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Not able to see libray ion in the result file and how to do the quantitation of the peaks
(3 responses) drrenugoel 2019-08-23

Kindly see the attached file. I have built the library in the peptide setting tab. However, I am not able to see the library ions in the attached file (skyline_doubt). some setting has been changed as I used to get it. Kindly see the attached (skyline2).

I have run these samples on 5600 sciex in MRM-HR mode and now want to do the quantitation. Could you please let me know how to take it out peak area for the same.

 skyline2.JPG  skyline_doubt.jpg 
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CE method export for application in MassHunter controlling an Agilent 6490 instrument
(5 responses) azad 2019-08-22

Hello support team,

I've searched the forum and exhausted my wit to find a solution, other than brut force which is borderline "manual" method development.

-I am running Skyline-daily and MassHunter version B.07.01 build 7.1.7112.4 SP1 which is controlling an Agilent 6490 instrument.
-skyline file consists of 2458 transitions corresponding to 389 peptides
-transitions per peptide varies from 2 to 10
-My intention is CE optimization for every transition and I have only included heavy stable isotope labelled peptides in the file
-In the transition settings tab - collision energy tab - Agilent QQQ is selected
-Regression values exist for charge sates 2 and 3 but not 1 and 4, I do have the latter for a subset of the transitions in the file
-Step size is 3 and step count is 2
-When exporting methods the max concurrent transitions is set to 60 which results in 20 methods each containing ~600 total transitions
-When I attempt to load a method in MassHunter the Agilent6490 switches from online to offline
-To attempt to diagnose the problem I manually shortened the number of transitions for a method to 450 and was able to run the method. However, when I used the same approach for a subsequent method the instrument once again went offline.
-I manually looked over the transitions in excel to ensure the maximum concurrent transitions were actually 60 or less and appears they are.
-At this point I can't think of anything else expect to manually add transitions in the QQQ tab until the instrument goes offline and then scale it back and run the method. However, I would prefer to avoid this if there is a "smarter" solution.

Thanks in advance for your assistance.


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Q-TOF .raw data search stops after certain retention time
(7 responses) Yao Chen 2019-08-13

Dear Skyline Team,

I am importing Waters .raw data generated by Q-TOF instruments into Skyline. The whole file collects data for 120 min (pic 1), however, the searches in Skyline seems stopped at around 60-70 min for almost all the transitions I was monitoring (pic 2 shows four examples).
I could see a very clear signal of a spiked-in standard peptide in the raw file, which eluted at 77 min (pic 3), however, its retention time was not reached in the search of Skyline (pic 2, upper left). How should I change my settings so I can do a full chromatographic search, please?

I have my partial skyline processing file attached.



 pic 1 _ base-peak intensity chromatogram.PNG  pic 2.png  pic 3_raw chromatogram of standard peptide.png 
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What's wrong with my Thermo RAW files
(1 response) solis 2019-08-19

Dear support team,

lately, some of my RAW files (independent of experiment or date) are not being imported by Skyline. This is the error message I always get:

Failed importing results file 'Ref_xxx_VS_090819.raw'.
[SpectrumList_Thermo::spectrum()] Error retrieving spectrum "controllerType=0 controllerNumber=1 scan=45461": [RawFileImpl::getMassList()] Object reference not set to an instance of an object.

Can you help me figure out what's wrong? If I convert the files to mzXML, Skyline does upload them successfully.


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Issue importing waters RAW files, Unhandled Exception: Invalid Function Type
(1 response) Allison Haase 2019-08-19

Hi All,

I have been getting error messages in skylines when importing raw files from a specific set of experiments on a Waters XEVO. Other files that have been imported from this instrument have worked fine in the past, and the raw files can be opened in MassLynx. The error is pasted below.

At 9:45 AM:
Failed importing results file 'C:\Users\allis\Desktop\redownload\20190809r001.raw'.
[pwiz::CLI::msdata::ChromatogramList::size] Unhandled exception: Invalid Function Type

Inner exceptions:
Exception type: System.Exception
Error message: [pwiz::CLI::msdata::ChromatogramList::size] Unhandled exception: Invalid Function Type
[pwiz::CLI::msdata::ChromatogramList::size] Unhandled exception: Invalid Function Type
at pwiz.CLI.msdata.ChromatogramList.size()
at pwiz.ProteowizardWrapper.MsDataFileImpl.get_HasChromatogramData() in C:\proj\skyline_19_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 844
at pwiz.Skyline.Model.Results.ChromatogramDataProvider.HasChromatogramData(MsDataFileImpl dataFile) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\ChromDataProvider.cs:line 365
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 194

I have also attached one of the raw files in a zip folder.

Thank you very much for your time,
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Unable to process chromatograms for the molecule
(12 responses) chinmaya k 2019-08-11
Dear Team,

I was trying to analyse 2 DIA files (technical replicates) using Skyline (version under proteomics interface. When I start importing DIA raw files, skyline come up with an error (please find the attached text file for the same). In brief, the error says "Unable to process chromatograms for the molecule 'AGEEGGSVGSGVFLIGR' because one chromatogram ends at time '' and the other ends at time '48.8989682017979' ".

What does this mean? How can I rectify this issue?

Request you to resolve this issue.

Thank you,
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Accurate Values for peak intensity
(8 responses) sa825 2019-07-23

Good morning,

I am using Skyline on Windows 10
I wanted to ask how you would accurately read off the intensity of a peak/peptide in skyline?
I can read it off by eye but II feel as though there is probably a more accurate method?
I have attached an image so you can see what I mean.

Also, how do I view values for the area of the peak?



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Order by document settings
(2 responses) mcqueen p 2019-08-19

I have been using Right-Click -> Order -> "Column Name" to order small molecule samples by specific variables within Skyline. The only options that seem to work are "Document" and "Acquired Time". I have previously been able to order by custom definitions that I made in "Document Settings" -> "Annotations" but they do not seem to work anymore ie when I select the custom options they do not affect the sample ordering. This seems to persist across different .sky files and I have tried starting from the beginning with brand new analysis files with no avail. I recently updated to Skyline v. (with Windows Pro 64-Bit) from the previous version, could this have something to do with it? I'm not sure what I'm doing incorrectly.

Thanks for any help,


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local FDR/PEP
Tobi 2019-08-19

Dear Skyline team,

could you imagine including a local FDR value to complement the Q-values in the results grid? (PEP) For me it seems nice beeing able to retrieve the actual chance of a specific peptide beeing a false discovery, but you might know more about limitations and relevance of this.

Looking forward to hear your opinion on this.

Best regards,

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Create Background Proteome
(8 responses) Richard Lam 2019-08-13


I followed the tutorial document 'MethodEdit-3_7' (see attachment, step on pg 6) to create the 'Yeast' background proteome in the 'MethodEdit' skyline document (see attachment). After I clicked 'OK" to digest the background proteome, Skyline stalled and could not proceed with the digestion (see the screen shot in the attachment (Building Background Proteome in Skyline).
I tried several time and restarted Skyline and my computer, the issue did not go away. Please help.



 MethodEdit-3_7.pdf  Building Background Proteome in Skyline.docx 
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Using Skyline to monitor glycopeptides in DIA
(14 responses) rmeccariello 2019-08-13


I am a newbie Skyline user and need some help using Skyline. I am monitoring glycopeptides with a DIA method. I'm looking at 3 different peptide sequences, and monitoring the same 13 glycans on each of those peptides. In order to calculate relative abundance, I'm monitoring the Y1 ion (full peptide backbone + GlcNAc) and the Y1+Fuc ion (full peptide backbone + GlcNAc+Fuc).

I'm not sure how to define my peptide settings and transition settings. Where do I input the parent mass (full peptide backbone+full glycan), and where do I specify the mass of the Y1/Y1+Fuc ions that I want Skyline to look for?

Thank you!


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ssl search results not being imported
(3 responses) solis 2019-08-16

Dear support team,

I'm trying to create a spectral library from an ssl file. Everything runs normally until the upload of the fasta file, where it says that the number of matches is 0. Can you help me figure out the issue?

So I'm doing this via: File -> Import -> Peptide Search. Please find the necessary files attached.

Thanks for your help!

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Large dataset processing, skyd silently deleted
(5 responses) Phil Charles 2019-07-31


We are processing a fairly large SWATH-type DIA experiment through skyline. Based on a fractionated spectral library, the .sky file contains 100,197 precursors; 970,600 transitions. We have 207 Lumos raw files to add to the project. I'm aware this is probably somewhat outside usual operating bounds. My ultimate goal is to export a custom report containing the XICs of each transition for further analysis and comparison with other processing workflows.

Because the skyline interface becomes unresponsive when dealing with this number of transitions, after setting up the .sky and importing from the spectral library, I saved and closed the document then used skylinerunner to do the raw file import on a fast desktop with --import-process-count=32, (and making sure to also add --save!). The machine thought about this for quite a while (10 days, although I think it would have been faster if I had used less threads to avoid cache swapping) and finally generated a corresponding .skyd that's just over a terabyte in size. The processing finished with no error messages logged to the console.

I backed up the whole project and then tried to open the .sky file. Skyline considered this request for about 15 minutes, then opened the file but without any results. In the background, I later discovered, it silently deleted the .skyd, which was Not Helpful. Thank goodness for backups!

I have restored from backup and tried again, with the same result. I've also tried setting the .skyd to read only / write access denied, in which case Skyline is unable to assist me by deleting the terabyte of cached chromatograms (representing quite a lot of processing time), but still reports no results attached to the document. When I try to export the report with skylinerunner, there's also no replicate information.

Is there any way to persuade Skyline to use the data in the .skyd, and salvage the progress so far? Also, the silent deletion thing ...isn't ideal.

Many thanks and best wishes,


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(1 response) dsullivan 2019-08-14

Hi all,

I'm trying to utilize some of the newer skyline cmd settings but I'm running into issues.

Specifically I'm trying to set the precursor resolution with full-scan-precursor-res-mz. I have tried:

Unfortunately I get no peaks found when I try to set this option. It seems to work fine if I don't set this option.

Any ideas about what's going on? I can't seem to find an example of how to use this option other than in the command line documentation which just has <m/z value> as a value.

Any help is appreciated.

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Issue with Skyline command runner
(5 responses) shridevi patil 2019-08-12

Hello Skyline Team,

We are using Skyline command runner in our application, we need to arrive at optimized method for data acquisition using Waters Xevo Tq instrument..
We internally use Skyline Daily runner command to execute the Step1: RT,Step2:CEFixed,Step3:CEOptimized and Step4:FinalMethod.

We are facing problem with execution of these command on runner.
--dir="D:\Bit_Dev\WorkFlowManager\TestData\SkyLine" --in="c:\" --import-all-files="C:\Users\Test\AppData\Local\Temp\zft5bmr3" --exp-method-instrument="Waters Xevo TQ" --exp-template="c:\template.exp" --exp-run-length=15 --exp-file="C:\Users\Test\AppData\Local\Temp\sjty44c0\CE.exp" --exp-method-type=scheduled --refine-min-peak-found-ratio=0.1 --tran-product-ion-types="p,y,b" --out="C:\Users\Test\AppData\Local\Temp\ofe4atzp.ftq\" --exp-strategy=Buckets --exp-max-trans=100

is there any mistake in this command. Because return type from execution of Skyline command runner is failure.

Please guide/ correct out understanding.
Thank you

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Odd NEM error when builidng library from MaxQuant results (but not Mascot)
(9 responses) Jason Held 2019-08-11

I've built (not quite literally, but close) a million skyline libraries with IAC and NEM modified cysteines from Mascot and MaxQuant searches. I haven't changed anything and my IAC modified cysteines are importing fine, but I'm getting the attached error when I try to import a MaxQuant MSMS.text file. A .dat file from mascot imports both IAC and NEM modified peptides just fine. Both NEM mods in Skyline and Maxquant are the same (H7C6NO2), supported by the happy imports from Mascot.... This is a MaxQuant-specific problem. I'm not sure why I'm getting the error and suddenly unable to build a library from the MaxQuant MSMS.txt file.

I've attached screenshot of the error and the msms.txt file.


 Screen Shot 2019-08-11 at 3.44.06 PM.png 
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X-intercept in linear fit
(1 response) bramachandran2 2019-07-26


Is there a way to display the X-intercept value on the calibration curve in skyline? I am doing a standard addition calibration method and it would be great if I could get the X-intercept as an output from skyline rather than going with an offline spreadsheet for calculating it.


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small molecule support for Agilent Chemstation data files
(4 responses) dgmccaskill 2019-08-08

Are there any plans to support working with Agilent Chemstation data files (not MassHunter) coming from an LC-MSD single quad system? This might be a small niche, but it would be very helpful (at least for me) in the small molecule arena to be able to work with SIM data from Chemstation using Skyline.

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Metabolomic isotope data- can only view chromatogram for the nominal mass
(2 responses) sarahmacpherson676 2019-08-06

Hi, I am looking at metabolomics isotope data using GC-MS with a QIT mass analyzer. I was initially able to get the results for each isotope using Skyline Daily. However, when I opened a new document with the same data, I was only able to view the chromatograms for the nominal mass. Not sure if there is something wrong with the software, as I was able to analyze the data before with the same settings.
Attached is the .sky file that works and doesn't work.
Thanks for your help

 gc-ms analysis 
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Export Transition List
(1 response) Richard Lam 2019-08-08


I would like to export the transition list for creating a Sciex 5500 QTrap MS acquisition method.
In the 'Prediction' tab under Transition Settings page (see attachment), what is the difference between the choice of 'SCIEX' and ' ABI 5500 QTrap' in 'Collision energy', between the choice of 'SCIEX' and 'ABI' in 'Declustering potential'?



 Export Transition List for Sciex 5500 QTrap.docx 
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Background Proteome?
(2 responses) Richard Lam 2019-08-08


I studied the 'Targeted Method Editing tutorial' already.
I have the following questions:

  1. I am still not quite sure what's the relationship among 'Background Proteome', 'Spectral Library', and the proteins/peptides pasted in Targets page. Can you give me an overview and if possible, with a simple example to explain it?
  2. How to view the protein(s) and/or peptide(s) of the 'Background Proteome' in Skyline?



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Chromatogram information unavailable
(6 responses) davis 2019-08-08

Hello Skyline team,

I am using skyline daily. However, when import my .raw file, there is no signal and "chromatogram information unavailable" is displayed. This is only the case for the files I generated as of today. My previous files (072419) have been imported and are visualized.

I appreciate for all of your help.
Best regards

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(1 response) rmoulder 2019-08-07

Dear Skyline team,

I would like to add citrulline as a post translational modification. Should I define the post translational modification by composition, or can I add a new amino acid. At present, as a work round, I have assigned the heavy synthetic citrulline containing peptide as deamidated.

Thank you in advance for your recommendations.

Best regards

Robert Moulder
Turku Bioscience Centre

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Question on Limits
(2 responses) Tobi 2019-08-06

Dear Skyline team,

we have an outstanding project and want to work with DIA and Spectral libraries on a atrget list generated from huge fasta files in the GB range. Are there still any (fixed) limits for the number of peptides and transitions in the target list or the number of peptides in a spectral library that skyline can hendle? Older posts here yield different answers.

Best regards,

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(5 responses) Tobi 2019-08-06

Dear Skyline Team,

could you please take a look at an issue with Avant-Garde DIA? Sadly the forum for Avant-Garde DIA does not seem to be maintained.

When trying to run it the execution is halted. The full error message is attached, but the main point seems to be

" cannot open file 'S:/avant garde test/AvantGardeDIA/DataFormatting/TempFiles/Report_GR_ColNamesName_Tag.csv': No such file or directory Execution halted"

The named file does not seem to exist. The preperation was done according to the AVG Manual, but it might still be an issue. Did anyone else experience an error like this one?

Best regards,
tobi  AvantGardeDIA.rar  Error MSG.txt 
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Illegal character found in sequence when building spectral library from MaxQuant results
(2 responses) Gqin 2019-08-02

Hello All,

I'm a new user of skyline, and trying to build a spectral library from MaxQuant results. I'm not sure whether this is a skyline problem or MaxQuant problem.

Mass Spectrometer: timsTOF Pro
MaxQuant version:
System running MaxQuant and skyline daily: Windows 10 enterprise
Problem: when using msms.txt file to build spectral library using skyline daily, an error message window popped up saying "ERROR: illegal character t found in sequence (Acetyl(protein N-term))AAA.... (line 3) ERROR: reading file msms.txt". I then delete this variable modification from the datafile and try to build the library again, however another similar error occurred "ERROR: illegal character d found in sequence AA...(Oxidation (M)) (line25) ERROR: reading file msms.txt". Error message screenshots are attached. It seems that the errors are due to modifications, unfortunately, I can't just delete them in my data file. Please let me know how to go forward.

Thank you very much.


 error.jpg  error2.jpg  msms.txt 
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Calculation of asymmetry factor and tailing factor of chromatographic peaks
Juraj_Lenco 2019-08-06
Hi, it would be great, and I guess appreciated by many users, if Skyline could determine asymmetry factor and tailing factor of chromatographic peaks. Skyline can determine peak widths at 50% intensity or at baseline so far. Unfortunately, the width at 50% intensity is not sufficient for assessing chromatographic peaks quality. The peak width at baseline is almost never used in LC-based analytical chemistry since it is challenging to determine real peaks boundaries . The peak width at baseline should be thus replaced by asymmetry factor and tailing factor that are very popular methods of measuring peak shape in chromatography.
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Variable modification
(1 response) dawn dufield 2019-08-05

How do you add a modification that you do not know which AA it goes on. For example a ribosylation that I know adds a certain MW, but fall off in the MS so doesn't change the fragments? or how do you look for a modification that can be on any AA's without creating it on all possible combinations.. Can you generate a list of the possible MRM's?


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Targeted Assays with Waters Synapt
(8 responses) msielaff 2019-08-01

Hi everybody,
Following problem: We acquired targeted runs on a Waters Synapt G2-S. Five MS2 channels were recorded, one for each precursor. When importing these raw files (screenshot of full-scan settings is attached), Skyline only extracts fragment ion information from the second channel, which is also the case when the MS/MS filtering method is set to DIA. The other four channels are ignored.
Is there a way/workaround to import the other channels in Skyline, too?


Skyline version
Windows 7

 Bildschirmfoto 2019-08-01 um 10.21.18.png 
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The quantification of MS2 ions in SWATH analysis
(1 response) sunbergsoon 2019-08-02


Recently we compared the DIA quantification results from PeakView and Skyline, we found that the results are quite different.

We analyzed protein extracts from different developmental stages of a fly species with two replicates for each stage. We used the same spectral library to do searching in both the software. In PeakView, we exported the results using default settings and q-value cutoff=0.05. However in Skyline, we exported the results with q-value cutoff=0.05 after training a mProphet model. In skyline we used the normalized area to do quantification where the normalized method is "none".

We compared the results by computing ratio of common peptides in common proteins yielded by the two software.The comparison result is shown in attached plot. Could you please give some suggestions on explanation about the results?



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Command line interface for peptide search
(3 responses) dsullivan 2019-07-31

Hi Brendan,

Thanks for developing skyline. I am trying to use it to perform some automated analysis for our lab. We have a small set of known peptides and modifications that we want to import and run in skyline.

I have been generating all combinations on my own in python and importing the respective m/z's to skyline as a transition list, however I see that in the recent release there is more support for peptides and the command line (and now included command line documentation). Great!

My questions are:

  1. Is the best way to get my peptides in to import as items in a FASTA? Or should I still use a transition list?
  2. I see I can import a "peptide search results file", but I can't find anywhere that details the format of this file. Can you provide an example?
  3. I see that modifications also go in this search results file, how would that look as well?
  4. There is now an option for charge state, but I don't see an option for specifying my digestion and missed cleavages, does one exist for the command line?

I'm very new to skyline so I apologize if there is an easy resource I have not been able to find.


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Unscheduled MS raw file
(1 response) aqassab 2019-07-31

I have uploaded a a raw MS files that was created using unscheduled SRM extending from 0-90 minutes. After I uploaded my file to Skyline I noticed that Skyline is showing my peptide in a scheduled time frame (for instance: 10 minutes or 20 minutes) instead of the whole 90 minutes. I tried to figure out whether it was some setting issue but I still couldn't make Skyline display the unscheduled run. I uploaded a zip file named "16 Glycopeptides" for that purpose. Could you please assist me to solve the issue?


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Skyline for small molecules - development plans?
(7 responses) takahashi18423 2019-07-29

Hello. I am a user of Skyline for proteomics applications and have been interested in where the development of the software for small molecules is headed. There are several approaches to filtering/processing HRMS data for identifying small molecule drug metabolites that will be great to have a vendor-agnostic data analysis solution for. In particular, algorithms such as background subtraction, isotope pattern filtering, or mass defect filtering would be key. Are any of these on the books for Skyline's development? I know it may be a long shot but I figured it is worth reaching out to find out.

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On-line Help Menu
(1 response) Richard Lam 2019-07-29

Is there any documentation of on-line help menu for Skyline, e.g. a description of what the setting(s) are?



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Not seeing 'small molecules' option for transition import
(1 response) Jason Held 2019-07-29


I'm new to small molecules in Skyline and following the tutorials it says to go Edit -> Insert Transition list. That works, but there is no radial dial for small molecules like in the tutorials. Ideas?

I'm using Skyline-daily (64) Screenshot of my import menu attached.


 Screen Shot 2019-07-29 at 3.19.20 PM.png 
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Data import problem
(2 responses) PeiLiu 2019-07-28

Hi Skyline team,

When I imported timstof pro MRM raw data (.d) into skyline, and the data contained 60min gradient time, but only ~25min chromatogram RT were imported, and no error report.

Data: timstof pro, MRM acquisition, TIMS OFF, raw data (.d)
Skyline: (64-bit), 4.2.0

Does anyone know the reason and how to fix it?
Thank you!


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(5 responses) dkueltz 2019-07-19

Hi Brendan, Nick,
Thanks for the new Skyline release!
It would be great if you could include another option under Refine - Advanced - Results:
Like filtering for a min dotp in the best or all sample(s) it would be great to have a similar filtering option for peak area (using the best or all samples);
This would greatly facilitate generation of assay libraries using a set of training samples. It would allow us to automatically remove peaks that are low abundance in all samples and likely to be very noisy.
We are now doing this by exporting peptide quant tables for training sample set result to csv and then identifying peptides with less than the minimal desired area, then select the ones that meet the criteria and use Refine - Accept peptides - peptides to keep. But this is cumbersome and there is a limit on how many peptides can be handled that way. It would be nice to have a more straightforward way to do this in Refine - Advanced - Results

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color mismatch
(2 responses) tkoller 2019-07-26

I am analyzing data in the ‘molecule interface’ and it seems that there is a color mismatch going on within the Peak Areas and elution plots. The most abundant ion in the elution is m/z 133, however, in the peak area it is shown as m/z238 (but it should be m/z 133). The other peak areas are also wrong: m/z 133 should always be the most abundant one. (Skyline (64-bit) on Windows 10 Enterprise).

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The difference between CV peak area and CV total area
(4 responses) Liangshuang 2019-07-24

Hi Skyline team,
When I look at the CV value of my data, why is the CV value of the peak area map in skyline inconsistent with the total area CV value I exported? I have tried non-normalization and normalization methods and none of them is useful. Is there any difference between “peak area” and “total area”? Or is there some problem with the settings of my skyline? Please see the attachment,thanks!


 The difference between CV peak area and CV total area.pptx 
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Peptide/ transition settings management
(4 responses) michel plisnier 2019-07-24


I have a question on the management of the peptide & transition settings.

To my understanding, when you receive a .zip Skyline data file (File/Share) the peptide and transition settings are saved within the data although the Settings .skys file is not part of the .zip compression... Is that correct?

Starting from those data, I would like to generate a Settings .skys file... to apply it on other raw data.
On my Skyline application the Settings/Share... menu command appears grayed out...
Do you have any suggestions on how to proceed to generate this .skys file?

Thank you,

Best regards,


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Global setting / adjustment of peak integration boundaries?
(2 responses) Matthias 2019-07-08

Dear Skyline-Team,

I was trying to get an idea of how reliable the quantification of peptides/proteins using different DIA Methods would be.
As a first and major read-out I tried to look at the points across peak value. If i understand this parameter correctly it should give me the number of points leading to the determined Intensity / area of each transition / precursor.
As a first start I modified the MSStats Input report by adding the PAP and filtering for "is not blank".

Using the Pivot Editor I focused on proteins with a max Detected Q value below 0.01 (see attached pdf showing screenshots).
Further pivot editing led to Mean Points across peaks per Protein for each replicate in each of the thee different conditions.
As a first and quick step I filtered for each replicate within each conditions and calculated the Average of the Mean Points across Peak values (shown in the table).

Although the values show the expected trend (higher values with shorter cycle time due lower max IT). The average values between 14 -16 seem to be a little high.

When going through the data I noticed that the integration boundaries for some peptides are nicely selected (e.g. AIGPHDVLATLLNNLK) but on the other hand for some peptides the integration boundaries seem to be too broad/ unspecific selected (e.g. LQTSSVLVSGLR).

Is there a way to specify global settings for peak integrations boundaries, e.g. more conservative or less conservative / more sensitive? Or does one have to go through all peptides manually and adjust the boundaries?

Best regards

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Include fragment ion annotation when "Copy Data" from Library Match tab
(1 response) jonasbecker 2019-05-08

Dear Skyline Team,

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

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

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

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

Thanks for your help in advance. Best,

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SKyline UI and Skyline runner dependent on each other?
(2 responses) shridevi patil 2019-07-23
Hello Skyline Team,

I need your help to understand behavior of Skyline UI instance and Skyline runner(command runner).
Use case is, I have a Test application which I would like to add to Tools menu in Skyline UI as external tool.

Now once I launch Test application from tool Menu, the Skyline UI instance is still running.
In my application, is an input accepted as command parameter from Skyline UI. That is the only input Test applications gets it from Skyline UI instance.
During execution Test application takes care of requesting data acquisition on MS system. Now once the raw data is ready Skyline runner(command runner) is invoked through the code to refine the raw data. (say during RT mode, CE mode and then to arrive at optimized method).

So **should Skyline UI instance be running till the Test application terminates? ** ( I am not using UI anyways after launching Test application).

Is there any dependency to keep Skyline UI instance running. Does Command runner needs it?

I did try closing the SKYLINE UI, and relaunching but previously running Test application will not be able to respond to SKYLINE(in case if I make it Interactive external tool) But in external tool is there any dependency between Skyline UI instance and command runner.?

I could not get much form documents(pdfs) So please help me understand it.

Thank you in advance. :-)

view request
Intensity in Skyline is much higher than Xcalibur
(2 responses) bin fang 2019-07-23

For the same peak, the intensity value in Skyline is much higher than the intensity shown in Xcalibur. However, if I click on the individual data points in Skyline, the spectrum will show the correct intensity.

view request
problems about quantity small molecular compounds
(5 responses) kaylee xu 2019-07-22


I have a question about skyline when I want to quantity the small molecular compound.

I want to get the concentration of SM d18_1-12_0 use the calibration curve of 18:1(d9)SM (Please refer to the attachment). Is it possiable?

Thank you very much!

 problems about skyline.pptx 
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Theoretical peptide z-ion mass appears to be incorrect
(10 responses) JHamey 2018-11-14

Hi there Skyline team,

I've come across a problem to do with the theoretical mass of z-ions in Skyline. They appear to be consistently off by 1 Da - they are predicted to be 1 Da lighter than they actually are. E.g. a z13 ion is predicted to be 1316.7 Da when it is actually 1317.7 Da. When I searched for threads related to this issue I came across this one (Issue 498):

It appears as if the "fix" that was made to make z-ions 1 Da lighter was done in error. The 1 Da heavier masses for z-ions I am getting from MS-Product match what I see experimentally for a normal peptide (it was noted in that thread that the peptide where the "error" was observed contained an abnormal gamma linkage at the position where the mass shift was thought to be wrong). C-ion masses are perfectly ok. I see this consistently for at least 5 different z-ions in the peptide.

Thank you in advance for your help.

view request
peptide RT prediction does not work.
(3 responses) wnstjr95 2019-07-20


I try to use iRT to predict the retention time of some peptides.
Without MRM data from QQQ, I tried to get the RT of some peptides.
But, I couldn't get any predicted retention time.

after importing MRM data, I could see the predicted and measured retention time.

I thought that I can simply get the predicted retention time since skyline uses the SSCalC.

Could you explain why it is not working? and If possilbe, let me know how to get the predicted RT without imporing MRM raw data.


view request
Problem during incorporating pep.xml in library creation
(1 response) Shalini Aggarwal 2019-07-19

I am facing issue while building the library using TPP analysed file (pep.xml file). The error it is showing is attached as screenshot below.

We have integrated this type of data earlier as well but did not face this type of problem. Kindly guide me.

Thank you,
Shalini Aggarwal

 skyline issue.JPG 
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Installation Issues: Skyline 4.2
(2 responses) leaptrkl 2019-07-10

I have been trying to install Skyline 4.2 64 bit (I also tried the daily install and the unplugged install files) and keep encountering the same error, "Your administrator has blocked this application because it potentially poses a security risk to your computer." I have attached a snip of the error box. I am running Windows 10 1809 Release.

I contacted our IT personnel for assistance. We have tried shutting off the firewall, app control, renaming the file (setup.exe), internet option reset, and none of these have worked. Also we signed in with domain account and still received the same error.

I tried looking through the support board for a similar issue, but didn't find another solution to try. Do you have any other suggestions?

Please and thank you for your time!

 Skyline Install Blocked.png 
view request
Import peptides AND charge states
(17 responses) gabe 2017-09-22
Is there a way to import a list of peptides AND charge-states into Skyline? I know I can import peptide sequences and have the charge-states auto-populated based on spectra in the reference library, but I can't figure-out a way to import specific charge-states into a document.


view request
error in VirtualBox: exception has been thrown by the target of an invocation
(2 responses) hec_vennbio 2019-07-19
Skyline (64-bit)
Oracle VM VirtualBox 6.0
Windows 7 Ultimate SP1
2GB allocated RAM, 2 processors

Hi, this is my first time posting, so please excuse me for any mistakes.
I run skyline on a PC emulator on my macbook. I had left a skyline document open, but "saved the machine state" in Virtual box and quit the program. The next morning, upon opening Virtual Box and the saved machine state an error popped up when I began using Skyline. Unfortunately I didn't save the error, but continued to use it and finished the analysis. I then saved the analysis and closed Skyline, but upon trying to re-open the saved skyline document, I am unable to open my document and the following error:

Failure opening Z:\190417_Transition List
Exception has been thrown by the target of an invocation.
OK More Info
System.Reflection.TargetInvocationException: Exception has been thrown by the target of an invocation. ---> System.Reflection.TargetInvocationException: Exception has been thrown by the target of an invocation. ---> pwiz.Skyline.Util.AssumptionException: Audit log is corrupted. Audit log entry time stamps and indices should be decreasing
   at pwiz.Skyline.Util.Assume.Fail(String error) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1918
   at pwiz.Skyline.Util.Assume.IsTrue(Boolean condition, String error) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1877
   at pwiz.Skyline.Model.AuditLog.AuditLogList.Validate() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\AuditLog\AuditLogEntry.cs:line 122
   at pwiz.Skyline.Model.AuditLog.AuditLogList.ReadXml(XmlReader reader) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\AuditLog\AuditLogEntry.cs:line 109
   at pwiz.Skyline.Util.XmlUtil.Deserialize[TObj](XmlReader reader, TObj objNew) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\Xml.cs:line 868
   at pwiz.Skyline.Model.AuditLog.AuditLogList.Deserialize(XmlReader reader) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\AuditLog\AuditLogEntry.cs:line 78
   --- End of inner exception stack trace ---
   at System.RuntimeMethodHandle.InvokeMethod(Object target, Object[] arguments, Signature sig, Boolean constructor)
   at System.Reflection.RuntimeMethodInfo.UnsafeInvokeInternal(Object obj, Object[] parameters, Object[] arguments)
   at System.Reflection.RuntimeMethodInfo.Invoke(Object obj, BindingFlags invokeAttr, Binder binder, Object[] parameters, CultureInfo culture)
   at System.RuntimeType.InvokeMember(String name, BindingFlags bindingFlags, Binder binder, Object target, Object[] providedArgs, ParameterModifier[] modifiers, CultureInfo culture, String[] namedParams)
   at System.Type.InvokeMember(String name, BindingFlags invokeAttr, Binder binder, Object target, Object[] args, CultureInfo culture)
   at pwiz.Skyline.Util.XmlElementHelper`1.Deserialize(XmlReader reader) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\Xml.cs:line 1121
   at pwiz.Skyline.Util.XmlUtil.DeserializeElement[TObj](XmlReader reader, Enum name) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\Xml.cs:line 885
   at pwiz.Skyline.Util.XmlUtil.DeserializeElement[TObj](XmlReader reader) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\Xml.cs:line 875
   at pwiz.Skyline.Model.AuditLog.AuditLogList.ReadFromFile(String fileName, String& documentHash) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\AuditLog\AuditLogEntry.cs:line 190
   at pwiz.Skyline.Model.SrmDocument.ReadAuditLog(String documentPath, String expectedHash, Func`2 getDefaultEntry) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2056
   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 305
   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

Does anyone know if I can salvage this document? Earlier saved versions of the skyline document (saved before the initial error appeared after shutting down VirtualBox), appear to be unaffected.

Thank you for any help.
 Screen Shot 2019-07-19 at 11.32.39 AM.png  skyline error.txt 
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Chromatogram information unavailable error with training files
(4 responses) Max 2019-07-18


i am a new user to Skyline - i want to use it for quantitative analysis of small molecule LC-MS data. The installation was no problem, however, i am walking through the webinar 16 and when I import the webinar training result files i do not get any chromatogram. It always gives me the Chromatogram information unavailable error. I am running on a german Windows 10 system - I have changed the settings for the decimal seperation to . an the number dividers to , as in the US. Can you please give me a hand.

cheers from germany,


view request
Notes, HMDB etc in report
(5 responses) henrik molina 2019-07-16

Dear Skyline Team,

For our analysis of polar metabolites we, in addition to names, composition RT, charge, also upload HMDB and KEGG info) [EDIT->INSERT->TRANSITIONS LIST].
However, we can not figure out how to get the HMDB and notes included when we export the report [FILE->EXPORT->REPORT].

Also, will it be possible to add a column for KEGG as well - so we can free up the 'Note' column (that we currently uses for KEGG).

Cheers, h

view request
Remove repeated peptides
dkueltz 2019-07-19

Another question: When using Refine Advanced Document Remove repeated peptides - does Skyline remove the extra copies of each peptide from the protein entries with the lowest number of peptides? This would be highly desirable to remove proteins with the lowest number of peptides from an assay library. For instance, if a protein is only represented by a single peptide that is also matching to another protein that is represented by multiple other peptides then it would make no sense to leave the protein represented by a single peptide and remove the peptide from the other protein that also contains additional peptides.
Thanks for any feedback on this question!

view request
How to add library intensities when exporting spectral libraries?
(1 response) Matthias 2019-07-19

Hi all,

I wanted to export a spectral library I generated using skyline.

I loaded the spectral library via
settings -> peptide settings -> library -> Explore -> Add all (with the box Associate proteins checked).

When trying to export the library now the column with the library intensity only contained #N/A (see screenshot).

How can I add the library intensity to the report?

Best regards and many thanks for any help

view request
Issue with Edit->Refine->Advanced->minTransitions per precursor
(1 response) Matthias 2019-07-18

Hi all,

I tried to import additional files into an already existing skyline analysis document.
Naturally, not all targets/transitions were found (see screenshot attached) in the new files which is why I was not able to use the library dotp when training the mprophet model.

According to a previous request ( I tried to use the advanced refinement settings and requesting for min 6 transitions per precursor (since I used also 3 MS1 precursor transitions).

However, the target list still contains peptides for which only 3 transitions were (only precursor transitions) were found and all the fragment transitions are missing.

This issue consists also in the latest skyline version (19.1) (tried it on another computer as well).

Does the Advanced -> min Transitions per precursor only check if at least 6 transitions are defined in the target list?
How can I filter those peptides out to enable library dotp in the mprophet model? (manually is not really an option since the document contains >150.000 peptides)

Thank you very much for any help

Best regards

 Peptide_missing_transitions.PNG  Refine_6transitions_2peptides.PNG 
view request
issue tools installing
(1 response) Wael 2019-07-17
Dear all,

I am trying to install the new AvantGardeDIA tool, but I always getting the following:

Downloaded failed.
Check your network connection or contact the tool provider for installation support.

System.Reflection.TargetInvocationException: Download failed.
Check your network connection or contact the tool provider for installation support. ---> pwiz.Skyline.Model.Tools.ToolExecutionException: Download failed.
Check your network connection or contact the tool provider for installation support.
   at pwiz.Skyline.ToolsUI.RInstaller.DownloadR(ILongWaitBroker longWaitBroker) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\ToolsUI\RInstaller.cs:line 187
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_19_1_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_19_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1868
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 180
   at pwiz.Skyline.ToolsUI.RInstaller.GetR() in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\ToolsUI\RInstaller.cs:line 137

I have tride to install other tools such as MSstats and QuaSAR, and I am getting the same error msg.
I have ran some diagnostics for my internet, netwrok, R and PC and all seem to be fine.

Can you please inform me if there is anything I have to do to get these tools downloaded.

best regards
view request
Bug in library generation
(1 response) Mike S 2019-07-15


I noticed that while building a spectral library from MaxQuant search results (v. and from Thermo Q Exactive HF raw files, the spectrum extracted by Skyline is off by one. For instance, MaxQuant matched scan number 28055 to a specific peptide, yet in the process of extracting the spectra from the raw file to build the library, it tried to match the spectrum with scan number 28056 instead (which obviously has no fragment ion matches). I double-checked that the match to the correct spectrum is very good with ProteinProspector and that my fragment ion matching tolerances are okay. It is quite evident that the wrong spectra are being extract based on the fragment ions themselves not matching.


view request
Need Cmdline runner commands for the following.
(1 response) kartik kundgol 2019-07-15

Hi Team,

We need Skyline Command Runner commands to refine the MRM transitions based on the following criteria:

  1. To identify RT of each peptide by looking for signal from multiple charge states for the same peptide overlaid.
  2. To identify the most intense precursors for each peptide.
  3. To identify a, b, y, z ions
  4. To rank orders each MRM/precursor charge state/peptide.( Selection of the top 2-3 MRM’s/ charge state/peptide for collision energy optimization.)
  5. To pick the best collision energy values for each MRM transition


view request
Issues in building library
(8 responses) nehasharma ns27 2019-07-11


I a new skyline user. I was able to run the software for the demo files that have been provided with the MS1 tutorial but now when I am trying to work on my data, I am having to issues:

  1. When I am trying to build spectral library from .mzid file, it is showing some errors (PFA: File No.1)
  2. When I am using .DAT format, although library is being built but chromatograms are not being extracted in the next step. There seems some missing files and even after processing the same, chromatograms are not being displayed in the final processing (PFA: File No. & 3)

Please help me with this.


 File No. 1.png  File No. 2.png  File No. 3.png 
view request
Min / Optimal Laptop Specs for Skyline 19.1 & MSStats
(1 response) bnewton2k 2019-07-15

Its Amazon Prime day. What are good specs for a new laptop?

view request
Results from collision energy optimization
(3 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
Failure during file import
(1 response) Matthias 2019-07-15

Dear all,

During the import of an mzXML file i received the following error:

At 13:35:
Failed importing results file 'E:\Skyline\DIA_variable_win\HEK_DIA_C3.mzXML'.
[SAXParser::parseAttribute()] Error at index 1025795:

However, this file was imported succesfully in previous skyline sessions.

Any idea why the import suddenly fails?

Best regards

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

Hello everyone,

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

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

Thanks a lot in advance for your time!

Roman (Heidelberg, Germany)

view request
Chromatogram information unavailable - metabolomics
(4 responses) sarahmacpherson676 2019-07-10

I am trying to analyze the metabolomics of a 13C-glucose labeling experiment using Skyline. The samples were run on Agilent 7890 GC and the .d directory files seem to upload successfully onto Skyline. However the Chromatograms are not visible with the message "Chromatogram information unavailable". I've increased the method match tolerance m/z, but still no luck.
The files are attached below.
Thanks for your help!

 U13C-glucose labeling 
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import peptide search from Mascot
(2 responses) io 2019-07-11
I am trying to import peptide search from MASCOT (.dat file and .mzid). I am able to see the library match but I cannot see the chromatograms.
With dat file I have an error of Missing results file.
With mzid file I have this error
ERROR: [References::resolve()] Failed to resolve reference.
ERROR: object type: struct pwiz::identdata::Contact
ERROR: referent list: 4
ERROR: reading file ESE10D2.mzid

OK More Info
System.IO.IOException: ERROR: [References::resolve()] Failed to resolve reference.
ERROR: object type: struct pwiz::identdata::Contact
ERROR: referent list: 4
ERROR: reading file ESE10D2.mzid

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

Could you help me?
Thanks in advance
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Cannot open wiff file from Sciex
(2 responses) kelvin chung 2019-07-11

I am totally new in Skyline, I am trying to open the wiff file by skyline,in "import peptide search", the software prompt that the file "is not a valid library input file"

I converted the wiff file to Mzml file, and do it again, however, the same error appear ("is not a valid library input file")

are all the wiff file be the same and shall be opened by skyline ?

Thank you very much

view request
Is there for me to create spectra library manually?
(14 responses) davidz 2019-07-08

Hi there,

We've compiled spectra library data ourselves, is there a way to build a spectra library for Skyline from tab delimited data?


view request
Insert Transition List for Small Molecule Analysis Problem
(2 responses) alejandro.cohen 2019-07-10

Hi Skyline,

I'm running into some problems inserting transition list for MRM analysis. Attached the file I am Edit>Inserting
Quite self explanatory. Four targets (RvD1, D2, LXA4 and Mar1), each target has it's own heavy (D5 labelled standard). I would expect for each precursor just two 'molecules', one light, one heavy with the corresponding transitions. Instead I get the following (Screen capture). The right precursors for the light precursors dont show up.

Any ideas???


 Capture.PNG  Skyline_LipidMediators_Final_Target-IntStd.csv 
view request
export isolation list: multiple scheduled methods for prm fails
Markus 2019-07-11

Hi Skyline team,

we are having issues when trying to export isolation lists for PRMs as multiple scheduled methods. We tried it in Skyline daily (versions & as well as in Skyline 4.2 but with the same result.

Here is what we are doing and what happens:

  • we have a skyline document with DDA MS1 data (196 precursors with according MSMS library), including custom retention time standards, which we would like to use to build a PRM assay
  • we have set the time window for scheduling to 5 min resulting in a max of 29 concurrent precursors per window
  • we would now like export multiple isolation lists with a max. of 20 concurrent precursors
  • when selecting Export Isolation List with the settings "Lumos, multiple methods, ignore proteins, scheduled" I cannot select less than 35 max. concurrent peptides. If I do there is no number of methods calculated and if try to export an error occurs saying: "An error occurred attempting to export. The number of required transitions 35 exceeds the maximum 15.".

What are we doing wrong? Why are 35 transitions required? (BTW, it might be a coincidence but 35 is the value of max. concurrent transitions for a 10 min window.)

Suggestions for solutions would be greatly appreciated!

Many thanks and kind regards,

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


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


 dotp label.pptx 
view request
Question about Autoselection 'Rules' for Peak Selection
mlane 2019-07-10

We use Skyline for targeted PRM experiments in less common approach: no library because we do HLA peptide screening. Since there is no spectral library, a list of peptides and most common transitions are supplied in the document, and we simply allow Skyline to include all matching spectra upon import and have it autoselect matching peptides. For autoselection, can you explain the prioritization Skyline puts on different factors such as transition intensity, number of transitions, shape, co-elution and mass accuracy in peak picking (if any or some of these are used)? There does seem to be a bias towards the most intense, but it also appears as if there is also requirement for some number of transitions and perhaps mass accuracy. If it uses these attributes or others, can you explain how? Thanks! Monica

view request
how to improve mprohpet model for difference between targets and decoys?
(4 responses) Matthias 2019-07-03

Dear Skyline Team,

I was analzing a DIA data set following the very nice and super helpful Webinar 14 (DIA large scale).

However when i came to the point to train the mprophet model I observed that in my data the difference between targets and decoys is not as nice as in the video and in the DIA Tutorial which was handed out during a course last year in Seattle.

Please see the attached pdf with some of the blots.

I was wondering how one could improve the target decoy discrimination?
Would this be achievable by e.g. demanding 6 transitions per peptide rather than 4?
(Like in the webinar where Brendan uses Edit => Refine => min 4 transitions per peptide) prior to adding the decoy peptides.

Best regards any thanks for any help.

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

Hi All,

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

Kind regards,

view request
Protein relative quantification
(1 response) tiziano brogna 2019-07-03


I have 2 biological conditions for which we monitore multiple proteins to check if they are up or down-regulated.
I have analytical replicates and we monitore multiple peptides for each proteins.

I would like to know how Skyline consider the variability for the different peptides used for protein relative quantification (at the end we obtain fold change ratio with error bar that are linked with the analytical replicates if I understood well)?

I based the method on this tutorial:

In other word, I have the variability for the analytical replicates and now I would like to know how Skyline use the peptides signals to give the fold change information for the 2 conditions tested.

Thank you,

Best regards,


view request
Failure opening ...\ - error on line X at column Y
(9 responses) vogelw 2017-04-21
Any thoughts on how I can recover from this error ?

Screen shot of ther error message attached: "The file contains an error on line X at column9

The XML section ending at that line is also attached.


      </precursor> # the line referred to in the error message

Nothing that I can see therein.

 Screen Shot 2017-04-21 at 11.31.35 AM.png  SylineErrorSection.xml 
view request
Analyzing PRM raw data files for small molecules
(2 responses) ssah9 2019-07-03


When working with PRM data, how do I make a transition list for multiple precursor-ion pairs? I followed the instructions given in the small molecule data tutorial and changed my settings according to the tutorial on targeted MS/MS data. I am not sure if that's how I should go about analyzing my prm files. the data is from a thermos qexactive


view request
DDA-light/heavy PTM on Lys
(5 responses) 2019-07-01

I have DDA data with samples containing light/heavy PTM on Lys residue. I don't have internal stds. I would like to see relative ratio of these PTM.
Q1. Is it possible to plot light/heavy of MS1
Q2. Can I make Skyline not to make C-term Lys-PTM? Skyline makes correct modification with light-PTM but with heavy-PTM it always makes on C-term Lys. I have to manually remove all of them.


 HYK_190701_Skyline question.pdf 
view request
Modifications file issue
(1 response) dbade001 2019-07-01

Im trying to build a skyline library with my maxquant search. All my other files have been fine, but for some reason whenever I try to use the files where the N-terminus was modified to have a heavy acetyl group, skyline says there is no matching mod for the N-terminal heavy acetyl group. The modifications file works fine for my other runs. It also appears that the heavy acetyl label on lysine residues is ok too. It only appears to have an issue with the N-terminus modification.
I will include a screenshot of the error message, as well as one of the area of the modifications file where this mod is listed.

Thanks for your help

 error 1.png  error 2.png 
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Displaying peptide mod in left target sequences
(2 responses) Erik de Graaf 2019-06-17

Hi all,

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

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

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


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No decoy peptides
(4 responses) sstoychev 2019-06-29


I'm trying to train an mProphet model using decoy peptides. These have been added to the document yet when trying to build the reintegration model it says "that no decay peptides are present?

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Panorama upload failure(s)
(2 responses) hans_vissers 2019-06-28

Dear Skyline team,

We are facing some small molecule and peptide document upload issues that appear to point to some common problems:

QC folders require that all documents use the same peptides, but they did not match. ...
For input string: "[M+H]"
Missing attribute drift_time for element measured_dt.

Could you advise please?



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Failed importing results- .wiff file from SCIEX triple Quad
(10 responses) sarahmacpherson676 2019-06-19

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

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

 error messages skyline.docx  cell_metabolomics.wiff  cell_metabolomics.wiff.scan 
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Skyline Spectral Libraries
(1 response) wei sun2 2019-06-28

I have a question regarding how to convert DDA .wiff file from Sciex TripleTOF6600 to build Skyline Spectral Libraries.

I know ProteinPilot can do it. But do you know any other ways without ProteinPilot?

We have the biopharmaView in house.


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Adding a heavy label to a modified peptide
(3 responses) zsu4 2019-06-27

Hello, Thank you very much for this wonderful program. I am trying to add a heavy 13C amino acid residue to a modified peptide but I haven't been able to do it. I've tried the settings tab --> peptides settings and I have also tried the edit --> refine --> advance --> add, but I don't see the I (C-term) bold or modified (+6). After adding the heavy 13C in settings, I go to the specific peptide do RMC --> Modify --> edit modifications, I can't see the heavy1 modification in the Isoleucine drop down list, created previously in settings.
Thanks a lot!

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TIC not importing/displaying correctly
(6 responses) thicks 2019-06-26

Hi all,

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



 skyline-import.PNG  skyline-import-zoomed.PNG  correct-chromatogram.PNG 
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issue with MSstats installation
(3 responses) seoyoung park 2019-06-26
I would like to install MSstats, but it showed me an error message as below. Can you please help me to resolve it?
Download failed.
Check your network connection or contact the tool provider for installation support.
OK More Info
System.Reflection.TargetInvocationException: Download failed.
Check your network connection or contact the tool provider for installation support. ---> pwiz.Skyline.Model.Tools.ToolExecutionException: Download failed.
Check your network connection or contact the tool provider for installation support.
   at pwiz.Skyline.ToolsUI.RInstaller.DownloadR(ILongWaitBroker longWaitBroker) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\ToolsUI\RInstaller.cs:line 187
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 232
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1854
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 180
   at pwiz.Skyline.ToolsUI.RInstaller.GetR() in C:\proj\skyline_4_2_x64\pwiz_tools\Skyline\ToolsUI\RInstaller.cs:line 137
Thank you,
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Document Grid - Reporting Options for Peptide Ratio Result
(1 response) davis 2019-06-25

Hi Skyline team!

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

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

Best! Sonnet

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

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

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

Dear Skyline-team,

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



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


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

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

I have tried the following so far:

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

Thank you in advance for the help!



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

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


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

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

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

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

Can you please help me figure out the problem?


ERROR: .mzid file contains an unsupported score type

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

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


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

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

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

Dear Skyline team,

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

First, some information about my experimental set up:

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

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

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

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

Thanks a lot for your help in advance!

Best wishes,

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

Dear all,

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

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

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

Looking forward a short reply.

Best regards,

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

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

Best regard!

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

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

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

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

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

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


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