]VATR[Label:13C(6)15N(4) (R)]
then only the first peptide is in the resulting list of peptide targets. If the second peptide is pasted again, it appears. Note that Edit ---> Insert ---> Peptides works correctly.

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Change Peak Integration Parameters
viragsagikiss 2019-04-12

Apologies if this question has been answered before and i missed finding it.

Using the small molecule CE optimization approach (skyline-daily version). The analyte peak wasn't picked up automatically in some of the repeats so i wanted to manually adjust integration parameters.

I receive an error message "failure attempting to modify the document, Duplicate or out of order peak in transition Ion [xxx]". As seen on attached screen.

Do you have more information what this error refers for? I have run the CE optimization method with a transition list that i manually transformed from the one i exported from Skyline (on a SCIEX QTRAP, the one i exported would give me errors, but i used the same CE values). Import the results files as you detail in the tutorial, so it would create a new file CE optimization. And the CE steps appear with different colours. Here no 5 different method file, only 1, but i injected 5 times to test repeats.

Thank you and let me know if you need more information.


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Difficulties creating a spectral library
(3 responses) t j f m voermans 2019-04-08


I have been trying to create a spectral library for several days now, but whatever I do I can't seem to get things right. What I did was, I downloaded a SpectraST file for Human Serum from Peptide Atlas (see picture: PeptideAtlas) and for Human from NIST (see picture: NIST). I have added the files I got as well for clarification.

I am sure I forget something obvious, but I can't seem to find it.


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Dilution factor per replicate
(1 response) waltteri hosia 2019-04-10

Hello the awesome Skyline team!

I am doing some impurity quantification, peptides. If I have made a calibration curve from pure target protein digest with ISTD concentration of, 400 µg/ml, and injected 10µl, and I have measured unknown series, but for few technical reason I had ISTD concentration of 100 µg/ml and injection volume of 3µl, then after merging files to same skyline document, how can I get quantification's of the unknown series? When trying to apply concentration multiplier in document grid, it applies automatically to all peptides with same sequence, i.e. also to calibration curve injections. Seems not be possible to apply it to unknown samples only?

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Modifications for this peptide do not match current document settings
(6 responses) michael plank 2019-04-05


I built a library in Skyline from a .pep.xml I exported from Proteome Discoverer 2.0 using SequestHT as search engine. (The MS-run was on a standard phospho-peptide and I exported only a single PSM. I also exported the .mzML from the same PSM).
The library build succeeded, but when I go to View - Spectral libraries I get 'Modifications for this peptide do not match current document settings.' and it is complaining about a S[+79.9799] modification. (Due to this problem I cannot add the library spectrum to the document.)

The monoisotopic mass for phosphorylation is 79.966331 (79.9799 is the average mass). I checked that this is configured correctly in PD.
The .pep.xml has a line:
2. Dynamic Modification: (...) DeltaMass="79.96633" DeltaAverageMass="79.97990"

(This is on Skyline 64-bit

Can you please help me to get around this problem?


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Small Molecule Untargeted Exploration of MS2
(1 response) bwidner 2019-04-09


I am new to Skyline and interested in using it for small molecules for targeted and untargeted analysis. Is there a way to click on a MS1 scan and see the MS2s that were collected for that scan? I looked through the tutorials, and I couldn't find this. I only found mention of MS2s for targeted analysis.


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List of peaks with the highest dotp value under the Find Results window
(4 responses) wen ding 2019-04-08

Hi Brian,
I would like to have 550. 2928++ (dotp 0.91) listed in the Find Results window instead of 550.2928 (dotp0.52) (please see attachments). I don't understand why Skyline highlighted the peak 550.2928++(dotp 0.52) at 17.3 min instead of the dopt 0.91 peak at 27.6 min.
How do I make Skyline highlight the peak with the highest dopt peak and have that peak listed in the Find Results window?

Thanks in advance!


 dotp question.rtf 
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Any way of overlapping chromatogram base peak with XIC of peptides in "targets" list?
Edu Zar 2019-04-08


Is there any way to overlap the MS1 XIC of selected peptides with the chromatogram base peak?
Beeing able to compare te XIC of selected peptides with the base peak would be very useful in order to quickly check if the peptides of a specific protein are the most intense peptides in the chromatogram.



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Fragment ion XIC intensity
(1 response) stephen swatkoski 2019-04-05


Thank you for the great support you provide. Your responses to my previous posts have been very helpful. I hope this is the last question I have for awhile. I am analyzing PRM data and noticed that when I click on a point along a fragment ion XIC the intensity of the fragment is different than the intensity of the fragment ion in the raw ms/ms data. This is observed consistently across multiple peptides and fragments of those peptides. The XIC intensity at a given point along the curve always appears to be higher that the fragment ion displayed in the ms/ms spectrum. Can you explain why this is the case? Please see attached example. The intensity at the apex of the XIC for y10 is about 2.7e6. However, the intensity of y10 in the raw ms/ms spectrum at that same time point is lower (7.2e5).

Thank you,

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Custom SSL format not correctly defined
(3 responses) Mike S 2019-04-04


Just a heads up that the custom ssl format described here: input and output file formats is not followed, at least not in Skyline 64-bit build

For instance, that page says the header should be "file" and BlibBuilder says it needs to be "filename". Also, that page says "retention-time" is optional, but BlibBuilder says the column "RT" is required.


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MSX data processing is prohibitively slow
(2 responses) Tim McCubbin 2019-03-31

Dear Skyline team,

We are presently attempting to assess the benefits of using multiplexing to assist in the label free quantification of complex samples, trialing this on a HeLa protein standard. The tutorial on how to do this was clear and simple to follow and we have acquired our MSX scans. However, the import of these result files into Skyline is incredibly slow. While we expected it to be slow given the tutorial said upfront that one user reported a 20 hour import time for a 200 MB file several years ago, a single result file is importing at a rate of ~2% per day (our files are just shy of 200 MB each). Because we expected it to be slow, we used slightly less windows than in the tutorial for our trial (4 windows of width 6m/z). Data is from a Q-Exactive HF-X and our library size is quite large as we are doing untargeted proteomics, so roughly 55,000 peptides and 680,000 transitions. We are currently running version of Skyline on a Windows 10 machine with 64GB RAM and a 6 core hyper-threaded Xeon processor. I thought our computer was of reasonable specs so was wondering if you had a sense of why the import is slow; are we simply not meant to be doing untargeted proteomics with MSX or is it likely we have messed up a setting? Please let me know if you need any files or more information.

Many thanks,


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Unsupported score in search output file generated from Peptideshaker and several different search engine output
(9 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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FIA of 3500 small molecules and scheduled method
(3 responses) a.petretto 2019-03-31

Dear developers
I have to inject in my QE 3500 compounds in FIA mode to collect the parent (MS1) and daughter ions (MS2). The final goal is to build a metabolic library. Now the best way to develop the project is to have 3500 different methods each of them have a dedicated compound, that is very hard to make manually.
I'm wondering if it is possible using skyline to export a scheduled method giving a template file with the same condition for HPLC e QE with the exception of monitored m/z for the different compounds.
Can anyone help me to avoid me to do manually :)
all the best

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library match spectrum
(1 response) stephen swatkoski 2019-04-02


When analyzing PRM data, is there a way to determine which replicate the library match spectrum came from? Also, is there a way to determine the exact scan number of the library match?


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How to assign report template name in Command-line argument
(4 responses) guoguodigital 2019-03-21

Hi There:
I'm trying to use Command-line interface to export a specified report:

C:\skylinedaily\SkylineCmd.exe --in=D:\ --report-name=Main\Name_of_report --report-file=D:\Name_of_report.csv --report-format=CSV --report-conflict-resolution=overwrite

But it always returned wit this error:
Error: The report M does not exist. If it has spaces in its name, use "double quotes" around the entire list of command parameters.

It seems like the name of report is in correct. But how Can I tell the software to use a specific report template in a specific folder?


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Thermo Altis method
(1 response) avizrosenberg 2019-04-03

When I try to export a method for a TSQ Alatis I get the following error.

System.IO.IOException: ERROR: Registry key (Software\Thermo Instruments\TNG\TSQAltis) not found. TSQAltis is not installed on this machine.

Any thoughts?

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Planned Functionality for Skyline Runner?
(4 responses) hogan 2019-04-02


First let me say thank you for all the work you do to keep this awesome software platform up and running and constantly making improvements!

I am not aware of a way to use skyline runner to upload a template file (.csv table) into the Document Grid for assigning analyte concentration and sample type to the imported replicates. So far I have been doing that manually every time I pull in a new batch of files from my run sequence (AbSciEx 4000 qTrap w/Analyst 1.6.2). I am attempting to set up a small molecule quantitative analysis workflow in skyline and was hoping to be able to automate most of the data processing using skyline runner.

Is this a planned functionality in the future?


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Precursor Ions
(5 responses) Richard Lam 2019-03-28


I was trying to use Skyline to predict the fragments ion of a known peptide.
I used the 'Import Peptide List' function and with the Transition Settings as shown in the attachment.
After the MRMs were generated in Skyline, I exported the MRM parameters as .csv by clicking 'File', 'Export', 'Report', then choose 'Peptide Transition List'.
Please see attachment for the report output.
The peptide is LCVLHEK, its neutral precursor is 822.4 and I expect the 2+ ion is at 412.2.
However, in the report, the peptide is shown as 'LC[+57]VLHEK' and the 2+ precursor ion is at 449.74.
Do you know why? Did I do something wrong in the settings?



 BSA Peptide Transition List_1.csv  Transition Settings.docx 
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CE Optimization Observation
(3 responses) mburgess 2018-01-31
I am using version of Skyline on a Windows 7-run PC operating a Waters Xevo TQ-S. I have utilized the CE Opt tutorial in the past and generated a series of methods with +/- 5 steps around a calculated collision energy value. The output methods from Skyline produce the proper number of transitions in the MassLynx MRM method and the data is collected normally. However, when I input the data from the runs into Skyline, I am only observing +/- 3 steps currently. Not sure if anyone else is seeing this. Am I missing a setting somewhere?
Many thanks in advance,
Mike B
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Purple rectangle on graphs
(2 responses) ecanessa 2019-04-01

I am trying to use the graphs for skyline as images in a publication but a large purple rectangle appears when looking at a peptide as seen below. It does not appear for all peptides. Is there anyway to eliminate this?

Also is there anyway to keep the y-axis stable so one graph isn't showing an intensity of 0 - 250 10^3 while another has an intensity of 0- 80 10^3?

Emily Canessa

 Purple rectangle.png 
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MS3 spectrum reading by skyline
(1 response) maotian zhou 2019-04-01

Dear Skyline team,

I am trying to use skyline to read MS3 spectrum. Seems skyline can extract the MS2 spectrum through the Raw file easily. However seems there is no way to setup the MS3 level product ions, and extract the MS3 spectrum. Could you please let me know if there is a method to analysis the MS3 spectrum like what is doing on MS1 or MS2 level (can calculate area under curve).


view request
Export MRM method into Sciex 5500 QTrap
(4 responses) Richard Lam 2019-03-27

I generated the MRMs from peptides using Skyline and experienced an error when trying to export the MRM parameters into a method for Analyst (Sciex 5500 QTrap) - see attachment for the error message.


  1. I am using Analyst 1.6.3
  2. Skyline (64-bit)

Any solution to resolve the Export to Method issue?

Best regards,


 Export Method Error.docx 
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How to create a file with default settings and how to see the precises signal intensity?
(3 responses) t j f m voermans 2019-03-27


I was wondering if there is a way to create a new file with default settings, like when you created your first file after installing Skyline. I am asking because I get different signal intensities for a peptide between two files while the same results where imported. So I must have done something with the settings that changed the signal intensity. I tried to create a new file, but I still didn't get the same signal intensity as the first file I created.

Also, is there a way to see the exact signal intensity? Above the peak, I only see the retention time and the ppm difference. I tried various options provided by the right mouse button, but nothing seems to give me some numbers.



 Skyline signal intensity.JPG 
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Issue with AutoQC upload of wiff files
(1 response) katherine glenn 2019-03-21

I have run into a problem with the AutoQC upload. I am uploading a lot of data that has been acquired over a period of time, and I think it may not be working because I tried to upload in the wrong order of acquisition. Throughout the log file it says that the file it is trying to upload was acquired before the acquisition date of the last uploaded file and is skipping.
However I tried to create new folders on Panorama, which are now all empty, but it is still skipping files. I tried removing the “AcquisitionTimes.csv” and log files but this didn’t work either. Are you able to identify the problem here? Is there an easier way to upload these files together?

view request
Endogenous peptide overlaps with minor peak of heavy
jjotto 2019-03-26


I am using a heavy-labeled peptide (13C(6)15N(2) on Lysine). The neat heavy-labeled peptide shows multiple peaks all within a few minutes of each other with one major peak. I believe some of the peaks after the main peak are the result of deamidations. (the peptide is not ideal because of these modifications, but it has been selected for other reasons) There are also a few minor peaks before the major peak. When spiking the heavy-labeled peptide into real samples, the endogenous peptide seems to consistently overlap with a minor peak before the major peak of the heavy-labeled peptide. Other spiked heavy peptides and endogenous peptides within the same samples show very clear and complete overlap. I am trying to compare the endogenous peak area to the known amount of spiked heavy peptide, but this is causing problems. I'm attaching a few images that I think will help illustrate what I'm seeing. Any help would be appreciated.

Thank you

 Showing heavy and light with IDs.png  Showing heavy transitions with IDs.png  Showing light transitions with IDs.png 
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how to add new elements?
(1 response) lvyayao90 2019-03-20

Hi Skyline team
I tried to add Tb, Tm, and Ho as new elements in the unimod.xlm file in folder Skyline_4_2_0_19009 as following, but Skyline software still pops-up that Tb is not a valid chemical formula.
<umod:elem title="Tb" full_name="Terbium" avge_mass="158.9253" mono_mass="158.925343"/>
<umod:elem title="Tm" full_name="Thulium" avge_mass="168.9342" mono_mass="168.934211"/>
<umod:elem title="Ho" full_name="Holmium" avge_mass="164.9303" mono_mass="164.930319"/>

 <umod:brick title="Tb" full_name="Terbium" mono_mass="158.925343" avge_mass="158.9253">
     <umod:element symbol="Tb" number="1"/>
  <umod:brick title="Tm" full_name="Thulium" mono_mass="168.934211" avge_mass="168.9342">
     <umod:element symbol="Tm" number="1"/>
  <umod:brick title="Ho" full_name="Holmium" mono_mass="164.930319" avge_mass="164.9303">
     <umod:element symbol="Ho" number="1"/>

Is there anything else I could do to get Skyline to recognize the three elements? Thank you!


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Total Area MS1 differs slightly from sum of MS1 areas
(4 responses) Tobi 2019-03-25

Dear Skyline team,

sry to add to this topic but I sadly could not find this exact problem in the forum. It is more for understanding than an actual problem.

When calculating the total Area MS1 myelf in Excel and via Pivot (Sum of Areas) the number differs slightly from the Total Area MS1 from the results grid but the difference is more than what I would expect from rounding errors.

Quant is set to MS1 and DDA and Product Ions are non-quantitative (and I excluded product ion areas from the pivot sum of areas).

Is this behaviour expected or are some settings wrong? Do you have a recommendation on how to retrieve the area of a peptide as the sum of all charges and isotopes?

Thank you very much for the great support

view request
[M+4] and higher carbon isotopes
(2 responses) Tobi 2019-03-22

Dear Skyline Team,

in the target list, the number of natural carbon isotopes [M+X] for each charge of a peptide can be nicely set via Settings/Transition Settings/Full-Scan/Peaks.

While changing the value between 1 and 4 (Precursor to M+3) the isotopes in the target list follow exactly, but any input value higher than 4 will result at maximum in [M+3]. Am I doing something wrong or is it limited?

Just in case it is limited, could you think of increasing the limit since this feature is automatic and fast and easy to control?

Looking forward to get a short comment for your side, and thanks for the great software.


view request
importing failed due to RT predictor linear regression
(14 responses) brlawson 2018-03-15
Hi Skyline team,
I am currently working on using skyline to analyze DIA data. All of our samples had PepCal mix spiked in so, I created an iRT document that I have been importing before importing my raw files. I am working with a very large data set so I have been importing in batches. Out of the whole data set I had 3 files which I received the error "unable to finish importing chromatograms because RT predictor linear regression failed." I have looked at all the files in Sciex software and the RTs seem to be fairly consistent across all of the samples. Do you think there is an actual issue with these particular samples or is there some type of setting that I may need to change? Thanks for your help!
view request
skyline 4.2.1 error
(4 responses) hgoldsc1 2019-03-22
when i try to open a file i get this message:

Failure opening \\Mac\Home\Documents\Lab\POSTDOC!\Experiments (HLG_000)\M_000-APEX mass spec experiments\ms_013-test on-bead\skyline peptide list\
The document format version 4.21 is newer than the version 4.2 supported by Skyline (64-bit)
OK More Info
System.Reflection.TargetInvocationException: There is an error in XML document (2, 2). ---> System.InvalidOperationException: There is an error in XML document (2, 2). ---> pwiz.Skyline.Util.VersionNewerException: The document format version 4.21 is newer than the version 4.2 supported by Skyline (64-bit)
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadXml(XmlReader reader) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 530
   at pwiz.Skyline.Model.SrmDocument.ReadXml(XmlReader reader) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2022
   at System.Xml.Serialization.XmlSerializationReader.ReadSerializable(IXmlSerializable serializable, Boolean wrappedAny)
   at Microsoft.Xml.Serialization.GeneratedAssembly.XmlSerializationReaderSrmDocument.Read1_srm_settings()
   --- End of inner exception stack trace ---
   at System.Xml.Serialization.XmlSerializer.Deserialize(XmlReader xmlReader, String encodingStyle, XmlDeserializationEvents events)
   at System.Xml.Serialization.XmlSerializer.Deserialize(TextReader textReader)
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass925_0.<OpenFile>b__0(IProgressMonitor progressMonitor) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 301
   at pwiz.Skyline.Util.ProgressWaitBroker.PerformWork(ILongWaitBroker broker) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\UtilUI.cs:line 123
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 232
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1854
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 180
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 132
   at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 295
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Large scale PRM data (msx count= 8)
(4 responses) Wael 2019-03-20

Dear Skyline team,
I have a question please regarding PRM analysis. In short, I have an inclusion list consist of about 300-500 peptides and I ran this in multiplex PRM mode (msx count = 8) without RT optimization, as a fast exploratory experiment.
I am getting the MS2 ions, but skyline is integrating the peaks for all peptides over 15 min (LC method 20 min), please find attached pic as an example.
My normal RT window for a pepetide is about 0.2 min. I have the resolving power 140,000 at 200m/z.

Is skyline able to process such a workflow and if yes how can I improve the peak integration, pelase ?

Please let me know if you would like me to share skyline file with you.

Many thanks

 Large scale PRM data.PNG 
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.NET framework
(1 response) Dan Itzhak 2019-03-21

Installation of Skyline 4.2 requested installation of .NET framework 4.7.2 on the instrument PC.

Following update of .NET to 4.7.2, there is no communication between the Lumos and the instrument PC.

The PC is windows 10 and the instrument is a fusion Lumos.

Is this a known problem? Is there any advice to resolve this issue.

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Peak area extraction from MS1 only data acquired in paralell with SRM data
(4 responses) wen ding 2019-03-12

Hi Brendan,
I can use Skyline to extract peak areas of ions from either MS only data or DDA only data without MS/MS being performed. But I can not do that from MS raw files with MS1 and SRM only data are acquired in parallel on LTQ or Quantiva. It appears that the results import process does take place since I can see the peaks being extracted very briefly , but eventually, no chromatograms or results are obtained. Is there a way to deal with that particular problem?

Thanks a lot in advance!


view request
Fixed integration/peak boundaries
seabiscuit 2019-03-21

Dear Skyline team,

I am doing regular SRM/MRM quantification of peptides on a Thermo TSQ Quantiva MS. We are testing a fast-and-dirty analysis process by eliminating the use of LC column for separation. So all the peptides we monitored with be introduced into the MS to form wide elution peaks that overlap with each other. The issue I have now is the algorithm Skyline uses to set the peak boundaries does not work well with this elution pattern, especially for peaks with lower intensities. So I am just wondering if you can add a feature letting the use set fixed peak boundaries for all peptides in all samples.
A typical elution pattern was enclosed for your reference.


Wenbo Zhi
Augusta University

 peak boundaries.emf 
view request
Share complete failure to open error
(3 responses) Brett Phinney 2017-11-01
When I share a complete skyline file and select current version and Try to open it again I get this error

Any ideas? I'm using the most uptodate version of skyline. I have repeated this several times

failure to open

System.Reflection.TargetInvocationException: There is an error in XML document (1, 1). ---> System.InvalidOperationException: There is an error in XML document (1, 1). ---> System.Xml.XmlException: Data at the root level is invalid. Line 1, position 1.
   at System.Xml.XmlTextReaderImpl.Throw(Exception e)
   at System.Xml.XmlTextReaderImpl.ParseRootLevelWhitespace()
   at System.Xml.XmlTextReaderImpl.ParseDocumentContent()
   at System.Xml.XmlReader.MoveToContent()
   at Microsoft.Xml.Serialization.GeneratedAssembly.XmlSerializationReaderSrmDocument.Read1_srm_settings()
   --- End of inner exception stack trace ---
   at System.Xml.Serialization.XmlSerializer.Deserialize(XmlReader xmlReader, String encodingStyle, XmlDeserializationEvents events)
   at System.Xml.Serialization.XmlSerializer.Deserialize(TextReader textReader)
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass128.<OpenFile>b__126(IProgressMonitor progressMonitor) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 257
   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 1940
   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 176
   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 131
   at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 261
 failure to open.jpg 
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fail to import results
(3 responses) chia-lin 2019-03-18


I failed to import results and I will paste the error test. Thanks for your help!
At 11:53:
Failed importing results file 'D:\sop\XX\PXXXXX\PXXXXXXX.raw'.
[ThermoRawFile] The system cannot find the file specified.

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MS or MS/MS scan numbers
(4 responses) mvillal1 2019-03-19

Hello Skyline team,

Is there a way to extract/show on the Document Grid the MS or MS/MS scan numbers per precursor?


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questions about install skyline daily
(7 responses) chia-lin 2019-03-11


The system of my computer is 32-bit .I meet some problems When I tried to install skyline-daily(32-bit) .It seems that the program need .NET framework(64-bit) .Look forward to your solutions.

Best wishes!

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Remove peptides without signal/spectra from view
(2 responses) Zac 2019-03-18

I am working with a large protein that has a number of potential tryptic peptides. Once i import my raw data i would only like to see those peptides with associated MS 2 spectra. Seems like there would be a quick way to do this but i am not finding it. I can manually go into 'pick children' and select the peptides with an orange or green label but would like to do this quicker. The peptide filter and autoselect don't seem to help with this. Probably something simple that i have missed

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Importing Agilent QqQ data with more than one time segment
(2 responses) Paige Riley 2009-07-24
Hi Brendan,

I was trying to analyze a sample set of scheduled results for the agilent qqq and have run into a snag. Skyline seems to only be able to display the first segment of the the schedule run. In my case the frist segments is from 0-10 minutes and has no pertinent information/data. I tried to attached a representative file for you to work with but because the instrument save all the data in a file I am unsure which part to send so I will attempt to email the entire folder to you.

Brendan responded: 2009-08-05 14:08
Hi Paige,
Investigation of this has been moving through email. There does appear to be an issue with segmented scheduled runs on Agilent instruments, or at least this one. Agilent has recommended an upgrade to dMRM, with which Skyline has been tested. But, I am still trying to get the segmented data to work better. Thanks for the report.

view request
An error of retention time
(14 responses) shin 2019-03-12


I tried to use slyline to analyze proteomic data.
Library making was succeeded, but extraction of chromatogram did not work.
Previously my setting have worked and I didn't change any saved settings.
Skyline showed an error that mentioned there is no valid retention time.
How do I show retention time in spectrum library?


view request
view spectrum
(4 responses) chia-lin 2019-03-11

Hi,skyline members

I build a peptide library with Maxquant results. I added all peptides by view library spectrum. But the spectrum did not show while the peptide list was ok. I will submit the image and hope to your reply. Thank you!

view request
Importing assay libraries having RT normalization with CiRT peptides
ankit1517.niper 2019-03-15

Hi Skyline team,

Actually I am trying to analyze the DIA data using the targeted extraction strategy i.,e, by using the assay library generated based on the DDA data.
During assay library generation I used the CiRT peptides for the RT normalization. Though I have used the Biognosys Standard peptides in the samples during the DDA data acquisition, but only one peptide was present in my DDA data after the statistical validation with iProphet. So, I could not used the Biognosys peptides for the RT normalization.
Now I am trying to import this library in the skyline, but as it contains the iRT information, skyline is asking for the iRT calculator.

I have few doubts now:

  1. Can I use the Biognosys iRT peptides for this purpose, by using the assay library for these peptides provided by your team alongwith the tutorial of Import assay library?
  2. If I could not use above option, can I use the CiRT calculator inbuilt in the skyline?
  3. Else I need to built a new calculator by using the CiRT peptides? If so, should I use only those CiRT which are related to human, as I have only human data?
    Also, should I use the CIRT_All or CiRT_SW only provided alongwith the paper of Sarah Parker on CiRT?

I will be very grateful for your help.

Thanks in advance.

view request
RT allignment and idotp filtering
(1 response) sigio86 2019-03-14

Dear all Skyliners,

I'm really a beginner in Skyline so perhaps my question will be very simple.
I have 5 samples, 2 replicates each and I would like to visualize in all chromatograms the same peptide at the same RT (please see attached what I have for a prototypical example)... do you know I could I do that?
A second question is if and how can I filter for "high" idotp (at least 0.8 based on previous posts).

Thanks a lot in advance.


 different RTs.PNG 
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Selective peak integration based on idotp
(2 responses) Fabian 2018-05-29

Dear all,

i would like to have the option to integrate only peaks above a certain idotp threshold.
At the moment, the option [refine_advanced_results] Min idotp: decides if a peptide is kept or not.
I would like to have the same option based on the peaks not on the peptide.

Did I miss sth. or are there workarounds (peak training model did not convinced me).

Best regards


view request
Importing Transition List Containing Lysine Succinylation
(5 responses) aaron robinson 2019-03-12


I'm not able to import a transition list into Skyline containing Lysine Succinylated peptides. See attached. I have the mod in my modifications and am importing it in as UniMod:64.

I'd like to publish a DIA dataset containing succinylated peptides and although it works fine if I use .pep.xml and use Skyline to build my DIA library I can't get it to go through importing a transition list to build my library.

Also attached is my sample transition list (1 peptide).

Please advise.

-Aaron Robinson

 succinyl.JPG  succinyl.tsv  succinyl_2.jpg 
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mProphet DIA model, retention time difference missing
(10 responses) anatoly.urisman 2019-03-10

Hi Brendan and Nick,
I have a DIA dataset with ~13K precursors. The mProphet model is not seeing retention time difference on a subset of precursors (snapshot 1). But I don't see any obvious problems with these precursors - they appear to be well measured (e.g. snapshot 2). Settings > "Integrate all" is checked.
I have a feeling that this has something to do with iRT, because some (perhaps the same) peptides are not calibrating correctly. In the regression curve (snapshot 3), you can see an example (selected/red) peptide which has 2 IDs (one from each sample) in the library. Both appear to be picked and integratedcorrectly, but I can't understand why the peptide is assigned an iRT score of -14. It also appears that most, if not all, of these peptides have an N-terminal modification - either Q[-17] or -M[-89]. I have "Use measured retention times when present" checked in the peptide settings (snapshot 4).
Have you seen this behavior before?
A reminder - I am using Protein Prospector generated blib libraries. If helpful, happy to share the entire document.
Thank you very much for your help!

 Snapshot1.PNG  Snapshot2.PNG  Snapshot3.PNG  Snapshot4.PNG 
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construct a self-made library from CSV output format
(6 responses) haidi yin 2018-06-18

Dear Brendan,

I am trying to use skyline to quantify glycoproteins. I am using pGlyco2.0 for glycoprotein identification. The output file is in txt file format, which can be easily transformed to csv file format for easy reading. The CSV file is shown in the attachment. My question is how I can construct a library from this CSV file? Thank you very much.


view request
Fail to import Thermo raw PRM data
(6 responses) haidi yin 2019-03-13

Dear Skyline team,

I used to import Thermo raw PRM data successfully. I use a portable hard disk to store my raw data. I can still open them with Xcalibur.

But these days I fail to import those old raw data to skyline daily. It did show chromatogram during importing. But after importing, no error sign or chromatogram is shown, as if the raw data was not imported. Attached is the skyline interface after importing one raw PRM data. Do you have any idea what went wrong?

Thank you very much,
Best regards,

 fail to import thermo PRM raw.pptx 
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Bug in TL and Method Export for CEOpt and DPOpt Sciex 6500
(2 responses) Will Thompson 2019-03-11

Hi Brendan and Brian

I recently was made aware of a bug similar to Waters TQ-S for the Sciex 6500 method and transition list export, for CE Optimization (and in the Sciex case, DP Optimization). Please see attached Skyline document for verification. There are two apparent issues, one generic and one specific to Sciex. The first issue goes back to apparently the storage of small molecule collision energies as a 'molecule' specific parameter, when in fact it should be a transition-specific parameter. Simply, the transition-level optimization function in Skyline is broken for small molecules. HOpefully this is not too difficult to be fixed (the bigger the methods get and the more often we have qualifier transitions for our small molecules, the more of a pain this is to correct manually, after the methods are exported). The second issue is with Sciex machines specifically; apparently whenever a CE Optimization or DP Optimization transition list (or method) is exported, the DP values for ALL transitions which are not set to the initial CE value are set to zero (i.e. as the CE iterates up or down, it does not carry the specified DP value along with it). Unless the user is paying close attention and manually checks the DP values for the new transitions, all of the MRMs for CE optimization will have extremely low intensity (because the DP value should be something like 70, and it is set to zero).

I know these are both Skyline issues and not vendor-specific issues, because the errors show up in the transition list export as well as the methods.

Thanks for looking at this!



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Failed installation for Skyline-daily
(1 response) Bing 2019-03-12

I was trying to reinstall Skyline-daily, there is an error occurred at the end of downloading, it said "Application validation did not succeed. Unable to continue". I attached the error log file. Do you have any idea why this happen? Thank you!


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Problems while installing Avant-Garde
(7 responses) molloylab2019 2019-03-05

Hi everyone,
after reading the new paper about Avant-Garde I wanted to install it and try it for myself. When I tried to install it from the Skyline store I got an error after R 3.4.3 was installed and before packaged were loaded (see pic. "Error while installing R packages for Avant garde"). I then installed the tool via the external tool options which worked. I installed all the relevant packages through the R console using devtools (which worked just fine I could generate a params file using R). When I now go back and try to run any of the options in Avant-Garde (i.e param file) I get the following error (see pic "AvantGarde error messages"). Another thing I noticed is when I try the "help" option in Avant-Garde it opens the command promt but closes it directly after.
I tried re-installing Skyline as well as R with all dependencies which did not help. I don't have any problems with other tools (MSStats or SProCop) so I don't think it's an issue with admin privileges. Any help would be appreciated.
Additional info:
Skyline version 4.2.0 64-bit
PC Specs (see pic "PC specs")


 Error while installing R packages for Avant garde.PNG  AvantGarde error messages.PNG  PC specs.PNG 
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Can we run metabolomics data recorded from Agilent Qtof mass spectrometer using Skyline
(3 responses) bhaswatic 2019-03-08

Can we run metabolomics data recorded from Agilent Qtof mass spectrometer using Skyline?
Please let me know the details about it that is how to do it as I am new to Skyline.
Thanks in advance.
Thanks and Regards,

view request
Selection of peptides based on the dot product
(2 responses) sunbergsoon 2019-03-09


After applying the library on the Skyline, we found that you also calculate the dot product for each peptide.
Actually, we want to do high-quality searching and only keep the good result, so can I select the searching
results based on the dot product? For example, filtering the peptides with dot product less than 0.8.

Thank you very much!

Best regards,

view request
(28 responses) qaporter 2019-03-01
Hi, again. I have some more problem and question when it comes to the document grid, how do I get them to calculate the concentration if left it unknown and blank? Also, how do I form the line? Because it said I have an error that all the external standards are missing one or more peaks for my small molecule. I attached picture below to show the problems that are still occurring with settings and graph.
 Screenshot (19).png  Screenshot (18).png  Screenshot (17).png  Screenshot (16).png 
view request
Define wich trace to proces
(7 responses) stsyp 2019-03-05

Hey Skyline

I was trying to use Skyline to analyze SmallMolecules. The .raw file I have consist of 3 traces: neg mode ionization, pos mode ionistation and a PDA trace.
I have the impression that Skyline is only analyzing the 1st trace (pos mode). Is there a possibility to define which trace skyline needs to process? That for example trace 2 is used to analyze the compounds in MoleculeListName "X" and trace one is used to analyze the features in list "Y"
I've added a ppt to make clear what I try to explain.

I hope this is already possible but I just don't find how to apply it.

Steven Vds

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Results from collision energy optimization
(2 responses) hober 2019-03-07

I am currently working with a Thermo TSQ Atis and have used the tutorial for collision energy optimization and this is truly an excellent tutorial.
I am as of now in the process of setting up a pipeline for screening a few hundreds of peptide standards we have in the lab and has run into some issues with the output from Skyline regarding the transition lists.

I have recalibrated the CE predictor based on my previous results and this works really well. However, when I run samples with longer peptides Skyline predicts collision energies up to 80, while the instrument only allows for collision energies up to 65. IS there any way of setting a global maximum value for Skyline?

An additional problem that I have run into is that Skyline occasionally chooses 0 as the collision energy for some peptides Even though I get good results for them in the CE optimization. Is it possible to tell Skyline to use the default value for the collision energy rather than 0 in these cases?

Another, more rare, issue that I've had is that Skyline sometimes provides me with duplicated Precursor-Product ion pairs which make Xcalibur complain. Is there any way around this?

I know that these are quite a few questions, but if you have any solution to any of the problems it would be much appreciated.

Best regards

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

I appreciate if you can provide the 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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Error while exporting methods on Thermo TSQ Altis
(3 responses) 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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Should Biognosys iRT FASTA be included in the MaxQuant database in order to work?
(7 responses) michelep 2019-02-28

Hi All,

I am doing some DIA analysis and recently bough the iRT Biognosys peptides to spike in for RT alignment.

My Library contains around 90 DDA runs and only 15 of them have actually the iRT peptides in.
ALL the DIA runs (10) instead have the iRT spiked in!
When I follow the DIA Webinar 14 for adding the iRT prediction etc, Skyline doesn´t find the iRT in any of the DIA runs, even tough I can see they are there from the Excalibur XIC.
Then when I do the mPhophet FDR calculation/peak picking the iRT prediction is greyed out.. and can´t be used for peak picking

Is this because the MaxQuant search I did to create the library does not contain the Biognosys iRT FASTA? So those IDs are NOT in the MSMS file?
Thanks in advance to anyone that can help me


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Using Excel or TSV files to create spectra libraries
(3 responses) anna kwasnik 2019-03-06

Dear Skyline Team, can anyone advice if it would be possible to use the Excel or TSV saved spectra to create spectra libraries in format for example BLIB or SPLC (SLC) in Skyline? Any advice will be greatly appreciated.

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Application of skyline with theoretical library
(6 responses) sunbergsoon 2019-03-04
Now we want to put some theoretical library on the skyline, however,
there are some issues with the application.

The theoretical library record each peptide with relative intensities
which are all no greater than 1. In the theoretical library, we put
the following information, for example, given this peptide:

<SPECTRUM charge="2" elution="56.82013" elutionpeakwidthfwhm="0.153"
msid="1" precursorelution="56.846" precursormass="766.4388"
precursorsignal="2379.753" precursorsignalacquisition="2085.837"
sumofms2counts="2514.072" xml:id=" " yscale="1" >
<MATCH charge="2" confidence="0.9999" confidence_prior="-1"
da_delta="0.005" eval="1.114007e-010" mod_prob="0" mz="766.4388" pid=" "
pm="1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16" score="15"
searches="SEARCH:34641" seq="ELYPIALPFLDIK" type="0" xml:id="34641" >
size="16" sp="0" >
243.13445 1 0.342857450246811
406.19778 1 0.0905757695436478
503.25054 1 0.0276164971292019
616.3346 1 0.00763198733329773
687.37171 1 0.0444362200796604
800.45577 1 0.0330323725938797
1289.75091 1 0.0753663927316666
1126.68758 1 0.983879029750824
1029.63482 1 0.122389301657677
916.55076 1 0.46008637547493
845.51365 1 0.207167357206345
732.42959 1 0.577380180358887
488.30842 1 0.0218868106603622
375.22436 1 0.0373408868908882
260.19742 1 0.0576274879276752
147.11336 1 0.0267378911375999

The issues are:
1, The retention time cannot be aligned correctly which only showed a
narrow range from around 60~80 min which should be from 3~80 min;
2, The ranked peak included "y1" fragment ions for most peptides with
theoretical library and some of the peak areas graph cannot be shown.

also, what is "sumofms2counts" in <SPECTRUM>? Could you please help me
to figure out what is the problem?

Thank you very much!
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Seek help building spectra library
(2 responses) xfbubu 2019-02-21
Dear Skyline Team,


I was trying to build a library from ip2 search result (mzldent file), but run into "ERROR: .mzid file contains an unsupported score type". I have set the score cut-off to 0 but still could not solve this problem. I am wondering if you could help me with this? I have also attached the file used.

Thank you and I am looking forward to your reply!

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Error on Line 1 Column
(6 responses) qaporter 2019-02-26
Hi, Im having trouble opening a raw file on skyline, as it said it pop up line 1 column 1 error. How do I fix the problem?
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cannot install skyline
(2 responses) yincool2008 2019-03-01

I try to install skyline ver 4.2 64 bit on my PC which runs window 10 pro 64 bit and failed many times.
Rebooting the OS will not solve the problem.

The error says "cannot start the application, contact the application vendor for assistance". The log file is attached.

Please help me with this problem. Thanks!

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"Library" peak area in replicate comparison graph
(1 response) stephen swatkoski 2019-03-01

Hi Brendan,

I acquired PRM data for 5 samples and I am using skyline to analyze the data. I used the option to load PRM database search results from the start menu page. I am very happy with the way the data looks but I am confused by the peak area that is labeled as "library" in the Peak Area Replicate comparison plot. How is the "library" area assigned? Is it the area of the best (most intense) peptide across the samples? Is there a document or tutorial that explains this?


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Calibration curve
(2 responses) rliyana 2019-03-01

I am just wondering how to set values for my calibration points for multiple compounds different set of concentrations. For example, I have A and B to quantify. Compound A has calibration points 10, 20, 30 and 40 ppb and B has 15, 35, 45, and 100 ppb.

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Exporting peak areas to compare multiple runs
(7 responses) 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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ion trap bruker and skyline
(4 responses) pucci biagio 2019-02-28

I m new in skyline. i want to analyse file from .d bruker ion trap but i don't known how load these files in the skyline.
can help me?

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Importing negative mode MRM scan on Skyline (small molecule)
(3 responses) jung165 2019-02-28

Hello Skyline team.

I have a one quick question about importing negative mode scan of MRM in Skyline software (small molecule)
I am performing metabolite analysis with Agilent 6490 MS devices and export the collision energy optimization method on Skyline.

After analysis and when I import my raw files on Skyline, it gave me the error message shown below:
[Component Microsoft .NET Framework 4.7.2 (x86 and x64) has failed to install with the following error message:
"A failure occurred attempting to install the Microsoft .NET Framework 4.7.2."

The following components failed to install:

  • Microsoft .NET Framework 4.7.2 (x86 and x64)

See the setup log file located at 'C:\Users\Allen\AppData\Local\Temp\VSD86B0.tmp\install.log' for more information.]

I also used Skyline-daily version and latest version of Skyline but, all of results shows same error.
Is there any solution for this error?? please help...
I also attached my Skyline and raw files for the analysis.

I really appreciate for your help and look forward for your answer.

Sincerely,  Raw 
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collision energy QTRAP 6500
(1 response) viragsagikiss 2019-02-28

Dear Skyline Team,

I find the collision energy optimisation option and tutorial very useful! Great work and looking forward using it.
Following your tutorial on I have 2 questions.

where do you get your data for converting CE between Waters and ABSCIEX instruments? I m looking for QTRAP 6500. And how do i upload them to the CE equation editor? Apologies, it might be obvious but i can't find it. In the Edit Collision Energy Equation you have 2 lines, one corresponding charge 2 and the below one for charge3.

Charge slope intercept
2 something something
3 something something

Again, apologies if this question has been answered.

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Potential mismatch peptide library and results
(3 responses) t j f m voermans 2019-02-25

Dear Skyliners,

When analyzing my QTOF results and having them imported in Skyline, I asked Skyline to match the spectra with the peptide library. Multiple matches were found (seeing the green dots), but after analyzing the spectra myself via various software, I came to the conclusion that the m/z values found by Skyline do match, but the number of charges doesn't. Therefore, the fragments from the library probably are probably not the fragments found by the QTOF.

Are there some settings that I need to change, or am I making mistakes with my reasoning?

Than you!

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Calibration Curve
(1 response) qaporter 2019-02-27

I apologize, for making so many requests, but it is difficult to follow instruction when you don't have any peptides in your experiment. So, I made a small molecule transition list and I was able to open my raw files. Then, I went through the steps for the small molecule targets, and then I went through another tutorial on how to create or make a calibration curve/line and I didn't get the line going across, instead, I got a line going up and down. It also classified my standards as unknowns and I don't know the reason for this problem. How do I create a calibration and what did I do wrong for it not to give me a proper line? I also attach a picture to illustrate the line and the problem.

 Screenshot (11).png  Screenshot (12).png  Screenshot (13).png 
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Error on Line 1 Column
(3 responses) qaporter 2019-02-27
Hi, again. I made the transition list with the small molecule, but then when I finish the list and tried to open the raw file, again it still wont let me open the raw file. The reason I doing is so that I can get my retention times on a calibrate curve/line. But, I keep having the same problem, I add the picture to illustrate the problem.
 error 1.jpg 
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Proxy Authentication Error
(2 responses) Sumeet 2019-02-27


I keep getting this error (attached) whenever I try to access the tool store. Is our company's firewall blocking the application?


 407 Proxy Authentication error.PNG 
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error importing from lumos to skyline
(1 response) sarah hennessy 2019-02-27

"Could not load file or assembly 'pwiz_data_cli.dll' or one of its dependencies. The specified module could not be found"

I uploaded the raw file, titled PFMS_003194 into the file sharing site

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Getting started, developing MRM's for the 1st time.
(2 responses) 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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CE optimize method XML parameter issue with Agilent 6495B qqq
(1 response) 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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MS1 full-scan filtering: missing one highly abundant peptide?
(7 responses) miruppen 2014-04-09
 we are having some trouble with the MS1 full-scan filtering procedure. In the lab we would like to start using Skyline to assess the DDA data quality from our Orbitrap Velos, based on ID-free metrics for 21 peptides of BSA.
We have created the spectral library from 8 .msf files and the corresponding .raw files for chromatogram extraction.
However, a highly abundant peptide of BSA we always identify (YLYEIAR; m/z 464.250++) is missing in the Spectral library explorer, and we cannot figure out why.

Please find the compressed .sky document attached.

This peptide elutes at Rt 28 min aprox.

Could anyone help us to find out why is this happening?
 MS1 Filtering_System 
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mProphet Modeling for MS1 XIC Analysis
(10 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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the installation has not gone smoothly
(1 response) danielhou 2019-02-21

Dear Skyline Team,

When I want to install the Skyline, an error always appear.
"Unable to retrieve application files. Files corrupt in deployment." This error always appear. So I reinstall the system, but an other error appear.
Details like this: An error occured trying to download "".
So I download the application from the web. Thus it can download Skyline, but the same error appear again. "Unable to retrieve application files. Files corrupt in deployment."

Best regards,

Zhanwu Hou

 error_1.PNG  error_2.jpg 
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