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

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

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

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

  • A detailed description of your problem or question, including instructions for re-creating any problem that you are encountering. Screenshots are often helpful.
  • Your operating system, and the version of the software that you are using.
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Showing: limited to 100 requests
5MRM not shown at the same time
(10 responses) xin huang 2021-01-20

Hello Skyline team,
I am working on peptide quantification with Water QToF-MRM method, I have five peptides and five MRM methods for one injection. From Masslynx, I could see the 5 peptides are detected, and they have been divided into two function channels (four MRM in Function channel 2, and one MRM in Functional channel 3). However, when I import the raw. file into Skyline, I could see the four peptides MRM very well, but the one peptide in Function channel 3 behaved strangely (it did not pick up the peptide at retention time 3.27 min). I was playing with the fdc. file to change the function number to the same, it did not work. I attached the chromatogram I obtained from both Masslynx and Skyline, appreciate if someone could help with the data import. Thanks!


 5 peptide MRM from Masslynx.pdf  Peptide in Function channel 3 from Skyline.png 
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CE optimization
hjl 2021-01-20

Dear, Skyline team

First of all, I wanted to send a huge thanks for you to support us by this far useful software.

On the way i am using a CE optimization option in Skyline, I got a question which turned out to be misterious for me.

In my study, I targeted 8 compounds for collison energy optimization and the process was described below.

  1. Specified 3 transition per compound by direct infusion
  2. Based on specified transition, set methods for 1st CE optimization( step size 4, step count 6) = 1st analysis
  3. Analyzed and clarified the best CE for each transition
  4. On the basis of 1st result, set methods for 2nd CE optimization( step size 1, step count 3) = 2nd analysis
  5. Analyzed and acquired the final optimized CE for every transition each
  6. Compared two methods( one = original method earned by direct infusion ; the other = obtained by CE optimization)

My question is on the 1st optimization, I could clearly see that the CE optimization is needed for every compounds
then, after setting the optimized method( on the comparison step) Original data showed far better results than the other.
If the original data is better than modified one, what made the optimization result (especially that of 1st optimization) different ?

Thank you for your help as always.

 1st optimization.PNG  comparison.png 
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Installing/enabling external tool in Administrator install of Skyline
(3 responses) m j noga 2021-01-14

Dear Skyline Team,
We are using Skyline in a very restrictive environment where Administrator install is the only way to make it run for regular users. I am currently exploring the possibility to also use external tools to better integrate it in our workflows. I see I can install and run a tool with the Administrator account but this tool is not showing up in the Tools menu for regular users.
Is there any way to make Skyline discover tools installed by admin?
I see I can enable the tool manually by filling in the form through Tools->External Tools...->Add...->Custom and changing the command path for absolute path to the executable in Tools folder within Skyline installation directory. So while it allows me to achieve the goal, this is a rather complex workaround considering what my users need (it also requires manual installation of report and annotations). I wonder if there is a simpler way.
I see there was also a similar call in 2018: and I wonder what changed since then as Nicks suggestion was not very encouraging.
I will very much appreciate your help!

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Optimal analysis workflow for 500 sample DIA experiment?
(1 response) becky carlyle 2021-01-19

Hi everyone,

Not necessarily a specific Skyline request here, but a hope that members of the Skyline community can help guide our workflows. We have 500 biofluid samples from a neurology clinic that are currently undergoing DIA. We are using an in-house generated fractionated library.

The Core running these samples uses Scaffold DIA but it's not capable of handling large sample sets. I also dislike the “black box” aspect and would prefer an open source solution. I was wondering if this community would be able to offer thoughts on an optimal pipeline for analysis of this data - preferably something with the potential for parallelization (I think ideally we’d use AWS to spin up some clusters to do this work - we have plenty of experience doing this with other 'omics pipelines). We are a biomarkers group and have moderate experience with MQ, Skyline, and X!Tandem, but we are definitely not experts in this particular field. We're also completely over-run with working from home and childcare, so it's very difficult to find the time to thoroughly research the huge number of developments in this field over the past couple of years. I hope the community can help point us in the right direction!

Thank you in advance!
Becky Carlyle (Buck course attendee from a few years ago)

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Libraries and targeted analysis
(1 response) Babak 2021-01-19

Hi Skyline Team,

I would like to ask you to answer my questions please.

  1. In the Proteome Discoverer, I uploaded raw data from MS, divided into three types and from these three types I created three libraries – each type had its own library. Is there any difference to upload all three libraries into Skyline, in which I evaluate the data of PRM analysis, or to upload them as one common library?
  2. We did targeted analysis on MS and the samples were measured in triplicates. Is it better, during the process of evaluation in Skyline, to do the average (total ratio) of all three replicates or just to select the most suitable replicate?

Thank you

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Error importing Percolator v3.05 xml
(5 responses) damon barbacci22098 2021-01-19
I receive the following error when I try to import Percolator pep xml output into SkyLine.

ERROR: boost::filesystem::directory_iterator::construct: The system cannot find the path specified: "../sequest/"
ERROR: reading file 2021012_MSB50697A.perc.xml

Command-line: C:\Users\dbarbacci\AppData\Local\Apps\2.0\LY2EZ3ML.RV9\JVPER9HM.HDE\skyl..tion_e4141a2a22107248_0014.0002_2f1cb11a037aa924\BlibBuild -s -A -H -o -c 0.95 -i MSB50697_98A -K -S "C:\Users\dbarbacci\AppData\Local\Temp\tmpFB5A.tmp" "S:\Process Dev\Analytical Team\Damon Barbacci\MSBioworks_HUD01_HCP\MSB50697_98A.redundant.blib"
Working directory: S:\Process Dev\Analytical Team\Damon Barbacci\MSBioworks_HUD01_HCP
OK More Info
System.IO.IOException: ERROR: boost::filesystem::directory_iterator::construct: The system cannot find the path specified: "../sequest/"
ERROR: reading file 2021012_MSB50697A.perc.xml

Command-line: C:\Users\dbarbacci\AppData\Local\Apps\2.0\LY2EZ3ML.RV9\JVPER9HM.HDE\skyl..tion_e4141a2a22107248_0014.0002_2f1cb11a037aa924\BlibBuild -s -A -H -o -c 0.95 -i MSB50697_98A -K -S "C:\Users\dbarbacci\AppData\Local\Temp\tmpFB5A.tmp" "S:\Process Dev\Analytical Team\Damon Barbacci\MSBioworks_HUD01_HCP\MSB50697_98A.redundant.blib"
Working directory: S:\Process Dev\Analytical Team\Damon Barbacci\MSBioworks_HUD01_HCP
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 156
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paired comparison in Skyline
(6 responses) zdarula 2021-01-15

I have a SureQuant dataset for 50 paired normal-tumor tissue samples and I would like to ask your advice how to set up a paired comparison (like "nested experiment" in Proteome Discoverer). As the biological replicates are not independent I should have the tumor/normal ratio per sample pairs instead of annotating the samples into a normal (50 sample) vs tumor (50 sample) groups. Can I do this in Skyline? Thanks a lot.

A minor issue: unfortunately I set up the Skyline experiment on the peptide level and now that I want to assign these to proteins so as to get a protein-level fold-change I cannot do it without losing my corrected peak boundaries, e.g. skyline reintegrates peaks if I associate proteins to the peptides. Can I somehow bypass this?

Thanks a lot,

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Small molecule (non-global) standard concentrations
(4 responses) jrenders 2020-09-01

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

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DIA analysis; unable to remove precursors
(10 responses) narjis fatima 2020-12-25

Hi ,
I have recently done a DIA analysis of whole proteome CLL cells after drug treatments. I could not resolve the issue of precursor removal in my analysis. I have unchecked the include DIA precursor window in transition settings --> filter and added "Start by precursor" in the document grid as recommended by one of the expert here.
I am still getting this error (mentioned below)
I am analysis DIA results for the first time, and therefore I think I am having some issues with the processing of results. Can you let me know how to fix this issue.

I am also observing precursor and product ions both in the target section. I saw you mention some one that either precursor or product ions should be there. Could you let me know if this is the issue, how to fix it ?
Thanks a lot.

** Loading the required statistical software packages in R .....

** Reading the data for MSstats.....
** Peptides, that are used in more than one proteins, are removed.
** Truncated peaks are replaced with NA.
Error in SkylinetoMSstatsFormat(raw, removeProtein_with1Feature = TRUE, :
** Please check precursors information. If your experiment is DIA, please remove the precursors. If your experiments is DDA, please check the precursor information.

Can't finish analysis.

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FDR control for DIA data
(2 responses) Z. Jiang 2021-01-13

Hi Skyline team,

I am currently using Skyline to analyze my DIA data. I was wondering if there is any way for Skyline to calculate protein and peptide FDR rate? I found there is a "Reintegratie..." function under the "Refine" menu. I was wondering if this one does the FDR calculations? If so, how does it return the results of 1% FDR? If not, what's the function of this "Reintegrate"?

Thank you,

view request
Error when importing .speclib spectral library from DIA-NN
(2 responses) levasseurmaxence 2021-01-11

Hi Skyline Team,

I am trying to import a DIA-NN spectral library (.speclib) but get the following error message:

ERROR: Unable to read peaks for redundant library spectrum 1, sequence , charge 2.

Command-line: C:\Users\maxen\AppData\Local\Apps\2.0\WXXH5JG8.6M9\H2BPMA06.WGC\skyl..tion_e4141a2a22107248_0014.0002_5cab26a4bb7d0a9e\BlibFilter -b true "C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test\test.redundant.blib" "C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test\~SK41BE.tmp"
Working directory: C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test
OK More Info
System.IO.IOException: ERROR: Unable to read peaks for redundant library spectrum 1, sequence , charge 2.

Command-line: C:\Users\maxen\AppData\Local\Apps\2.0\WXXH5JG8.6M9\H2BPMA06.WGC\skyl..tion_e4141a2a22107248_0014.0002_5cab26a4bb7d0a9e\BlibFilter -b true "C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test\test.redundant.blib" "C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test\~SK41BE.tmp"
Working directory: C:\Users\maxen\Documents\Proteomics\bnpage_ms\skyline_test
   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 62
   at pwiz.BiblioSpec.BlibFilter.Filter(String sourceFile, String destinationFile, IProgressMonitor progressMonitor, IProgressStatus& status) in C:\proj\pwiz_x64\pwiz_tools\Shared\BiblioSpec\BlibFilter.cs:line 52
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 211

After reading that post I am pretty sure Skyline supports .speclib files but I nonetheless tried to convert the .speclib library to a .blib library using BiblioSpec. However, I get the same error message when I tried to import that .blib file instead (I assume that is how Skyline imports .speclib files in the background?).

Am I doing something wrong here? I am using the latest version of Skyline (

Thanks for your help!


 20210105_bnpagemsms_O7_speclib.tsv.speclib  20210105_bnpagemsms_O7_speclib.tsv 
view request
parse rules MS Amanda
(4 responses) kguehrs 2021-01-05

Hi Skyline team,

First of all: Happy New year with good health and ongoing success in all things of your personal and professional life.

I have tried to generate a spectral libraries from DDA runs with MS Amanda integrated in the newest Skyline version. In my first approach, I have used a limited but refined (adapted to UniProt headers) database. MS Amanda performed well and created a library as expected. In my second approach, I used the same DDA files but a larger but not refined in-house generated database. MS Amanda did all the searches and created the mzID files but failed to create the library due the failure to parse the database entries. The database was generated in our institute and has a header format that is obviously not recognized by MS Amanda in its default state of integration into Skyline. Unfortunately, the standalone version of MS Amanda is not very handy for generating results of multiple searches and I actually have no idea how to change parse rules in MS Amanda.

I there any documentation about the parse rules MS Amanda applies and which type of database headers are supported. This would be helpful to adapt the databases if necessary. The other way would be to have a possibility to adapt/change the parse rules when using MS Amanda in Skyline to generate libraries.

Best Karl-Heinz

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Peak Scoring with heavy peptides
(1 response) tilman werner 2021-01-08

Dear Skyline Team,

Happy new year - thank you for this amazing tool!
I have a question regarding peak scoring. I have run a PRM experiment with spiked in heavy peptides to detect several cytokines. Some we could clearly detect, but with others the results are shaky and we are not really sure if we should count the peptides as detected in our sample or not. The main issue is which peaks to already count as a fragment pattern and which ones to discard as noise.
I was wondering if there is a tool in Skyline that allowed us to calculate something like a Q-value for the peaks by using the heavy peptide peaks as input/template instead of a seperately generated peptide library? Or is there another way to discern signal from noise in the presence of heavy peptide standards?

Thank you for your great work: Skyline is by far the fastest way to go from measurement to shiny result!
Best wishes,

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Protein abundance issue
(1 response) francescodemetrio lofaro 2021-01-13

Hello there,

I'm a new user of Skyline. I imported my DDA search from Mascot and I'm able to quantify proteins in two different conditions using MSstats. However, using this tool i can't quantify protein inside samples, but I obtaine only log of fold chande between conditions. So, is it possible to obtain protein quantification for each sample using MSstat integreted in Skyline?

Furthermore, i tried to quantify protein using protein abundance report integrated in Skyline. However, there are different missing values (#NA) for proteins that show peptide intensity in Skyline interface. How can resolve this issue?
I hope to find a solution to quantify in MS1 my samples thanks to your help.


view request
Importing results does not show peaks (which I know are there)
(4 responses) barryh 2021-01-12

Hello there,

I am creating a very simple project for absolute quantitation, I selected my one and only peptide then I imported the file with the results (and while uploading I can visualize the peaks at around 2.5 min) and then when it is done importing the results the chromatogram window shows from 0 to 1.2 min and shows that there are NO peaks (See attached screenshot). I have tried everything to zoom out of that region and see the 2.5 min peaks (LC method is 6 minutes long) and I can't seem to find a successful way. Any advice would be greatly appreciated.

Thank you very much!

view request
Modifying Molecule Name in Target List
(1 response) davis 2021-01-12

Hi Skyline team,

When I try to rename ( right click on molecule in target list >modify>name) a molecule in my target list, the chromatographic vanishes following name change. Is there work around to avoid this?

Best! SSD

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PRM-quantitate with precursor or fragment?
(1 response) sallym8 2021-01-10

Hi Skyline Team!

Your software and resources are amazing- thank you for all your hard work! I recently ran my first PRM experiment on an Orbitrap Fusion and have been analyzing my data with Skyline. In the past I have run DDA experiments, but my protein is in a buffer with albumin and my peptides of interest have been getting drowned out. I have been able to use a large amount of protein and the DDA method in order to generate a list of peptides that I could include in my PRM experiment, but moving forward I will be limited in the amount of protein I can use. The goal of my experiment is to quantitate peptides in two regions of the protein in order to monitor a cleavage event. Currently, I am trying to compare the PRM and DDA experiments in order to determine how much the new method improved my signal. I am confused about whether I quantitate the PRM data using the precursor ions or the fragment ions and why. If you could offer some clarification that would be amazing!

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Different results from raw data that are nearly the same in MassLynx
(3 responses) anna walke 2021-01-07

Dear SkyLine Team,

I'm working with a simple method on a waters Premier Pro QToF to weekly check system performance by analyzing a standard of angiotensin II. Measurement is performed by Full Scan (function 1) and MS/MS scan (function 2) in parallel over the entire time range of the method.

Unfortunately when analyzing my raw data with Skyline (version I get very different results compared to manually analyzing the same data in MassLynx. I added screenshots of this and also attached the corresponding information of my LC-MS experiment. Within the experiment report I highlighted the different paramters in yellow, but there are only little differences.

I do not understand why the same SkyLine Document (also attached below) with same molecule and transition settings gives that different results after integration.

Could you please help me or give a hint towards a possible solution?

With kind regards,

 AGII Std 01_Experiment Report.pdf  AGII Std 16_Experiment Report.pdf  SkyLine AGII  Screenshots for SkyLine Support.pdf 
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Not able to install Skyline software on my computer (Windows 7)
(1 response) jeon 2021-01-07

Skyline suddenly stopped working a day ago.
We re-installed all skyline software and tried re-installing but it didn't work.

I've attached a word file with the error details and out computer operating system.

Please help up resolve this issue.


Marlene Thaitumu on behalf of Ju Eun (

 Error Message when installing Skyline software.docx 
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Analysis Strategy
(4 responses) klemens froehlich 2021-01-05

Dear Skyline Team,
First of all happy new year and thank you for your great work !

Yesterday a collegue approached me and asked about detection qValues with Skyline for some peptides he wanted to quantify and now wanted to have some numbers for his collaboration partners how sure we can be numerically that we detected the peptides with our PRM experiments.
I only had experience with peak scoring with DIA data, so I did some reading in the forum and the advanced peak picking tutorial: Similar problem I think is described here:

I came up with some general workflow and I would really appreciate it if you could share your thoughts:

I would first of all generate independent spectral libraries and retention time libraries for all measured targets. For this I would use the integrated PROSIT feature (awesome integration btw!)
Then I would go to reintegration and build an mPROPHET model based on second best peaks
However, when I try to do that, Skyline gives me an error saying I do not have enough targets (0 and 374 decoys), which is ironic because when I check the use decoys then Skyline says I do not have any decoys and I should uncheck the use decoys box......

Since I cannot measure the samples again and we are stuck with the PRM masses isolated during the measurements I was wondering whether I can get Skyline to accept decoys without the 10m/z mass shift usually wanted by mPROPHET. I would not like to use that in the final analysis but I would at least like to look at the comparison between this decoy scoring model and the second best peak scoring model.
I therefore generated decoys, exported them in a transition list, subtracted 10m/z from the precursor, added a "decoy" column filled with "true" and reimported it. But Skyline does not recognize the peptides as decoys but only adds them as normal targets (at least they are not red like my decoys normally are).

I think I screwed up the document somehow, but before I continue deeper into this rabbit hole I wanted to ask whether this is the right direction or whether I should try something else.

I will attach the skyline document.

Best Klemens 
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iRT on non linear gradient
(6 responses) benoit fatou 2021-01-04

Dear Skyline team,
I was wondering how I can use the iRT peptides for retention time adjustment when using a non-linear gradient.
Thanks for your help and Happy New Year !

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Grey dotted lines?
(18 responses) henrik molina 2020-12-16

I am using Skyline to extract MS1 signal of small molecules (Q-Exactive +).
Once in a while I come across cases where what I think is an obvious peak has not returned an area THOUGH Skyline have clearly marked - with grey dotted lines - what the correct integration should be. What does the grey dotted lines mean?
I have attached an example: the same sample injected 2x. For sample B, the area was ok integrated. No area for sample A. Both sets of dotted lines appears to have marked the correct peak.

I can easy manually mark what should be integrated - but I am curious why the software only integrated this molecule in Sample B.

I am running Skyline

Cheers, henrik

 Auto-Skyline -Dotted grey lines - no signal.pdf 
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(4 responses) ziyang zhang 2020-12-22

Hi again skyline gods,

I recently had an issue when importing a DDA search (done by MaxQuant). The peptides carry a custom modification "ARS", which is defined in the modifications.xml file. When importing, Skyline prompts that there is no matching modification for "ARS".

I did the following:

  1. adding modifications.xml file to the data folder containing msms.txt
  2. adding modifications.xml file to the spectral library folder under the user home folder
  3. adding modifications.xml file to replace the skyline installation folder (with new App installation tool, this is now at C:\Users[user]\AppData\Local\Apps\2.0\\\skyl..tion_xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
  4. adding a modification in peptide settings named "ARS" with the same formula as in modifications.xml

But still cannot import the search result - the error persists. Could I please get some help...

Many thanks,

 Skyline_Mod_NoMatch.png  Mod_Def.png  modifications.xml  msms.txt 
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transition detection issues
(3 responses) clemence balty 2020-12-17


I am trying to quantify peptides in a biological sample using the light / heavy ratio (using the Sure quant method by thermo). I have designed heavy peptides corresponding to the peptides that I want to quantify in the biological sample. First, I created a spectral library with heavy peptides. Then I added these heavy peptides to all samples (potentially containing the light version of each heavy peptide). When I opened the raw file in Skyline, I detected heavy peptides and their transitions except for two heavy peptides (SGYSSPGSPGTPGSR and TPSLPTPPTR). These peptides are present when I manually check the raw file. I don't understand why skyline can't detect the transitions of these two peptides.

Can you help me or give me advice to solve these problems?
Is the library required to perform quantification?

I can share my raw files and the library I created with you.

Thank you,

 peptides list.xlsx  SQ_Method_Development_20201120.skyd  transitions issues.png  Tau_Aqua_skyline.blib 
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Must have MS1 scans in PRM methods?
(4 responses) ziyang zhang 2020-12-16

Hello fantastic people at skyline,

I am quite new to quantitative mass spec and have very limited sources to learn - hope you don't mind these very basic questions...

  1. Is it mandatory that MS1 scans be include in PRM methods for skyline to perform quantification? My current method only has an inclusion list for precursor masses to cycle through. When I analyze the data acquired this way, Skyline is able to extract the ion chromatograms but cannot quantify the results. Also giving red tags for all the transitions and dotp=0. Screenshot attached.
  2. I have gone through the tutorial ( without much issue. I do wonder if there is a way for plot the extracted chromatograms for a particular transition on the same plot (overlaid)?

Again, I know these questions may be due to simple lack of understanding of skyline features or mass spec fundamentals. If there are tutorials somewhere that can save you some frustration with my ignorance or time explaining simple concepts, please do point me to them!

Many thanks in advance!


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Target non-monoisotopic ion
(1 response) Zac 2020-12-17

I want to target a non-monoisotope peptide ion. I have used mono and average before, is there a way to work with ions outside of these two options. The isolation width will be close to 0.4 m/z

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Cannot load Daily installation file onto any pc's that do not already have it installed
(1 response) jgb2 2020-12-16

Every time I try to download a daily installation file onto a pc that does not already have it installed, I am prevented from completing the installation and shown the attached prompt. Sent email to Brendan as prompted then redirected to support board.

 Screen Capture Daily install 202012161.PNG 
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Peptide Atlas Download for human library build
(1 response) daraghob 2020-12-16

I am attempting to generate a new spectral library to achieve better target peptide coverage for a transition list I am generating for a future MRM experiment.

My issue is trying to discern which file to download on the peptide atlas builds page (
There are many options for the human build but I can't seem to find the appropriate data format (.pep.xml) for the library build.

Any help with this would be greatly appreciated.
Many thanks,
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A fixed and variable modification on the same amino acid
(10 responses) laura declerck 2016-07-13

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

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

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

Kind regards,
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Issue With Retention Time Selection
(3 responses) ed3 2020-12-11


I am trying to import a pepXML file from PEAKS Online into Skyline to generate a peptide library. However, I am having an issue when doing this as for some reason Skyline is not selecting the appropriate retention time for the peptides in the pepXML file. I have attached a picture of this issue to illustrate what I am talking about. In the attached picture, Skyline has selected the retention time to be 0.02 minutes for the selected peptide; however, the actual retention time of this peptide is 15.20 minutes. Is there a way to resolve this issue?

Thank you in advance for your help, and please let me know if any clarification is needed.


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Error when importing iproph.pep.xml
(1 response) ztflaten 2020-12-07

I am trying to build a spectral library with an iproph.pep.xml document I've generated and I keep running into various forms of this error
ERROR: index 17017 out of range in mzXML file '.\P2.mzXML'

Command-line: C:\Users\pc\AppData\Local\Apps\2.0\5PYCWJGV.XX2\M6EMW1MM.TJV\skyl..tion_e4141a2a22107248_0014.0002_be619ba2d44825a4\BlibBuild -s -A -H -o -c 0.99 -i fragpipe_dda_2 -S "C:\Users\pc\AppData\Local\Temp\tmp79D2.tmp" "C:\Fragpipe_Skyline\fragpipe_dda_2.redundant.blib"
Working directory: C:\Fragpipe_Skyline
OK More Info
System.IO.IOException: ERROR: index 17017 out of range in mzXML file '.\P2.mzXML'

Command-line: C:\Users\pc\AppData\Local\Apps\2.0\5PYCWJGV.XX2\M6EMW1MM.TJV\skyl..tion_e4141a2a22107248_0014.0002_be619ba2d44825a4\BlibBuild -s -A -H -o -c 0.99 -i fragpipe_dda_2 -S "C:\Users\pc\AppData\Local\Temp\tmp79D2.tmp" "C:\Fragpipe_Skyline\fragpipe_dda_2.redundant.blib"
Working directory: C:\Fragpipe_Skyline
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 156

I am not sure I understand what the error is telling me but I think it has to do with the location of the files it's trying to find (which are located in the same directory in a sub folder). I've tried moving files around in my directory but keep having issues, even when they are all in the same folder as the saved skyline document.


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"resolving protein details" problem AND lost library problem upon install of newer Skyline version
(3 responses) cabarnescabarnes 2020-12-05

Hi all,
Thanks for the great software. I am using the EncyclopeDIA work flow to get DIA data into Skyline. I imported an .elib library out of EncyclopeDIA that seemed to be working and everything was working great until one day I got the "resolving protein details" problem. I was using Skyline, so I uninstalled that and then downloaded Skyline and installed that. Now, I'm getting a library problem. Everything else seems to be working. Here's the error code:

Skyline version: (64-bit)
Installation ID: 5b133551-e4fe-4406-b30c-a9e78485df9a
Exception type: Exception
Error message: Failed loading library 'C:\TPP\data\smORF_Experiments\Mouse_plasma_inflammation_young_old_smORF\Mouse_plasma_inflammation_young_old_smORF_spectral_library_w_plasma_fractionations_Joan_chromolib_quant_report.elib'.
SQL logic error or missing database
no such table: LibInfo
Format: BiblioSpec
FileRevision: 0
SchemaVersion: 0

System.Exception: Failed loading library 'C:\TPP\data\smORF_Experiments\Mouse_plasma_inflammation_young_old_smORF\Mouse_plasma_inflammation_young_old_smORF_spectral_library_w_plasma_fractionations_Joan_chromolib_quant_report.elib'.
SQL logic error or missing database
no such table: LibInfo
Format: BiblioSpec
FileRevision: 0
SchemaVersion: 0 ---> System.Data.SQLite.SQLiteException: SQL logic error or missing database
no such table: LibInfo
at System.Data.SQLite.SQLite3.Prepare(SQLiteConnection cnn, String strSql, SQLiteStatement previous, UInt32 timeoutMS, String& strRemain)
at System.Data.SQLite.SQLiteCommand.BuildNextCommand()
at System.Data.SQLite.SQLiteDataReader.NextResult()
at System.Data.SQLite.SQLiteDataReader..ctor(SQLiteCommand cmd, CommandBehavior behave)
at System.Data.SQLite.SQLiteCommand.ExecuteReader(CommandBehavior behavior)
at pwiz.Skyline.Model.Lib.BiblioSpecLiteLibrary.CreateCache(ILoadMonitor loader, IProgressStatus status, Int32 percent) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLite.cs:line 605
at pwiz.Skyline.Model.Lib.BiblioSpecLiteLibrary.Load(ILoadMonitor loader, IProgressStatus status, Boolean cached, Exception& failureException) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLite.cs:line 949
--- End of inner exception stack trace ---
at pwiz.Skyline.Model.Lib.BiblioSpecLiteLibrary.Load(ILoadMonitor loader) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLite.cs:line 927
at pwiz.Skyline.Model.Lib.BiblioSpecLiteLibrary.Load(BiblioSpecLiteSpec spec, ILoadMonitor loader) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLite.cs:line 150
at pwiz.Skyline.Model.Lib.LibraryManager.LoadLibrary(LibrarySpec spec, Func`1 getMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\Library.cs:line 295
at pwiz.Skyline.Model.Lib.LibraryManager.LoadBackground(IDocumentContainer container, SrmDocument document, SrmDocument docCurrent) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\Library.cs:line 124
at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
Exception caught at:
at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
at System.Threading.ExecutionContext.RunInternal(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state)
at System.Threading.ThreadHelper.ThreadStart()

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Trouble reading wiff files. Does not contain SRM/MRM chromatograms.
(1 response) stephan kamrad 2020-12-05

I am trying to quantify peptides using our Sciex QTOF 6600. I have made a document of my targets in Skyline and exported an acquisition method. The acquisition method looks good to me and contains multiple 'Product Ion' experiments, one for each of the precursors. There is no MS1 full scan experiment as this was not included in the template. I think this shouldn't be an issue as I only want to quantify on the MS2 level. When I try to load the data in Skyline, I get the following error. This persists after converting to mzML with MSConvert.

The file xxxxx.wiff does not contain SRM/MRM chromatograms. To extract chromatograms from its spectra, go to Settings > Transition Settings > Full-Scan and choose options appropriate to the acquisition method used.

Does anyone have any clue where the issue could lie? Is there any reason why Skyline shouldn't be able to deal with these data files in principle?
The same seems to work when my acquisition method also includes an MS1 experiment (as I have done previously)

Thank you,

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Is there any availiable tool for export skyline results and deal with the results?
(1 response) 16622080827 2020-12-02

I find the exported results format is just liake this:
Protein Name Protein Accession Peptide Modified Sequence Point Count Protein Gene Replicate Name
sp|A0AVT1|UBA6_HUMAN A0AVT1 YQC[+57]VVLTEMK 0 UBA6 500ng_1
sp|A0AVT1|UBA6_HUMAN A0AVT1 YQC[+57]VVLTEMK 0 UBA6 500ng_2
sp|A0AVT1|UBA6_HUMAN A0AVT1 YQC[+57]VVLTEMK 0 UBA6 500ng_3

But actually,I want the result like this:
Protein Name Protein Accession Peptide Modified Sequence 500ng_1 500ng_2 500ng_3

This format is more friendly for my data analysis.So is there any .skyr file for exporting the quantification in both protein and peptide level?I know that skyline support exporting the results mannuly.But there're too many informations here.So I want some useful information by default. And for the data format transformation,is there any avalible tools here?

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Hide from View
(2 responses) Jan Sklenar 2020-06-09

Hi Brendan,

I would like to be able to hide some chromatograms from view, without completelly removing them from document. Is it possible? The reason is, I like to keep all data for future purposes, but wish to compare only some runs in my QC data document.


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MS1 filtering mode for lipids
(6 responses) Eric Merkley 2020-12-01

Is there a way to use Skyline in an MS1 filtering mode similar for what you would use for peptide untargeted DDA data?

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Issue with Skyline transition list export
(7 responses) Shalini Aggarwal 2020-10-27
Dear Skyline team,

I am facing an issue with the export of the transition list from skyline daily software. It is showing the below shown error. I cross-check each step but still unable to figure out the issue. My colleagues tried the same thing on their PC and it worked. Kindly guide me with the error rectification.

thank you in anticipation,
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Specifying Integration Boundaries
(20 responses) jrenders 2020-11-09

Hi there,
My small molecule analysis methods produce some pretty ugly peaks for a few analytes. Skyline does its best, but I do not expect any integration algorithm could figure out some of my data. What I have been searching for to fix the problem is some place to specify explicit integration boundaries. In reading back on support, I understand that "explicit retention time" and "window" will get me close but these are more "suggestive" to skyline (and in fact do not work for my peaks). Integrating a peak and then rt-clicking and hitting "Apply Peak to All" also seems more suggestive than explicit and does not work. I would like to have skyline integrate all peak area between two specified RT's without any consideration to inflection points or anything else and then apply this setting across the whole batch for that particular molecule. Is that possible at this time? Thanks!

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Sequential protein extraction analysis
(1 response) francescodemetrio lofaro 2020-11-30


I would like to performe a DDA analysis on three sequential protein extraction on each sample comparing two different conditions. My idea is to import DDA search from Mascot engine merging all technical replicates of all fractions together and import the dda search on Skyline.

  1. How i can specify that .raw files are fractions of the same sample?
  2. Is it possible to consider the total amount of the same protein combaning peaks of the three fractions of the same sample?

Thank you for your help


view request
Adding peptides manually in spectral library
(1 response) mdshadman abid 2020-11-30

I am using software PEAKS for the peptide search in my samples and also using these database search from peaks as the spectral library in skyline. However there are some peptides present in the sample but peaks could not detect somehow. That is why when I am generating a spectral library, I am missing those peptides. Is there any way to add/input peptide sequence manually in the spectral library?
I tried by going to Edit- Inset-peptide- then just manually typed the peptide sequence to add the peptide in the library, but I think it's not proper way of what I want to do.
In case I am unable to ask my question, I am giving an example. I know the peptide LYQNKPRRPYIL is present in my sample but PEAKS software couldn't detect it, so it is also missing in spectral library as I am generating library from PEAKS. How can I manually add this peptide in my spectral library?

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CE optimization on Agilent QQQ
(4 responses) Sarah Michaud 2017-11-06

I getting some strange results when optimizing collision energies for an Agilent 6495 QQQ using Skyline. I am generating the transition list for CE optimization using skyline, then using the method to acquire the data, and importing the data back into Skyline. After importing the data, sometimes the transition results look fine and as expected, but other times the pattern in the peak area graph is really unusual (almost random instead of a curve shape), and other times the results are missing completely. When I check the same data file in the Agilent MassHunter software, the data appears to have been acquired correctly and the peak area pattern is normal. I've attached some slides with examples to try and show what I'm seeing.

I think the problem might be that for some transitions the product m/z value is for some reason different in the acquired data file compared to the method generated by Skyline, and when that data is imported back into Skyline the peak areas associated with each CE step gets jumbled.

Thank you for the help!

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NIST library with Unimod, but untypical modifications
(2 responses) solis 2020-11-26

Dear Skyline team,

thanks for your continuous improvement of the software and the tools within it.

I have an issue with Skyline recognizing "Propionyl" modifications in a NIST library as well as Methy and Propionyl modifications co-occurring on the same site. As you may know, these PTMs are relevant in the context of some protocols for histone PTM analyses. Could you please let me know if indeed this is a current limitation of Skyline or if something is wrong with my attached NIST library instead?

And if the former is the issue, you could then maybe make Skyline able to recognize any modification as long as its name is in the Skyline Modifications list.

Thank you very much and my best regards.

Victor Solis

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Incorrect modification (TMT) mass detected in the spectrum file
(1 response) ab 2020-11-25

I am trying to build a spectra library from a TMT tagged sample to evaluate and select peptides for a PRM experiment. I defined the peptide and transition settings as recommended in Webinar #17 and included the modification information for TMT at K and N-term. When I explore the built library, I see at he N-term modification is marked as [+228.1511?], the precursor m/z is accordingly 1 Da less than what is reported by my peptide search in proteome discoverer (SEQUEST) and therefore I am not able to evaluate results for any of my proteins/peptides of interest. I am wondering why this is the case. I am including an image as an example.

Thank you,
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Quantifying with surrogate standards
(1 response) Joerg 2020-11-24

I am tring to quantify different lipid classes using several surrogate standards. Although I set the respecitve molecules as "surrogate standard" and set the "Normalization Method" to the respective standards for the other molecules, there is no option to normalize the area view to these standards. Nor is it possibl to use the surrogate standards in the Quantification tab of the "Molecule Settings". I am obviously missing something...
Many thanks in advance!

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Chromatogram information unavailable after importing mzML files
(3 responses) Andreas 2020-11-24

I am analyzing Lipid data in the Small Molecule Mode of Skyline. I can import the Sciex MRM wiff files. However, importing peak boundaries changed the first sample of the batch only. Therefore, I converted the wiff file to mzML files using Msconvert (ProteoWizard). Unfortunately, after importing the results as mzML some lipids could not be imported with certain samples (Chromatogram information unavailable, see attached files). Are there special parameter for Msconvert required for usage with Skyline?  LPX_STDs_mzML_ajh.png 
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ppm-level calculation
(2 responses) laura nieder 2020-11-23

Hi, thank’s for the great software and the really nice tutorials. Is there any way to let skyline calculate the ppm-levels of a certain protein (using for example 3 peptides for quantification) in sample A regarding to a housekeeping protein in the same sample?
I always have the same two Proteins in my samples. Protein X with a steady concentration, protein Y with changing levels. Usually we do absolute quantification with an external calibration curve. This time I would like to get the ppm levels of Y regarding the concentration of X within the same sample. Similar to HCP analysis.
Is there a possible way, that Skyline calculates this for me?
Thank you,

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Trouble finding spectrum files - can they be input through a command line interface
(2 responses) jharrison 2020-11-23

Hi Skyline team,

Thank you for making such a useful software. I want to do some ms1 filtering in some data and I have been having some issues when I try to open the .pep.xml file. I keep getting the error message below and I do have the .mzML file in the appropriate folder. I think the issue is that I am using VMware fusion on a mac to run windows and I think it is somehow frustrating your auto search tools. I am wondering if there is a way I can pass the spectrum file into skyline through a command line interface like skyline runner. Or maybe I am doing something else silly that is giving me an issue. I am grateful for any help you can offer.

System.IO.IOException: ERROR: Could not find spectrum file 'NA[.mz5|.mzML|.mzXML|.ms2|.cms2|.bms2|.pms2]' for search results file 'WT_Roos_1.pep.xml' in current directory, ../,../../.


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Annotation of Light peptide fragments instead of heavy fragments
(3 responses) nyalwijo 2020-11-22

Dear colleagues,
I have PRM data for a SIL peptide. THe MS2 fragmentation data is perfect but Skyline annotates fragments for the Light peptide which are not in the MS2 spectrum instead of the Heavy peptide fragments. How can I correct the issue? Thank you. J

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Precursor Report: Duplicate lines?
(4 responses) alejandro.cohen 2020-11-19

Skyline people... again requesting your help. Here a curve ball:

I'll try to summarize:

I'm developing a targeted SIM (tSIM) method for 18 steroid hormones using a LC-QExactive and Skyline's Small Molecule interface. For each molecule, I have a light and deuterated standard. ALSO, for each molecule, I'm monitoring both the [M+H] and [M+Na] precursors. Reason being, these samples are ocean water derived, so I'm concerned Na might not be easy to completely removed during sample prep, and signals will likely be split (I'm clearly seeing this in the data I've analyzed so far, even with the standards).

I've followed the following tutorials which were very helpful:

So, I have one question and one concern.

Q: For the calibration curves for any of the molecules, I see that they remain constant regardless whether I select the [M+H] or the [M+Na] (Curves DO change selecting different molecules, of course) . Does that mean the areas of both [M+H] and [M+Na] are summed up to make the final areas? Does this also apply to their Internal standards and the ratios? I'm finding it hard to navigate the tables to see how the data is compounded together.

Concern: in my precursor-Quant table, for each Replicate, I see TWO lines having the same column content (Rep, Rt, Precursor, Molecule, Ion Formula, Precursor Adduct, etc etc) EXCEPT the Area, Area Ratio and Background. Attached a screenshot.

Could you help me out with this? Thanks again for your fabulous work with Skyline.

 Screenshot 2020-11-19 151723_double entry.jpg  Screenshot 2020-11-19 153841.jpg 
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Question about iRT prediction in skyline
(2 responses) 16622080827 2020-11-15

I have used iRT kit(Biognosis)in my experiment and I have also detected all the iRT peptide in my data both in PD software or check mannually.But when I try to import the PD result, it always shows that RT prediction failed.Maybe some peptides are in low intensity.Faced with this problem,what can I do for this condition?

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Small molecule: calibration curves for individual targets
(2 responses) alejandro.cohen 2020-11-18

Hi !

I'm developing a targeted SIM method for 18 steroid hormones using Skylines Small Molecule interface. So far so good.
I have prepared calibration curves for each standard (3 concentration levels) separately and run them on a LC-MS (QExactive). What I mean is that I did NOT pool all standards together in the same tubes, rather, have a separate calibration curve for each standard.

Replicate 1: Std X at 0.1ng
Replicate 2: Std X at 1ng
Replicate 3: Std X at 10 ng
Replicate 4: Std Y at 0.1ng
Replicate 5: Std Y at 1ng
Replicate 6: Std Y at 10 ng
Replicate 7 :Std Z at 0.1 ng, etc etc

In the Replicate Tables, I have already classified them as Standards and their concentrations as in tutorial.

Is there any way to inform Skyline which Molecule each Standard corresponds to? By default, Syline tries to find ALL Molecules in each each Replicate. Manually removing peaks from each Replicate is very time consuming.

All tutorials I have read so far combine all standards into single replicate injections.



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Train Drift Time predictor - "Skyline Drift Time Predictor Training" tutorial
(5 responses) s1899812 2020-11-09


I am following the "Skyline Drift Time Predictor Training" tutorial and when I was going to train the drift-time predictor on page 17/26, the Prediction tab under Peptide Settings is only showing me a "Retention time predictor", but not a "Drift time predictor". I have attached an image to show this.

Do I need to do anything to add the Drift time predictor?

The link to the tutorial can be found:


 Skyline Peptide Settings.png 
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MPPARRReport tool not working
(7 responses) roman koziy 2020-11-16


I have constructed a library from DDA SpectrumMill search results in order to evaluate MS1 spectra. I further wanted to export the Skyline results into Agilent MPP software for analysis. However, whenever I try to run the MPPAPRReport tool I get an error message (please see attached). Could you please suggest what could be the problem?

Thank you very much for your help,

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Skyline external tool using SkylineTool not able to import small molecule blib from directory using AddSpectralLibrary command
(3 responses) Chris Ashwood 2020-11-13

Hi Skyline Support Team,

I hope you are doing well.

I am currently trying to fix a new error that I have tried to fix with no avail. Has the new Skyline updates (daily or stable) changed how they handle external tool directories and paths? Or the SkylineTool?

The C# app I am developing loads small molecule spectral libraries using the SkylineTool with the following command:
_toolClient.AddSpectralLibrary("NGlyCat_Library", NGlyCatBlib);

Skyline appears to be unable to find the blib file (as shown in red in the attached image) despite the blib being in the tool directory. To my knowledge, this command has not caused any issues until I updated to the newest Skyline versions. I also get an error (shown below) that the document must be fully loaded so I cannot share the full Skyline document with the Share button.
System.IO.IOException: Settings.PeptideSettings.Libraries: null library
at pwiz.Skyline.SkylineWindow.ShareDocument() in C:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1185

I rolled back my daily version but it did not seem to fix the issue. And I also tried an older version of my program that worked fine and it also failed.

Any help would be appreciated because I'm going a little crazy trying to find what I changed on my end to cause this issue. I have included the Skyline assay and spectral libraries in the uploaded file (


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peptides with mixtures of modified and unmodified residues
(24 responses) Keri 2018-05-09

Hi Brendan,

I have attempted to use the peptide settings > modifications feature to quantify samples that contain both fixed and variable modifications (e.g. some but not all cysteines in a peptide are isotopically labeled (+521 or +527) and all cysteines are carbamidomethylated +57). I input both the structural modification for carbamidomethylation and the variable modifications for the additional isotopic label, but when I apply those settings, I see multiple modifications, including the sums of multiple modifications (e.g. 1022, 1028, which are not correct). Can Skyline handle samples with mixed modifications and, if so, how?

Thank you


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Variable MRM time windows
(1 response) benoit fatou 2020-11-13

Hi Skyline Team,

I was wondering if there is any way in Skyline to generate variable MRM time window for each peptide when exporting the method or transition list.
I am asking you because some of my peptides have a broad peak width so if I set a very short time window, I would crop them out. On the other hand, making a short time window for the peptides with very small peak width might increase their sensitivity because we decreased the number of concurrent transition pairs.

Let me know if you need more information or if you have any question.
Thanks for your help,
All the best,

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Silicone isotope pattern: is it possible to add it?
(2 responses) marcel reimann 2020-11-13

Hi Skyline Team,

I´m using Skyline in a kind of "off-label-use" for my GPC-MS. For that purpose it would be great to add also the silicone isotope pattern. Is there a way to add this to the skyline "automated isotop calculation"?
Adding it to the transition list import would be a nerve wrecking work.

Thank you in advance,

best Marcel

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MS1-Filtering small molecules: Threshold - "empty samples"
(1 response) marcel reimann 2020-11-13

Hi Skyline Team,

I´m using Skyline in a kind of "off-label-use" for my GPC-MS.
Sometimes it happens, that I dont have a Signal at all when using the MS1-Filtering. No noise, no error, just nothing. Using the same transition list on the next sample (more concentrate) it works.
Could it be that there is a "hidden" threshold? I couldn´t find anything, would be great to know where it is.
Could be also the case that I have to much precursors with one name. For example I have 47 precursors with 3 charge states each. Is this too much for skyline? In some cases I got "signal" when I removed some precursors.

Thank you in advance!
best Marcel

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spectral library from Peaks Online pepXML
(4 responses) Tharan Srikumar 2020-11-11

Hi Skyline team,

I'm trying to use exports from Peaks Online v1.4 to create a spectral library in Skyline daily (yesterday's release). I've exported pepXML, MzIdenML and a mgf. If I rename the pepXML file from the original .xml to pepXML, Skyline begins to read the file, but throws an error looking for the spectra file. It seems like Skyline is not looking for the mgf file but rather several other formats (see screenshot attached). I've also tried to rename the mgf file to the same filename Skyline is requesting with no success either.



 Screenshot 2020-11-11 123329.jpg 
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typing sequence name
(2 responses) gabriele philipps 2020-11-09


when I paste an entire amino acid sequence into the left pane in order to identify peptides according to my peptide settings, I am always struggling with filling in the sequence name, i.e. replacing the "sequence 1" name with my protein name. By some strange reason the name flips back to "sequence 1" after some seconds and I have to type my protein name a second time an then it will remain.
Is there any trick to keep already the first entry?

Thanks a lot for your help!

view request
Set Pause Time and Dwell Time
(6 responses) benoit fatou 2020-10-27

Dear Skyline Team,

I am working on the LCMS 8060 from Shimadzu and I was wondering if it is possible to set the Pause Time and the Dwell Time when exporting the methods from Skyline.
Thanks for your help,

view request
peptide prediction tool (stability and signal strength)
(1 response) gabriele philipps 2020-11-09


I have a rather general question which is not necessarily related to the Skyline software:
is there a software that can predict which peptides are better or worse suited for targeted proteomics (with respect to stability, retention time and above all signal intensity of the fragments which to my knowledge correlates with their ability to ionize)?
At the moment I know I should avoid cysteine and methionine and N-terminal glutamine and asparagine. But a tool which gives an overview on the peptide properties (when pasted in ) together with a judgement on the suitablility would be very helpful.

I hope you can help. Thanks for every suggestion.
Best regards,


view request
predict peptide from the first amino
(1 response) spklongz 2020-11-09


I want to predict peptide starting from the first amino acid (N-term) of a protein. How to set parameters of Skyline?

I varied Skyline parameters, while it failed to do that.

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Spectral Library Creation
(1 response) pg23 2020-11-06


I would like to create a spectral libray for a targeted method set-up similar to the one that is included in the Targeted Method Edit Tutorial (from Yeast).
I have been searching for potential spectra at PRIDE, but I fail to download the right format to upload in the library (Peptide Settings). Where can I find suitable files for spectral library creation?

Thank you

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Error 0x800F0954 when installing Skyline
(1 response) alvaro sanchez 2020-11-06

I could not install Skyline because of that error.
It says:
The following feature could not be installed:
.NET Framework 3.5

Any solutions?

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Drif time predictor
(7 responses) mariangela valletta 2020-10-30

I have skyline 20.2 64 bit on my Windows 10. To get familiar with skyline and ion mobility data , l followed the tutorial 'Skyline Ion Mobility Spectrum Filtering' and I downloaded zip file. I read that with new version of skyline IMS filtering settings moved to Transition Settings - Ion Mobility. My problems is: 'Use Results' button is not clickable. Why?

Thank you

 Drif time Predictor.pptx 
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Why Can't Peptide Search Be Imported Without Full-scan MS1 Filtering?
(3 responses) philip remes 2020-11-03

I'm importing search results into Skyline from a DIA chromatogram building experiment. After the search results are imported, and Skyline asks to Configure the Full-Scan Settings to Import the files, I want to select 'None' for the MS1 Filtering. I thought part of the advantage of DIA is that the parent ion may not be visible in the MS1 Full Scan or may have a high probability of having interferences, so why require that it be processed? There may be no MS1 scans, or they may be of such low resolution that they are not helpful. I realize I can then cancel and import the data the usual way after setting up Transitions. But I wondered if this MS1 filtering requirement could be waved for DIA and PRM peptide search imports.


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Need Skyline 32bit (f09d9bed5)
(2 responses) wnc4 2020-11-04

Hi, I am working with a collaborator and they are using an older version of skyline. I would like to import the current transitions everyone else is using but I do not have the same software version. I have the newer software version. Where can I download the older version from?

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Error on Importing after installing a UV/VIS detector
(1 response) chris brown-2 2020-11-02

Hello -

I received an error message when attempting to import data to Skyline

Up until I configured a UV/Vis detector on the instrument I had been able to import data just fine. After installing that detector, Skyline is unable to import the .raw files. If the work around is just to de-configure the UV/Vis detector, that is fine.

I am attaching the error message that is returned in the import box below.

Thanks, CJB

At 8:15 AM:
Failed importing results file 'D:\Studies\Lumos-DailyQC\Data\100ppb-HypersilGold_20201031014802.raw'.
error reading spectrum controllerType=4 controllerNumber=1 scan=1

Inner exceptions:
Exception type: System.Reflection.TargetInvocationException
Error message: error reading spectrum controllerType=4 controllerNumber=1 scan=1
error reading spectrum controllerType=4 controllerNumber=1 scan=1
at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1909
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.NextSpectrum() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 915
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatogramsLocked() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 258
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatograms() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 238
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.SetRequestOrder(IList`1 chromatogramRequestOrder) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 575
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 381
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252

Exception type: System.Exception
Error message: error reading spectrum controllerType=4 controllerNumber=1 scan=1
error reading spectrum controllerType=4 controllerNumber=1 scan=1
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.ReadSpectrum(Int32& i) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1125
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.Read() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 952
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.<RunAsync>b__33_0() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 809

Exception type: System.Exception
Error message: [RawFileThreadImpl::getTrailerExtraValue(SPS Masses Continued:)] GetTrailerExtraValue
[RawFileThreadImpl::getTrailerExtraValue(SPS Masses Continued:)] GetTrailerExtraValue
at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, DetailLevel detailLevel)
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetCachedSpectrum(Int32 scanIndex, DetailLevel detailLevel) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1056
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetMetaDataValue[TVal](Int32 scanIndex, Func2 getValue, Func2 isUsableValue, Func2 returnValue, DetailLevel& detailLevel) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1116 at pwiz.ProteowizardWrapper.MsDataFileImpl.GetMetaDataValue[TVal](Int32 scanIndex, Func2 getValue, Func2 isUsableValue, Func2 returnValue, DetailLevel& detailLevel) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1123
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetMsLevel(Int32 scanIndex) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1128
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.LookaheadContext.GetMsLevel(Int32 index) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1359
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.ReadSpectrum(Int32& i) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1036

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Import Library Assay for small molecules process?
alejandro.cohen 2020-10-29

HI Skyline people

I'm developing a PRM (Targetted MS/MS) method on Skyline from data acquired on LC-QExactive setup on the latest Skyline daily (20.2.1)

I have already:

1- Generated the molecule list and imported it into Skyline (Edit>Insert>TransitionList). I have so far the Precursors but NOT selected the Fragments.
2- I already acquired DDA files on all my standards (I have a single .raw file for each standard with good MSMS spectra for each one)

I'd like now to add the 'transitions' (Fragments) to the PRM method on skyline. I've looked at tutorials and webinars, and I cant find one that specifically explains the process to Import Assay Library for PRM. I'm hoping this could be done automatically from the DDA experiments on the standards (.raw files) instead of manually inserting the PRM transitions through the Edit>Insert>TransitionList option.

I notice the Import Assay Library requires a .csv,.tsv file as input. Could you take me through the generals steps from going from the .raw files (DDA of my stds) to the Import Assay Library for small molecules and which programs to use at every step? I can handle the rest of the steps such as importing the results of the PRM acquisition, etc etc.

Thanks again




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Calibration curve data export
(2 responses) Zac 2020-10-28

I want to reproduce the calibration curve data in graphpad prism, when i export the data i see two sets of data one 'Standard' and one 'Calibration Curve' each with columns 'Analyte concentration' and 'Normalized Peak Area'. What i am confused about is that there are more rows of data for the 'Calibration Curve'. 68 rows for Standard and 100 rows for the Calibration curve. Based on the number of calibration points and replicates i expect 68 rows of data. Should there be extra for the Calibration Curve, are means for replicates included? Thanks for all your great help and support.

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wiff2 file from Sciex Echo MS fails to import to skyline
(4 responses) michelle robinson 2020-10-26

Hi Brendan,
I'm unable to import MRM data generated on the Sciex Echo MS into skyline (wiff2 file format). I am using Skyline daily. The error message says, "[Experiment2ImpI::ctor()] Object reference not set to an instance of an object"
Thanks for your assistance,

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Peaks of light and heavy peptides have unequal peak boundaries
(9 responses) fcsigloch 2020-10-22


In an MRM experiment, I came across the problem that Skyline picks non-coeluting peaks for the light endogenous peptide and the heavy labelled reference peptide (screenshot attached). I never observed this behaviour in PRM data, where I am used to perfectly matching peak boundaries.
I could not determine a specific setting that caused this behaviour. Can I somehow force Skyline to choose the same peak boundaries for light and heavy trace?

Also, I tried to train a default or mProphet model to improve the peak picking. Training the model worked, but when I want to apply it to the data, I get the following error message:

Failed attempting to reintegrate peaks.
The index 6 must be between 0 and 2
OK More Info
System.Reflection.TargetInvocationException: The index 6 must be between 0 and 2 ---> System.Reflection.TargetInvocationException: The index 6 must be between 0 and 2 ---> System.IndexOutOfRangeException: The index 6 must be between 0 and 2
   bei pwiz.Skyline.Model.Results.ChromatogramInfo.GetPeak(Int32 peakIndex) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromHeaderInfo.cs:Zeile 2773.
   bei pwiz.Skyline.Model.TransitionGroupDocNode.UpdateResults(SrmSettings settingsNew, SrmSettingsDiff diff, PeptideDocNode nodePep, TransitionGroupDocNode nodePrevious) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\TransitionGroupDocNode.cs:Zeile 1487.
   bei pwiz.Skyline.Model.TransitionGroupDocNode.ChangeSettings(SrmSettings settingsNew, PeptideDocNode nodePep, ExplicitMods mods, SrmSettingsDiff diff) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\TransitionGroupDocNode.cs:Zeile 1116.
   bei pwiz.Skyline.Model.PeptideDocNode.ChangeSettings(SrmSettings settingsNew, SrmSettingsDiff diff, Boolean recurse) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\PeptideDocNode.cs:Zeile 979.
   bei pwiz.Skyline.Model.SrmDocument.<>c__DisplayClass142_5.<ChangeSettingsInternal>b__7(Int32 i) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:Zeile 1080.
   bei pwiz.Common.SystemUtil.ProducerConsumerWorker`2.Consume(Object threadIndex) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProducerConsumerWorker.cs:Zeile 186.
   --- Ende der internen Ausnahmestapelüberwachung ---
   bei pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:Zeile 1944.
   bei pwiz.Skyline.Util.ParallelEx.LoopWithExceptionHandling(Action loop, Action`1 catchClause) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:Zeile 2246.
   bei pwiz.Skyline.Util.ParallelEx.For(Int32 fromInclusive, Int32 toExclusive, Action`1 body, Action`1 catchClause, Nullable`1 maxThreads) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:Zeile 2190.
   bei pwiz.Skyline.Model.SrmDocument.ChangeSettingsInternal(SrmSettings settingsNew, SrmSettingsChangeMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:Zeile 1068.
   bei pwiz.Skyline.Model.MProphetResultsHandler.ChangePeaks(IProgressMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\MProphetResultsHandler.cs:Zeile 172.
   bei pwiz.Skyline.EditUI.ReintegrateDlg.<>c__DisplayClass12_1.<OkDialog>b__1(IProgressMonitor pm) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\EditUI\ReintegrateDlg.cs:Zeile 133.
   bei pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:Zeile 254.
   --- Ende der internen Ausnahmestapelüberwachung ---
   bei pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:Zeile 1944.
   bei pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:Zeile 202.
   bei pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:Zeile 140.
   bei pwiz.Skyline.EditUI.ReintegrateDlg.OkDialog() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\EditUI\ReintegrateDlg.cs:Zeile 135.

Thanks for your help!

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MRM triggered MSMS
(4 responses) heyang 2020-02-04

Dear Skyline team,

I want to compare MRM triggered MS2 with library spectra. Could I do this in skyline? if yes, how?



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Precursors per sample injection thinks it is transitions per sample injection
(2 responses) philip remes 2020-04-04

Hi Brendan,

This is a repost of issue 723. Sorry I didn't know to first post to Support and not Issues.

I've attached the skyline file. To reproduce what I saw,

  1. File->Export->Isolation List
  2. Instrument Type -> Thermo Fusion. Interestingly, if I use the first Instrument in the list, Agilent QTOF, and enter 216 in max precursors, the number of methods is correctly set to 9
  3. For Fusion, if 216 is set to max precursors, the number of methods is 138.

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MS1 filtering: is manual XIC window mass tolerances possible?
(5 responses) alejandro.cohen 2020-10-23

Hi Skyline,

I'm using Skyline for a targeted metabolomics project on an QExactive (running at 70K resolution @ 200m/z according to Thermo). I inserted this value in the Transition Settings> Full-Scan>MS1- filtering>Resolving Power tab.

My standards are all showing at the expected Rt and with mass errors <2ppm. However, Skyline is also labeling other peaks between + - 2 to 9ppms. Is there any way to manually reduce the XIC mass windows, or set mass error thresholds to reduce the 'labeling clutter' in the chromatogram windows? Or do I just have to increase/fake the Resolving Power value on the Transition Settings pane.

Thanks again for you continuous support, Skyline rocks!


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Import Peptide Library from PEAKS Online from TIMS TOF data
(12 responses) ed3 2020-10-09


I have been trying to import a peptide search from PEAKS online into Skyline but I keep getting an error and I was hoping somebody could please help me understand what is going on. The spectrum was acquired with a Bruker TIMS TOF, and when I try to import the pep.XML file in the "Peptide Search" option, I get an error saying "The .pep.xml file is not from one of the recognized sources." I have the IMS-TOF spectrum in .mzXML format in the same folder as the pep.xml file, and I am not sure why I get this error.

Thank you very much for your help.


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Problem library creation for cross-linking
(1 response) tatianibl 2020-10-22

I am trying to build a cross-linking library using an output created by proxl from plink 2.0. However, I am having a problem. Follow bellow the error message.

All the best.

ERROR: No spectra were found for the new library.

Command-line: C:\Users\tatiz\AppData\Local\Apps\2.0\49BTDOGX.MMZ\O1B3EWR8.PN0\skyl..tion_e4141a2a22107248_0014.0002_e354297bfe67025e\BlibBuild -s -A -H -o -c 0.95 -i teste -S "C:\Users\tatiz\AppData\Local\Temp\tmpA7B.tmp" "C:\Users\tatiz\Desktop\Arquivos_desktop\sample_input\teste.redundant.blib"
Working directory: C:\Users\tatiz\Desktop\Arquivos_desktop\sample_input
OK More Info
System.IO.IOException: ERROR: No spectra were found for the new library.

Command-line: C:\Users\tatiz\AppData\Local\Apps\2.0\49BTDOGX.MMZ\O1B3EWR8.PN0\skyl..tion_e4141a2a22107248_0014.0002_e354297bfe67025e\BlibBuild -s -A -H -o -c 0.95 -i teste -S "C:\Users\tatiz\AppData\Local\Temp\tmpA7B.tmp" "C:\Users\tatiz\Desktop\Arquivos_desktop\sample_input\teste.redundant.blib"
Working directory: C:\Users\tatiz\Desktop\Arquivos_desktop\sample_input
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\pwiz_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 156
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Confusion with Skyline and Proteome Discoverer
(3 responses) Mark Athanason 2020-10-20

Hi all,

First off thanks for providing the support that you do, hope I don't bother you all too much.

For right now I'm fine using the proteome discover software, but ultimately I want to perform DIA quant on similar data using skyline. Within PD, I perform a search using sequest and peptide validation with percolator with an FDR of 1%. Using the "import DDA search" wizard in the most recent version of skyline daily, I use the .msf file with a cut off score of 0.99. What I get in skyline doesn't seem to match what I see in PD at all. For example PD says I have 411 protein groups, but in skyline when I check remove repeat and duplicate peptides, I'm left with 222. Shouldn't these be the same since percolator is filtering for high confidence peptides? additionally, for some peptides PD does not correlate an MS1 trace where skyline does ... in my opinion incorrectly.

I'm uploading the .msf file, "Ibu1_DDA_1.raw" "Ibu1_DDA_2.raw" and "Ibu_DDA_3.raw" to

Any help would be greatly appreciated!

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Some peptides show incomplete peaks but in raw data they are okay
(17 responses) yingzhu1992 2020-10-05

Hi Skyline team,

I imported my result into the Skyline, some peptides show incomplete peaks, I checked from the raw data, they look fine. Can you please check and explain it for me and is there anything I can do to solve this?

And I also upload the raw data.

 20201001 H+L all  Capture.PNG  Raw data.PNG 
view request
HMDB-msp file import problem
(7 responses) Joerg 2020-10-14

we tried to import the HMDB for small molecule quantification into Skyline. We downloaded the msp-file from Oliver Fiehn´s website. Skyline import part of the database, but just "forgets" the majority of the data. Is there any way to change the msp-file in a way that Skyline recognizes all entries? Or is import of the "original" xml-file from the HMDB website possible?
Many thanks in advance!

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Storage location for custom PTMs
(2 responses) dkueltz 2020-10-07

Hi Nick, Brendan,
I have generated quite a few custom PTMS for one of my skyline documents (peptide settings/ modifications/ add) and would like to have them available for future documents. Where are these added PTMs stored? With only the Skyline document I entered them for or somewhere central?

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installation problem
(1 response) niyatti 2020-10-17

Hi, I tried to install Skyline (64 bit) on 64 bit Dell home basic desktop with Windows 7 through google chrome. The install.log is attached. Any idea what is wrong?

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Problem building spectral library
(3 responses) itv005 2020-10-15


I am a farily new user of Skyline, and I am struggling to build my spectral library. Aside from some of the tutorials, my training is very limited. The source of my spectral library is a search from Proteome Discoverer 2.4 that I exported to a pep.xml file. I also have a used MSconvert to generate mzml files from my raw files.

I suspect that my issue is not due to any of the settings I made when setting up skyline, therefore I have not included those in this request. As seen in the attachment, I get an error saying the wrong directory is used, but I cannot seem to find out how it chooses this directory, or how to change it. I have put the skyline file, the raw files, and the converted spectrum files all in the same folder. The only thing missing, as I see it, is the program itself. However, I struggle to see the logic if this is the missing link, as this would be impractical for further projects.

I used the 20.2 version of Skyline and Windows 10 Home. The data I have is a non-targeted HPLC-MS/MS of CSF. I wish to use this library in a PRM project which is ready to be analyzed.

Thanks in advance! Let me know if I should add more info.

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Problems with import of peak boundaries in smallmolecule mode for .wiff data
(4 responses) Max 2020-08-24


i am trying to import peak boundaries for a number of data sets based on .wiff data. We have set up some small molecule transitions for small molecules and peptides (due to qunatifiaction porpuses). I have used the allready discussed workarounds for small molecules (see which is perfectly working for data from e.g. waters were a single filne represents a single injection. However, in the case of AbSciex 5500 the data is stored in a single .wiff file. I added therefore the sample name (alias Sample identifier) column to the peak boundaries list as stated in the FAQ for importing peak boundaries (see here: Nevertheless, skyline gives me an error message back stating that the sample names are not fitting to the sample names in the raw file . We have not changed the names of the single sample upon importing in Skyline thus i am looking for help to resolve this. I know i am missing something but i can not figure it out....

Looking forward to hear from you guys!


PS: I have example files ready but can only send them confidentally.

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Skyline using questions
(5 responses) chaoxue 2020-10-05
  1. Can skyline show the same precursor and same product, but with different transition settings, such as CE and RF lens?
  2. Can skyline show different monoisotopic transitions, such as M+1, M+2?
view request
Reduction in the number of true peptide identification as the number of DIA raw files increases in the spectral library search
(13 responses) Chinmaya k 2020-09-28


I have carried out a Spectral Library search for one of our DIA data generated with a 25m/z isolation window from 400-1000 m/z range. The spectral library search of single DIA.Raw file is able to identify 8000+ peptides with Q-Value < 0.01 from mProphet model. However, when multiple replicates of the same sample (Which includes the DIA file used for the first search) were searched against the same spectral library keeping all other parameters as the same is resulting in a drastically less number of peptides (<5000) with Q-value < 0.01 from mProphet model. And this goes on as the number of files increases.

What might be the reason for this? Why the number of true peptide identifications is decreasing as the number of DIA files are increased?

Prior to applying the mProphet model, the peptides RT was calibrated using Pierce iRT standard spiked in the sample. The screenshots of parameters used for RT prediction and calculation are attached below for your references.

Please let me whether any parameters have to be changed and if so, why? How does it will effect the DIA Spectral library search and statistical validation?


 Peptide_Setting.PNG  Edit_iRT_Calculator.PNG  Edit_Retention_Time_Predictor.PNG 
view request
Identifying a significant protein in Skyline but it's not found in raw files.
(1 response) renad almahdali 2020-10-15

Dear Skyline team,

First, thank you loads for this amazing efficient software!
I am now working on constructing human proteomics profile from PBMC samples using Sciex TripleTOF 5600 and DDA method. I happened to come across a significant (highly expressed) protein in Skyline but when I wanted to check it in the proteins list exported from ProteinPilot raw file I didn't find it. My question is, is it possible to find a protein in Skyline software only but not from the original files?? I even tried to look it up by its protein name and accession including it synonyms.

Waiting for your support guys,
Renad Abdulrahman

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Unable to retrieve application files. Files corrupt in deployment. Skyline-64_20_2_0_286 unplugged version
(1 response) h a ebhardt 2020-10-15

Hi Brendan & team members of the Skyline team.

I tried installing Skyline-64_20_2_0_286 unplugged.

I first downloaded the zip file,
unpacked it,
ran setup.exe as administrator

Then: the setup.exe still accessed the internet and after downloading 3/4 of the program, the "Unable to retrieve application files. Files corrupt in deployment" occurred.

I attached a screenshot of the error plus the txt of Details...

There is a two problems here: first, the error itself.

Second, and more important, my IT dep't does not allow me to use programs which access the internet. Hence, the unplugged version. Is it possible to get an unplugged version which does not require the internet for installation?

Maybe the error is related to the fact that programs here are not allowed to access the outside world.

Kind regards,
Holger Alexander Ebhardt.

 skyline202error.jpg  26BB66Q3error.txt 
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fail to recognize spectral library from NIST
(3 responses) 851813663 2020-10-13

Dear nick
I have downloaded the Rat's spectral library from NIST, it was in msp format, and when I wanted to use the skyline to build the library, I found that the software could not recognize this format, and I was very eager for your help.
Thank you sincerely

view request
manual selection and change of ion mobility range for prm-PASEF
(2 responses) RBl 2020-10-14

Dear Skyline Team,
thank you very much for your constant efforts and all that you provide to the mass spec community! The software developed very much over past few years when i did not use it a lot!

I have started using Skyline in combination with the TimsTOF Pro recently. I needed to create a prm-PASEF method also with the ion mobility dimension. I noticed that ion mobility range that is showed in the Skyline was in many cases on the edge of the mobility peak/ion cloud and thereby a big proportion of the peak was not included. In some cases the ion mobility range in Skyline was in the middle of the mobility peak/ion cloud but edges were missing. I prepared some pictures so you can get the impression of what I am talking about. Please see the file attached.
I tried to set the ion mobility ranges in Skyline but I failed. Is there an option available for manual selection of ion mobility range or is it on me and I can not find it?
If there is no option for manual selection of ion mobility range, would it be possible to get something similar as manual chromatographic peak selection for Ion mobility peak/ion cloud in the future? And in the more distant future something like automatic peak peaking for ion mobility dimension if that is possible at all?

May you need more information from me, please do not hesitate to contact me.

Thank you very much for your efforts and your answer.
Best regards,

 ion mobility in skyline.pdf 
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Transition settings ion mobility "use spectral library mobility values when present" cannot be unchecked
(1 response) pierre-olivier schmit 2020-10-14

While unchecking the corresponding radio button and choosing "OK" : the radio button is automatically checked again if we re-open the transition settings ion mobility tab.

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ERROR: No Spectra were found for the new library
(1 response) lake n paul 2020-10-13


Excited to see the DDA search implemented into Skyline. However, I tried unsuccessfully to run a search. The search goes to completion however I don't get any spectra for the detected peptides. The XIC for the peptides are present but no MS/MS spectra. An error (see attached) is given.



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Skyline CMD help
(7 responses) smanda 2020-08-13

Hi Brendon/Kaipo,

I am trying to build a spectral library all using command line as we are trying to automate some of the steps at our lab. To this, I am unable to figure out, how can I choose peptides standards for RT calibration (after I import the library). My current steps are:

  1. SkylineCmd.exe --import-search-file=searchresults.mzid --import-fasta=20190717_Uniprot_concat_decoys.fasta
    This step creates a nr library and adds the peptides as targets.
    I do not specify RT peptides here.
    I then import a report format

  2. SkylineCmd.exe" --report-add=skyline.skyr

  3. SkylineCmd.exe --report-name=OpenSWATH --report-file=test_report.tsv --report-format=tsv

I see that the exported reported as several NAs in the iRT column. We have around 40 endogenous peptides that are added as iRT standards in all experiments, can you please let me know, how to I add them at RT peptides and export the report with iRT values.


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Some bugs shown in software
(4 responses) fenglinshen yzu 2020-10-13

Some peptides can't show the intact peak when we analysed MRM HR results using skyline which was the lastest released. Only three peptides were analysed with a 15 min gredient and 7.5 min-window was set. We also found the same scan data imported to the latest released skyline software showed only three transitions which was set as "full scan", and when the data was imported to earlier version, all of the transitions were shown. The details are shown in the attachments.

 新建 Microsoft PowerPoint 演示文稿.pptx 
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Unable to see any fragment ions in Skyline
(2 responses) clgunth2 2020-10-13

Hello - I created a test transition list with ions I can see in the raw mass spectra and imported into Skyline. When I imported the data, only the precursor mass is identified with the green circle next to the precursor name, even though the ions in my transition list were extracted from the raw file. The fragment ions don't have a red circle for being not detected but rather say "Chromatogram not available" if clicked on. I'm not sure if it's just not reading my data correctly or not. Again, these fragment ions were chosen directly from the raw spectra to test the workflow in Skyline, so they are present in the raw data.

My data file contains only MS2 scans for one precursor. My goal is to be able to filter through a much longer list of theoretical fragments for one complex structure. I'm following similar to what I would do if I was filtering through a transition list with MS1 data except the data file contains solely MS2 scans.

Crystal  SkylineError.PNG 
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Skyline 20.2 refuses to install
(5 responses) Tomas Vaisar 2020-10-13

For some reason, the manual install of 20.2 does not want to go - after Microsoft Store message - clicking Install Anyway does not do anything. It does not matter whether I run setup.exe normally or as Administrator?
My Windows 10 problem or Skyline setup.exe problem?



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MS Amanda search result files?
(3 responses) dkueltz 2020-10-12

Hi Nick,
Are the msamanda search results stored somewhere in a format that can be imported into Scaffold or other viewers? It would be useful to have those search result files for comparison with other search engines.

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Customise reports
(3 responses) giulia lambiase 2020-10-09


I happen to analyse lots of data and would like to automate the data analysis process. Is it possible to customise Skyline reports to make them run scripts for you?


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Question New Protein Abundance Report Feature
(4 responses) roman sakson 2020-10-12

Dear Skyline team,

thank you for providing us with the possibility to get numbers representing protein abundance per replicate directly from Skyline, which is also supposed to be part of the next main Skyline release. I intend to use this option rather frequently in my research and would like to understand it a bit better. I am mainly interested in the normalization case against heavy (each light signal must have a heavy counterpart to be considered).

In the release notes, you state that this protein abundance value is the same that the Group Comparison (GC) framework uses. I thought that, as default, on protein level GC sums up all transition areas that belong to the same protein (also across different peptides) that are quantitative and have a heavy counterpart and then normalizes this sum to the sum of heavy signals. This is slightly different compared with calculating normalized ratios transition by transition and then averaging those ratios, since strong signals dominate the sums of values, as previously described on several occasions. However, the explanation in the reporting window for the "Protein Abundance" feature now states that "The Protein Abundance is calculated by taking the average of the normalized areas of all of the Transitions under the Protein." This now sounds to me exactly as the latter option to do it and the question is, whether protein abundance numbers really are exactly what the GC framework uses?

Thanks a lot,


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