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.
  • Any other information that may help us to answer your question, including whether you are working with proteomics or small molecule data.

If you are including text output from a tool, please attach files to your message, rather than pasting in long text.

If you are including a Skyline document, please use Skyline's File | Share menu item (choose "Complete" if asked), which prepares a single zip file with your document and all the needed supporting files in it. Then upload that file to the Uploads page. If the actual raw data files are needed to illustrate a problem, those will need to be zipped up and uploaded separately.

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Showing: limited to 100 requests
Skyline export to Waters TQ-XS limited to 32 transitions?
(1 response) superstic 2018-06-08

When I export from skyline to waters TQ-XS method, if I have more than 32 transitions for a peptide, it will only put first 32 transitions into one function. The rest of transitions will not show in the method. I found out Waters method have upper limit of 32 for one function. Is there anyway to get around this?

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Slow importing of raw files
baezaj 2018-06-18

Dear Skyline team,

I am trying to import 4 thermo raw files into skyline. The skyline document has about 7000 proteins and the raw files being imported were collected using an overlapping window (24 m/z total, 12m/z overlapping). I began importing the data on Friday (6/15/18) and I could immediately see the chromatograms as they were being imported. After about 5 minutes, the full chromatogram for 1 raw file was displayed and the import had reached 40%. I returned on monday (6/18/18) and the progress for the same raw file has only reached 41%.

I am using Sklyine Daily (64 bit) on a windows server 2016 Datacenter v1607. The processor is an intel(R) Xeon(R) Silver 4114 CPU @ 2.2GHz 32 processors and 110 Gb of Ram. I also copied the raw files to the virtual desktop on the server.

I read a previous post that mentioned the issue stemming from unmapped network drives. All of the networks mapped to the server seem to be in working order. can you provide some guidance on how to fix this issue?

Many thanks,


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Conversion to mz5 or mzml in centroid
(8 responses) sstoychev 2018-06-16

hi guys,

In webinar 14 on DIA data you mention that for Sciex files use of centroided data is best. Can you share the settings for MS Convert to get a SWATH wiff file converted to centroided mz5 or mzml?

Thank you.

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Reading Fasta is slow on importing MetaMorpheus results
(3 responses) rmillikin 2018-06-14

Hello from the MetaMorpheus dev team!

I'm trying to get our .mzID search results to import correctly into Skyline. The .mzID imports successfully with Skyline Daily (but not Skyline 4.1 release, which is fine). However, when I add my .fasta database, the process gets stuck.

To reproduce:
Download .mzID, .fasta, and .mzML from
In Skyline Daily, Import DDA Peptide Search
Add .mzID, click Next
Spectra get read
Click thru menus to extract chromatograms, select mods, etc
On "Import Fasta" Browse and select fasta, 2 missed cleavages, click finish
Progress bar appears and proteins are added, but extremely slowly. No real progress is made even after >1 hour

Let me know if this is an issue with our .mzID format, etc. We'd be happy to change code to get this to work.

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small molecular targets QTOF
(3 responses) colorzhang 2018-06-14

I imported a small molecule transition list and imported 1 run from QTOF5600. It showed chromatogram information unavailable.
I tested the Tutorial Files , it works well.
Thank you!

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How to categorise proteins according to their functions
(3 responses) hanh t nguyen 2018-05-31

Hello everyone,
I am working on proteome of bovine fetal fluids and found there are more than 3 hundred proteins in these fluids.
I would like to categorise the 300 proteins according to their functions. Could you please advise me with a solution to deal with my objective?
Is this going to check the functions of each protein using the database of Uniprot, one by one?
Thank you very much for your time and all the best wishes,

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installing skyline daily error
(1 response) baezaj 2018-06-14

Hi Skyline team,

I get an error when trying to install skyline daily and I've included the install.log file.
I am installing skyline daily onto our lab server. If the error is stemming from our end, please let me know so I can tell my IT person.



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Trouble Importing Searches from IP2
(4 responses) lougee 2018-06-13

Greetings Skyline Team,

Whenever I try to upload a spectral library from data searched on IP2 I receive the following error message. The file is saved as an MZID file and has only probability and P-value as a primary and secondary score respectively. It is a SILAC data set and we hope to utilize Skyline in analyzing the ratios. I'm not exactly sure why skyline is having an issue with the searched data. Thank you!


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Discrepancy issues with Peptide Search using .msf or .pdResult
(1 response) Vera 2018-06-12

Hello Skyline team,

Currently, I analyze my data with Proteome Discoverer first and import the msf. file into skyline peptide search, then accept FDR<0.1 peptides only. However, recently my file is getting too big so I tried to import the pd.file (which suppose to contains only FDR<0.1 peptides only) instead. I noticed that less number of peptide and protein were identified when pd.file was imported (prot: 5689 pd. vs 8871 msf. ), but the number was not as small as I expected (about 50 protein). I was wondering is there any explanation for the difference between peptide search imported form msf. and pd. ? And is there any way to import FDR<0.1 peptides from Proteome Discoverer results (either msf. or pd.) only?


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retention time (RT) alignment or read XCMS processed files for small molecule
(5 responses) zhijcao 2018-06-12

Hi skyline team,
I am trying to analyze metabolomics data using skyline. There is no RT calibration or alignment function for small molecules. I am wondering:
1, whether RT alignment or calibration function can be added in Skyline
2, whether skyline can read files which RT have been aligned by XCMS
Thank you,

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Integration of multiple isobaric peaks
(9 responses) victor nesati 2018-06-03

Hi, I am trying to figure out whether it is possible to automatically integrate peaks with different retention times corresponding to the same
isobaric mass. In the enclosed example I have 5 of them, and skyline automatically picks only 1. I can manually adjust integration boundaries and have them all included, though the question is whether options existing in Edit>Refine>Reintegrate, or Advanced could be of any help to automate this process. I specified quite broad RT window ( around 6 min) to help program to get all peaks at once but that did not help much.

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How to prepare the Transition list that was imported into the Skyline?
(7 responses) zzhang9 2018-06-07

Hi there,

I am wondering how to produce the transition list( or Assay library?) that was imported into the Skyline? As shown in the Tutorial. Thank you!


 Step1.png  Step2.png  Step3.png 
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Support for isotope labeling studies
(1 response) manohar_dange8 2018-06-11

Hi Skyline team,
I wish to know if skyline can support targeted analysis of 13C labeled metabolites. Basically if MS1 precursor along with its isotopic distribution is specified can skyline do the MS1 peak integration for unlabeled and labeled peaks? I am interested because skyline already supports MS1 and MS2 peak integration for proteomics studies and as there are very limited options available for metabolite studies in this regard, any help from your side would be appreciated.

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MSstats: data processing -> add detection qvalue column in the input
(1 response) sas28 2018-06-11

I was wondering about the following error message when running reintegrated data through MSstats. It gives this error when using "data processing" in the MSstats menu. Group comparison works fine. I previewed the file through export -> MSstats input and it shows a column with Qvalues (except for iRT peptides which contain an N/A but these are removed anyway). Is there a setting in the document that is not correct?

** Reading the data for MSstats.....
** iRT proteins/peptides are removed.
Peptides, that are used in more than one proteins, are removed.
** Truncated peaks are replaced with NA.
** For DDA datasets, three isotopic peaks per feature and run are summed.
Peptides, that are used in more than one proteins, are removed.
Error in SkylinetoMSstatsFormat(raw, filter_with_Qvalue = filter.qvalue) :
** DetectionQValue column is needed in order to filter out by Qvalue. Please add DectionQValue column in the input.

Can't finish analysis.

Many thanks.


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Reintegrate -> Peak scoring error using mprophet
(5 responses) sas28 2018-06-08

I was wondering if you could help with the following error:
System.IO.InvalidDataException: Insufficient target peaks (0 with 5718 decoys) detected at 15% FDR to continue training. ---> System.IO.InvalidDataException: Insufficient target peaks (0 with 5718 decoys) detected at 15% FDR to continue training.
at pwiz.Skyline.Model.Results.Scoring.MProphetPeakScoringModel.CalculateWeights(String documentPath, ScoredGroupPeaksSet targetTransitionGroups, ScoredGroupPeaksSet decoyTransitionGroups, Boolean includeSecondBest, Boolean nonParametricPValues, Double qValueCutoff, Double[] weights, Double& decoyMean, Double& decoyStdev, Boolean& colinearWarning) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\Scoring\MProphetScoringModel.cs:line 405
at pwiz.Skyline.Model.Results.Scoring.MProphetPeakScoringModel.<>c__DisplayClass7.<Train>b__4(MProphetPeakScoringModel im) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\Scoring\MProphetScoringModel.cs:line 255
at pwiz.Common.SystemUtil.Immutable.ChangeProp[TIm](TIm immutable, SetLambda1 set) in c:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\Immutable.cs:line 201 at pwiz.Skyline.Model.Results.Scoring.MProphetPeakScoringModel.Train(IList1 targetsIn, IList1 decoysIn, LinearModelParams initParameters, Nullable1 iterations, Boolean includeSecondBest, Boolean preTrain, IProgressMonitor progressMonitor, String documentPath) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\Scoring\MProphetScoringModel.cs:line 196
at pwiz.Skyline.SettingsUI.EditPeakScoringModelDlg.<>c__DisplayClass13.<TrainModel>b__b(IProgressMonitor progressMonitor) in c:\proj\pwiz_x64\pwiz_tools\Skyline\SettingsUI\EditPeakScoringModelDlg.cs:line 269
at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action1 performWork) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 228 --- End of inner exception stack trace --- at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Util\Util.cs:line 1842 at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 176
at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 131
at pwiz.Skyline.SettingsUI.EditPeakScoringModelDlg.TrainModel(Boolean suppressWeights) in c:\proj\pwiz_x64\pwiz_tools\Skyline\SettingsUI\EditPeakScoringModelDlg.cs:line 272
at pwiz.Skyline.SettingsUI.EditPeakScoringModelDlg.TrainModelClick() in c:\proj\pwiz_x64\pwiz_tools\Skyline\SettingsUI\EditPeakScoringModelDlg.cs:line 191

The Skyline file contains 447 target proteins with the same number of decoys (shuffled sequence).
When performing -> Reintegrate -> Peak scoring, it gives error when mprophet option is used, default is fine but then Skyline integrated MSstats gives an error as it is also missing q-values:
** Reading the data for MSstats.....
** iRT proteins/peptides are removed.
Peptides, that are used in more than one proteins, are removed.
** Truncated peaks are replaced with NA.
** For DDA datasets, three isotopic peaks per feature and run are summed.
Error in SkylinetoMSstatsFormat(raw, filter_with_Qvalue = filter.qvalue) :
** Please check annotation for Condition and BioReplicat column. There is missing information.
Can't finish analysis.

What can i do?

Many thanks,

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Scheduled PRM Global Peptide standard issue
(3 responses) sivapira 2018-06-07
Hello Skyline Support Team,

I'm working on a PRM experiment where I'm planning to use BSA as global standard (spiking BSA to sample before digestion). I have run a preliminary experiment with an unscheduled method and I have refined the method. Now I'm ready to run a scheduled method but it seems like I have to do at least 3 scheduled injection due to the cycle time limit (I'm using a QExactive where a max 39 precursors can be monitored simultaneously).

My question is do I include the peptides for BSA for all 3 scheduled injections and if yes how do I standardize the peptides from Injection #1 with BSA peptides from Injection #1?

Please correct me if my thought processes are wrong and thank you for the help.

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Natural sorting of Results
(1 response) paramon 2018-06-04

Currently, imported results are sorted lexicographically by their names. However, that breaks the expected sort order in common case of short cardinal numbering, e.g.


come out in Results Manager/Results Grid as


It is proposed that Natural Sort algorithm is used for replicate names:

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Targeted analysis on MS1 only
(6 responses) paramon 2018-06-05

For small molecules, it's not uncommon to perform targeted analysis on MS1 only (high-throughput). However, currently user is required to specify at least one product m/z. At import time:
You must add at least one target transition before importing results.

It is proposed that this restriction is lifted, so that only precursor m/z or formula may be specified (product m/z is not required) for a target.
Probably a warning should be issued in this case, but the program should continue if accepted.

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Thanks Brendan for the answer to the question
lourdes thevanayagam 2018-06-08

Thanks Brendran ,

For your answer and a great help
Best regards

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peptide setting on enzyme characteristics
(1 response) lourdes thevanayagam 2018-06-07

Hello Brendan,

Thanks for the training on skyline at Buck Inst,
After the training I started working on protein quantification using peptides cleaved by different enzymes and it is working things getting better.

I have a question on peptide settings creating my own enzymatic digestion and on “edit current enzyme” I would like to have a clear understanding of choosing

  1. Cleave C-terminal to
  2. Cleave N-terminal to

I know where the enzyme cut the peptide

Example -----SGRSD------ the enzyme I chose cut RS, so in that case could you please advise me for the cleave C- terminal to and cleave N-terminal to.

Hope you understand my question.

Thanks for your help.


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MS optimization for small molecules
(1 response) 120887548 2018-06-06

Hi there

I used to use skyline for peptide optimization, it is very useful. I recently start targeted analysis for small molecules. I am wondering if I also can use skyline for their MS parameters optimization such as CE, DP, CXP and etc. Which MS instrument is supported for that ?

Many thanks in advance!

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MS1 analysis_Lipidomis_DDA data
(1 response) SMohan 2018-06-07

Hi there,

I have a DDA lipidomics data (acquired both in negative as well as positive mode). I know the m/z I am interested in and I want to quantify these m/z's in my data. Is there a way that I can have XIC (for MS1 species) for these features ?

I am trying to import the data in targeted fashion ( Transition setting --> Full scan --> isotope peak included --> count; orbitrap; MS/MS filtering --> None) and inserting the list of m/z but I am unable to get the XIC for these ions (while Xcaliber gives good XIC for these features).

Thanks a lot for the help!. It will save us from a lot of manual analysis in Xcaliber


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the prespecified isolation windows are different from that I set in the MS method
(1 response) zzhang9 2018-06-01

Hi there,

I tried DIA on Q Exactive HF. The following inclusion list was used for defining the isolation windows:
15 entries
Mass Formula Species CS Polarity Start End (N)CE MSX ID Comment
[m/z] [M] [z] [min] [min]
377.50000 Positive
423.50000 Positive
459.50000 Positive
493.00000 Positive
526.50000 Positive
560.50000 Positive
595.50000 Positive
633.00000 Positive
673.00000 Positive
718.00000 Positive
772.00000 Positive
839.00000 Positive
933.00000 Positive
1064.00000 Positive
1469.00000 Positive

However, when I import the data into the Skyline, I got the following results:
347.5 407.5 7.00
393.5 453.5
429.5 489.5
463.0 523.0 13.25
496.5 556.5
530.5 590.5
565.5 625.5
603.0 663.0
643.0 703.0
688.0 748.0
742.0 802.0
809.0 869.0
903.0 963.0
1,034.0 1,094.0
1,439.0 1,499.0

It seems something wrong. Do you have any idea about that? Thank you!



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MSstats error
(1 response) jung165 2018-05-31

Hello Skyline team.

I have a one quick question about running MSstats.
It keeps me showing error in dataProcess step and do not know why it cannot process the data

I already posted a question about this issue on this support board but, I got a reply that I have to ask it on support board on external tool.
So I posted another question on external tool of Msstats support board and sent e-mail to its developer (Mina Choi on Google groups) but, could not get an answer

I attached Skyline zip file and if you run Msstats through tool tab, it is possible to see my error.
Where should I ask about this problem??

Thank you for your help

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QuaSAR error
(6 responses) jung165 2018-05-30

Hello Skyline team.

I have a one quick question about running AuDIT through QuaSAR (external tool) on Skyline.
However, I got LOD and LOQ calculation error during analysis and I am curious about solving this issue.

I already posted a question about this issue on this support board but, I got a reply that I have to ask it on support board on external tool.
So I posted another question on external tool of QuaSAR support board about 4 months ago but still could not get answer.
I also posted my question on GenePattern, developer of QuaSAR (I guess) and they said QuaSAR running through Skyline is not a tool they support.

Where should I ask about this problem??

Thank you for your help

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MSstats installation error
(3 responses) Vera 2018-05-31

Hi Skyline Team,

I am trying to install MSstats as an external tool in Skyline, but I keep run errors like Failed attempting to extract the tool from, The system cannot find the file specified. Detailed errors were as below:

System.ComponentModel.Win32Exception (0x80004005): The system cannot find the file specified
at System.Diagnostics.Process.StartWithCreateProcess(ProcessStartInfo startInfo)
at pwiz.Skyline.ToolsUI.RUtil.RunRscript(String pathToR, String pathToScriptFile) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\ToolsUI\RInstaller.cs:line 438
at pwiz.Skyline.ToolsUI.RUtil.WhichPackagesToInstall(ICollection1 packages, String pathToR) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\ToolsUI\RInstaller.cs:line 406 at pwiz.Skyline.SkylineWindow.InstallProgram(ProgramPathContainer programPathContainer, ICollection1 packages, String pathToPackageInstallScript) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Skyline.cs:line 4938
at pwiz.Skyline.Model.Tools.ToolInstaller.UnpackZipTool(String pathToZip, IUnpackZipToolSupport unpackSupport) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Tools\ToolInstaller.cs:line 393
at pwiz.Skyline.ToolsUI.ToolInstallUI.InstallZipTool(Control parent, String fullpath, InstallProgram install) in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\ToolsUI\ToolInstallUI.cs:line 133

I tried uninstalled skyline and R several times but none of them worked. Could you help me with it?

Thanks a bunch,

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Selective peak integration based on idotp
Fabian 2018-05-29

Dear all,

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

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

Best regards


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Quantification small molecules, pivot editor...?
(1 response) anne 2018-05-28


I calculated CV`s using the pivot editor. But I could only calculate for all Replicates. In one batch I normally work with three to five replicates per sample or calibration standard. (see in my file e.g. three replicates His1-His3) I want to group those and calculate mean area and CV´S. How can I manage that?

Is it possible to see the ratio of transtions of one precurser. I mean it is visualized in plots, but can I also see it in my result table.

Best regard,

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Skyline runner - DIA
(7 responses) bart van puyvelde 2018-05-23

Dear Brendan,

We are currently testing SkylineRunner as we are more and more confronted with large DIA/SWATH datasets. Last weekend, one of our data pc's crashed when it was importing multiple .wiff files (using a very large library with a few million transitions)
Specs: Intel Xeon, CPU E5-2640 v3 2.6 GHz (160 GB ram/Windows 10 Enterprise)

I have been testing SkylineRunner on a rather simple dataset (files attached). The user interface needs approximately 10 seconds to import the .wiff file. Though when using SkylineRunner, the command prompt never seems to import the data (screenshot of command prompt attached)

Probably we are doing something wrong, as I am not an expert in writing .bat files.
Is there a command making it possible for us to follow the progress during import?

If extra information/data is required, please let me know.

Kind Regards,
Bart  TEST.slc  runskyline_5.bat  F003252.dat  Outpuntcommandprompt.PNG 
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Error building spectral libraries from Comet/percolator output
(7 responses) timothy acker 2018-05-23

Hi All,

I hope this message finds you well. I am having a recurring problem building spectral libraries or importing search results from Comet/percolator output.

In peptide settings when I try to create a spectral library or import results, I am given an error informing me that the pep.xml file is not from one of the recognized file format and it references the first line in the search results within the pep.xml file. I am running comet from the crux interface in Cygwin.

crux v, percolator v 3.02.0, Skyline v, Comet version "2017.01 rev. 4"

I cannot find another example in the support forums.

Thank you,


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Transition List for SRM
(4 responses) rsabnis 2018-05-23

I made a spectral library with all heavy phosphorylated peptides using msms file from MaxQuant. When we import results, we see very few transitions, most of which are b ions. We think this improper y ions assignment is because the software is not recognizing the heavy peptides. Any help to get the transition list for SRM method would be appreciated.

Thank you!
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mzTAB File Type for Spectral Library
(5 responses) melissa martinez 2018-04-27

Despite the many types of data that are acceptable to build a spectral library using Skyline, I've recently run into some compatibility issues with mzTAB files. A couple years ago HUPO selected mzTAB as the standard data file type for proteomics data sharing. Some data repositories, like MASSIVE from UCSD, require converting all peptide files into mzTAB files and that is all that's available to download.

Could you please consider making Skyline capable of using this file type for the peptide IDs? If this is the chosen file type for proteomics data sharing, I imagine it'll be seen more and more.

Thank you very much!

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Quantification of small molecules
(2 responses) anne 2018-05-23


I watched the webinar 16, which is about small molecule quantification. They showed how to get values of accuracy after building up a calibration curve, but is it also possible to visualize the values for mean, standard deviation and realtive standard deviation for replicates of each transition of one precursor within the table? I could not find it in the results grid.

Best regards,

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"Peak Areas" graph
k valgepea 2018-05-23

Dear Skyline team,

I hope you can help me with two short questions:

  1. I am double-checking if it is somehow possible to plot the "rdotp" value for a precursor above the bar showing its peak area in the "Peak Areas - Replicate comparison" graph? I can plot "dotp" in my Skyline version (, but not "rdotp".

  2. Could I simultaneously have two "Peak Areas - Replicate comparison" graphs opened within one Skyline document? For example, one grouped by "Replicate" and showing non-normalised peak areas while the other grouped by "Condition" and showing CVs of pear areas normalised to heavy?

Thanks in advance!

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MS1 XIC QUAN without MS2 Library
(23 responses) victor nesati 2018-05-17

We are trying to configure Skyline to do MS1 XIC based quantitation based on precursor masses alone without prior results of database search.

We were able to create sequences of desired peptides with custom modifications in the Skyline. But getting Skyline understand that we want to use precursor masses of those peptides as "pseudo-library" for consequent QUAN was a challenge. Actually raw file was not loaded. We found work-about through building SSL file then converting it into blib, importing those library along with mzXML file, and then getting desired info, but this still requires presence of fragmentation in data acquisition, which we dont really need, as we do know the masses we are looking for and put it simply we are just trying to use Skyline instead XCalibur to get peak Area data. Any suggestions on how to handle this situation ?

Interestingly enough I also found that Panorama could be used to create Chromatographic Libraries which might be applicable to what we are trying to do. In addition I found Topograph software which from the looks of it might be able calculate chromatographic peak areas extracted from MS1 scans in DDA data, which sounds exactly what we are trying to do.

I would appreciate your pointing me into right direction.



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Single amino acid substitutions in mascot error tolerant search
(3 responses) michelle dubuke 2018-05-21


I am working on a peptide-based project with single amino acid substitutions at specified locations in the sequence. I am able to build a fasta file that contains all of the possible peptide sequences and do the analysis that way, but an error-tolerant search in mascot is faster and allows me to look for "off target" amino acid substitutions. I updated to skyline-daily to get the error tolerant searches to populate properly, but it appears that the single amino acid substitutions are not pulling from unimod like the rest of the options/chemical modifications are for that data.

Is there any reason why the amino acid changes wouldn't be included in skyline during error tolerant searching?

Michelle Dubuke
UMass Medical School

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'Share' feature saves without prompting
gabe 2018-05-21

If you have an un-saved Skyline document, and you use the 'Share...' feature from the File menu to create a minimal/share-able Skyline document, it will save the current changes without prompting. This can result in unanticipated losses. A simple prompt to confirm that the original .sky file will be over-written would fix this bug, but as it stands it can be pretty dangerous.


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Do you have experience in building SRM method with theoretical digestions?
VJ 2018-05-18

I'm interested in few plant proteins where there is no spectral library. Unfortunately, I cannot digest vague Skyline tutorials.

Does anyone have experience in building an MRM method with theoretical digestions in Skyline?
Any guideline with a possible example protein, if available please (i.e.Protocol)?

Would appreciate your help with this.

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RT issue when one peptide and it's own modified peptide co-exsit
(7 responses) qi tang 2018-05-15

Dear Skyline Group,

I have one peptide and it's own modified peptide. One RT is 68.21 and the another is 45.71(See attached picture 1.png and 2.png). The extraction window is set to 3 min(See attached picture 3.png). I found the actual extraction window is from 42.71 to 71.21. Why? Can I avoid it? I just want extract the peptide peaks as it's been set to(3 min window separately).


 1.PNG  2.PNG  3.PNG 
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Skyline spectral library export
(2 responses) Helian 2018-05-16

We often use the option in Skyline to export spectral libraries based on experimental data, but we're wondering how does it work when there are multiple raw files imported. In most cases the peptide is found only in one of the samples. When I export the spectral library does it base it on the best peak (the sample where the peptide was actually found and should be found in) or does it combine all results into one?


view request
Any way to plot residual precursor in PRM data?
(1 response) joshua nicklay 2018-05-16

Hi Skyline team,

I'm wondering if there's any way to include the residual precursor within my PRM/MS2 spectra as an additional ion type. I'm using "p" to see my precursors from my MS1 scans, but I'm hoping there's a way to be able to see how much precursor is left over within my MS2 spectra.


view request
Unique peptide selection
(9 responses) Alex Zhu 2016-06-20
Hi Brendan,
I need to quantitate an endogenous protein from human plasma. Is there an easy way to create a background proteome containing all human plasma proteins except the target protein in skyline so I can select the unique peptides for the target protein?
view request
SRM - Force Skyline to integrate scans with different/multiple headers
(4 responses) v delcourt 2018-05-11


I'm currently working on optimization in MRM/SRM using a quantiva. Let's say I didn't want to miss any transition and I asked skyline to produce a,b,y transition list which I imported into a quantiva.
After analysis, skyline integrates only a few ions which seemed surprising in the first place as i knew that some ions would be produced by CID but were missing from Skyline integration. After looking a little bit deeper into the data, I noticed that the Quantiva renamed my precursor groups of 10 transitions

Example : Precursor = 803.37 -> 18 transitions

Group 1 : 803.370 -> 10 transitions

Header : + c ESI SRM ms2 803.370 [105.065-105.067, 136.075-136.077, 164.070-164.072, 202.118-202.120, 207.112-207.114, 235.107-235.109, 354.180-354.182, 365.181-365.183, 382.175-382.177, 411.202-411.204]

Group 2 : 803.371 -> 8 transitions

Header : + c ESI SRM ms2 803.371 [422.202-422.204, 439.197-439.199, 569.271-569.273, 574.265-574.267, 602.260-602.262, 640.308-640.310, 671.318-671.320, 699.313-699.315]

The problem is that Skyline only takes into account the first group as the only group for integration and that most intense ions are contained within second group. (see capture)

Maybe there's some quick workaround for this but I wasn't able to find it. Playing with Peptide and Transition options didn't help so far. I also tried to trick skyline and asked to import it as DIA which wasn't successful either (as expected :( ).

Is there anything I'm doing wrong ?

Btw this is a sticky peptide so no surprise it has such a wide rt.

Best regards,

view request
Incomplete MS1 XIC in case of additional ms/ms filtering
(2 responses) Fabian 2018-05-08

Dear all,

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

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

Many thanks in advance


 MS1XIC_DLGEEHFK_487_7325_MS1_only.PNG  MS1_and_MS2_XIC_DLGEEHFK_487_7325_MS1_only.PNG  Only_one_MS2_CCTESLVNR_569_7526.PNG 
view request
Apex biotin-phenol modification spectral library
(4 responses) daniswan 2018-05-11

I have done some APEX proximity labeling experiments in which I enrich for Y-biotin phenol sites using anti biotin. I would now like to build a spectral library to perform targeted quantification of these sites. I have searched the data with MaxQuant and tried importing the peptide search as usually done, but it is not recognizing the Y-biotin phenol modified peptides in my msms.txt file and these are being excluded from the spectral library. I have added the Y-biotin phenol modification to the list of modification in the peptides settings. Is there some text from the msms.txt and skyline settings that needs to match in a certain way for these peptides to be imported properly?



view request
Training a peak scoring model with a large-scale of DIA
(6 responses) lihaikuo 2018-04-29
Hi, when I do the procedure (reintegrate--add--train) and want to edit the peak scoring model, I get the figure shown in the attachment.
You see, I can not see the detailed distribution. The horizontal coordinate is too large, and I can not distinguish targets from decoys. This is my question.
Maybe it is due to that there exists some targets or decoys with a very low score? If so, how can I delete these targets?

Here, there are more than 200 DIA raw data imported and more than 200,000 transitions.
view request
(2 responses) amey593 2018-05-10

After putting the FASTA sequence in Skyline for the Osteopontin; I am not getting the respective peptides/ transitions we regularly get in other cases, how to troubleshoot it?
Please help me with this.
 Osteopontin SKYLINE 1.JPG  Osteopontin SKYLINE 2.JPG  Osteopontin FASTA seq.txt 
view request
peptides with mixtures of modified and unmodified residues
(9 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


view request
Graph alignment in skyline
rle041 2018-05-10

Hi Skyline team,

Sometimes the graphs in split graph mode is not aligned. This can be a bit confusing, is there any way you can fix this in a later release?
I have added a screenshot of an example for the retention time windows of precursor and fragment ion signal without zooming, and one where I have zoomed in on the retention time boundry,

Best regards,

Ragnhild Reehorst Lereim

view request
Need to get normalized area for SRM data
(3 responses) kerry hassell 2018-05-09

The software is calculating the peak area in replicate comparison but not in the results grid (showing #N/A). It is SRM data from the triple.

view request
Missing peptides pop-up pick list
(2 responses) carmen.gonzaleztejedo 2018-05-09

Dear Brendan,

I have a doubt regarding the list of peptides that are shown in the pop-up pick list for a specific protein. Are they unique peptides? Sometimes, it's not showing the whole list of peptides for the protein (see attached). In my case, for protein Q96BR1. peptides contained between 42 and 102 positions are not available to pick. As you can see, I have used a Human background proteome, but I haven't enforced any type of uniqueness. Why is that happening then?

Many thanks in advance,


view request
Small molecule retention time assignment problem
(2 responses) george dubay 2018-05-08
Good Afternoon,
I am using Skyline to evaluate my data. There seems to be a problem with the way it is handling the assignments of the peaks that are isomeric, even though the retention times submitted are in close agreement with the observed times. We would love to see the system assign these peaks automatically without any curation.
Notes of significance:
1.    The chromatography peaks are reproducible within +/1 0.02 minutes for all compounds.
2.    The assigned RT’s are always the best match for the observed RT’s.
3.    The intensity of the peaks makes little difference.
4.    The signal to noise is generally very good.
5.    Another anomaly that I am encountering is:
i.    group of samples consisting of my calibration curve and quality controls is imported to Skyline and curated to match retention times and created the curve things look good.
ii.    A second set of data for samples from plasma and fecal material is added.
iii.    When the added data goes into the system the RT assignments for the initial work are lost. This is easily observed for 2-methyl butyric acid, but others suffered the same loss of the RT assignment values.
iv.    As a result, the data for several of the compound requires reassignment of the retention times.
Please, look the data over and see what you can tell me about the assignments.
Data specifics:
1.    All data is coming from a Waters Xevo TS-Q.
2.    The SIS used is 13C6.
3.    The fatty acids (derivatives) fragment in the negative ion mode so the [M-H]- ion yields product ions from the fragmentation.
4.    The sample id # are related as given in the spreadsheet attached(page 3).
5.    The transition list used is in the first window of the Excel file attached.
6. I am working with Will Thompson here at the Duke GCB.
7. Raw data is in the folder attached as well.
8. Samples are run with the standard Waters MassLynx software.
view request
Importing decoys
(2 responses) bart van puyvelde 2018-05-07
We would like to use our own decoy spectra (generated using an in-house script) in the mProphet model.

Is there a possibility to do this?

Kind regards,
Bart Van Puyvelde
PhD student
Lab of Pharmaceutical Biotechnology
Ghent University
view request
MSX-DIA (or overlapping?) in Lumos
Kavee 2018-05-07


I try to implement MSX-DIA in Orbitrap Lumos by generating isolation list from Skyline 4.1. However, I could not find a way to import them into the method. When I select DIA in Xcalibur 4.1 and checked the multiplex option, the isolation table is only readable. Do I need to choose PRM mode instead of DIA?

Another question is do you recommend overlapping isolation scheme or MSX for Orbitrap Lumos? My understanding is that orbitrap take some time for mass analysis, so it should benefit from using multiplexing (less number of scan?).

Thank you very much.

view request
Product ions filtering in DIA
(3 responses) Kavee 2018-05-06


I'm interesting in doing DIA experiment and have watched the skyline webinar 14,15. In the transition setting, you suggest to use product ions from '3' to 'last ion'. Could you explain the reason to exclude y1,y2 and b1,b2 ions? I try to find the answer for quite some time but still don't understand. Another question is could I include p ion in the ion types?

Thank you very much.

view request
Score type of xQuest scores for building .ssl files
(7 responses) kmk 2018-05-02

I am creating a ssl file according to:

The software I use for identification is called xQuest (

xQuest exports a (linear discriminant) score for each peptide based on the xProphet metrics. I'd like to import these scores into the Skyline library I'm building, so I have to choose the correct score type. Since there is no xQuest score type mentioned on the linked web page, I am wondering which score type to choose.

Right now I am using "PEPTIDE PROPHET SOMETHING" as a type since that software also uses a (different) kind of ld-score describing the probability of a positive identification.

Also, I am quite curious how Skyline exactly uses these imported scores.

Thank you very much for any help!
view request
Auto-zoom - retention time window is inactive
(1 response) hregut 2018-05-05
Please tell me why retention time window is inactive? Maybe it should be pre-configured somewhere? (I am using Skyline

 RT window.jpg 
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iRT Retention Time Issue
(2 responses) johnny perez 2018-05-03
Hi Brendan,

One more question about how skyline predicts peptide retention times. I've attached a chart to highlight my question. In this scenario, the same peptide elutes at different times with considerably varying ion intensities. The user selecting the correct peaks and recalibrated the iRT database shows near perfect linear regression, however, my question pertains to the yellow prediction box. How is this time predicted? It does not appear to correlate with the SSRCalc times.

All the best and thank you for your help!

 iRT Prediction Question.jpg 
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Refine Results: 1) filter based on rdotp ?& 2)how to remove peptides where heavy & light pair is lost
tcj11 2018-05-03
I have data with heavy isotope labeled brain tissue standard and am evaluating SRMS. In the end I want to reduce the SRMs to 3 peptides per protein and 3 transitions per peptide. So far I've used the Refine Results, filter based on target value 0.85 and min dotp of 0.7 (I know in an ideal world these values would be set higher ...).
1) is there a way to filter on rdotp ?
2)I noticed in many cases I lose either the heavy or light. Is there a way to remove peptides where the "complete" heavy & light pair is lost ?
3) I seem to have forgotten how to automatically reduce it to the top 3 ranked peptide also . 
view request
Q-Exactive ppm mass error question
(1 response) johnny perez 2018-05-02
Hi Brendan,

I ran a targeted PRM assay method for a set of peptides with an inclusion list that was produced using skyline. The method takes a full MS spectrum followed by 32 MS/MS spectra for 16 peptides with two precursors each all listed in the inclusion list. When importing into skyline, it filters the Thermo .raw files beautifully and gives great looking data. I had a question about the ppm error measurements. I've attached a screenshot for your review. Is the ppm mass error measurement the linear combination of mass errors for the skyline determined fragment ions? I cannot imagine it has anything to do with the precursor due to the Q-Exactive fragmenting all ions within the + or - m/z window.

Thank you!

 High Resolution Skyline ppm question.emf 
view request
reverse external calibration curves
(3 responses) chiva cristina 2018-04-26

I was wondering if Skyline supports reverse external calibration curves in which you inject different concentrations of heavy peptide and you use the light peptide (at fixed concentration) to normalize.


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

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

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

Many thanks for helping.

Best Regards,
view request
Skyline results - background column
(1 response) dspiciarich 2018-04-30
I'm running small molecule samples and I see a column with "Area" and one one with "Background" and wondering if you could explain what the background refers to? How is it calculated and how is it generally used?

For example (see attachment)

Thank you,
 Screen Shot 2018-04-30 at 10.18.05 AM.png 
view request
Can skyline filter the DIA results by Protein FDR?
(2 responses) rbbsky123456 2018-03-27
Hi, skyline team

   Some other software,like spectronaut can filter the DIA results by both peptide and protein FDR. I'm wondering if the skyline can do the same thing. Thanks.
view request
Plotting correlation between two transition of same peptide or two peptides of same protein
(3 responses) abasit 2018-02-23
Hi Skyline,

I am wondering, can I plot the peak area from the two transition of the same peptide (say fragment 1 versus fragment 2) to see the correlation between the peak area of the two transitions.

In the same way, can I see the correlation between the peak area of the two peptides of the same protein in skyline.

Right now I am plotting the correlation in excel after exporting the data from skyline but it will be useful if I can do the same in skyline.

Thank you.

Abdul Basit
University of Washington, Seattle.
view request
(1 response) alexandra antunes 2018-03-13
I am running DIA method for the identification of modified peptides. I was trying to use a DDA library for RT alignment but using peptides matching filter (using the modifications I am searching for) to choose the peptides of interest. In this way I was trying to use skyline to do a untargeted identification of modified peptides. However when I try to load the FASTA file I got an error message (see attached). Can you please tell me what to do to solve this problem? Is it possible to use skyline to identify peptides that are not present in the library (just using filter settings)?
Thanks in advance,

 Skyline error.GIF 
view request
Cannot install Skyline
(1 response) mmarx 2018-03-21
I am running Windows 7.
I had an old version of Skyline that may not have been uninstalled properly. It still appeared in the control panel list of installed programs, but there were no program files that I could find. There was copy of the old program in my user's Installed Programs directory. I removed it. Unfortunately, I didn't make note of what version it was, but I think it was 2.1. The last time I installed it was back in 2014.

I get the following error when I try to install 4.1, either the normal version, or the stand-alone one.

When I run 'setup.exe', I see a dialog with the title 'Cannot start Application'. The text reads 'Application cannot be started. Contact the application vendor.' There is a 'Details...' button.

The following text is displayed when I click it.
    Windows             : 6.1.7601.65536 (Win32NT)
    Common Language Runtime     : 4.0.30319.42000
    System.Deployment.dll         : 4.7.2558.0 built by: NET471REL1
    clr.dll             : 4.7.2563.0 built by: NET471REL1
    dfdll.dll             : 4.7.2558.0 built by: NET471REL1
    dfshim.dll             : 4.0.41209.0 (Main.041209-0000)

    Deployment url            : file:///C:/Users/mmarx.AGILENT/Documents/Skyline-64_4_1_0_11796/Skyline-64_4_1_0_11796/Skyline.application

    Below is a summary of the errors, details of these errors are listed later in the log.
    * Activation of C:\Users\mmarx.AGILENT\Documents\Skyline-64_4_1_0_11796\Skyline-64_4_1_0_11796\Skyline.application resulted in exception. Following failure messages were detected:
        + The system cannot find the file specified. (Exception from HRESULT: 0x80070002)

    No transaction error was detected.

    There were no warnings during this operation.

    * [3/21/2018 5:46:21 PM] : Activation of C:\Users\mmarx.AGILENT\Documents\Skyline-64_4_1_0_11796\Skyline-64_4_1_0_11796\Skyline.application has started.

    Following errors were detected during this operation.
    * [3/21/2018 5:46:21 PM] System.IO.FileNotFoundException
        - The system cannot find the file specified. (Exception from HRESULT: 0x80070002)
        - Source: System.Deployment
        - Stack trace:
            at System.Deployment.Internal.Isolation.IsolationInterop.GetUserStore(UInt32 Flags, IntPtr hToken, Guid& riid)
            at System.Deployment.Internal.Isolation.IsolationInterop.GetUserStore()
            at System.Deployment.Application.ComponentStore..ctor(ComponentStoreType storeType, SubscriptionStore subStore)
            at System.Deployment.Application.SubscriptionStore..ctor(String deployPath, String tempPath, ComponentStoreType storeType)
            at System.Deployment.Application.SubscriptionStore.get_CurrentUser()
            at System.Deployment.Application.ApplicationActivator.UninstallApplicationAndRedirectActivation(Boolean& isShortCut, Uri& deploymentProviderUri, String textualSubId, Uri activationUri)
            at System.Deployment.Application.ApplicationActivator.CleanApplicationReInstall(Boolean isShortcut, Uri deploymentUri, Uri activationUri, BrowserSettings browserSettings, String textualSubId, String errorPageUrl, String deploymentProviderUrlFromExtension, String shortcutFilePath)
            at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivationWithRetry(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl)
            at System.Deployment.Application.ApplicationActivator.ActivateDeploymentWorker(Object state)

    No transaction information is available.
view request
Merging of FTLs
dkueltz 2018-04-28
Hi Brendan, Nick,
I have two manually curated filtered target lists (FTLs) for the same cell type that I would like to merge into a single FTL. I tried importing one document into another, which generates a cumulative FTL. However, protein ACs are not merged but duplicated. How can I merge the different sets of targets that are listed as multiple protein entries into a single protein entry? I would like to merge the FTL such that I have unique (and not duplicated) protein IDs without loosing any peptides/ transitions from either of the separate documents. Can you let me know how such merging of 2 FTLs (2 separate skyline documents) can be achieved?
Thanks for your help!
view request
Bruker tdf 2.0 format and memory consumption
(18 responses) tobias.kockmann 2018-04-17
Dear skyline support,

I am experimenting with the new Bruker tdf 2.0 file format that is now supported by the latest skyline-daily release and realised that the memory consumption is pretty high. I am running skyline-daily on a Windows Server 2012 R2 std incl. 32 GB RAM and skyline seems to occupy >36 GB when importing a few targets (123 transitions, see screenshot). Is that possible and why? I am only importing MS1-level data acquired in PASEF mode (one TIMSMS heat map every 1.1 s). So only around 15% of the available scans data should be coming from the MS1 level. The tdf_bin is around 3.8 GB on disc. That would correspond to something like 0.5 GB of MS1 scan data on disc.

 Screen Shot 2018-04-17 at 11.47.37.png 
view request
Export analysed results in report csv files - separate precursors in individual tabs/sheets
(1 response) elisabeth janssen10128 2018-04-25
Is there a way to export the data so that the file when opened in Excel would create a tab (=Excel Sheet) for each precursor in the transition list?

Say, I have 40 peptide precursors in my transition list and I read in 30 data files. It would be great to export the data so that I get 40 tab/sheets in Excel, one for each precursor, and each tab the information for this precursor is listed for all 30 results/data files.

Currently, we do that manually in Excel or R. If this would be a “simple” feature to include, it would be quite handy. Or is it already possible and I missed the option?

view request
issue with normalization ratio
(11 responses) jfidelin 2018-04-25
Skyline Daily v., Instrument: Thermo QE+.
For all our metabolites (analyzed using polarity switch), we have defined a normalization method: Ratio to surrogate. The ‘normalizer’ is a heavy labelled amino acid.
When working with many samples (40) we have observed that for ONLY some metabolites NO normalized ratio are reported.
We believe we see the following pattern: normalized values are not available for metabolites that only appear in one polarity mode. The Ratio to surrogate is measured in both positive and negative modes.
Using only one raw file (one sample), we cannot reproduce the problem.

We have attached an .csv file with an data example: Look for '02_carnitine' (line #2148 to #2221) as an example of missing normalization but signals in positive mode.

Will be glad to share .sky file

Very best, Justine and henrik
 Rockefeller_Transition list_Untargeted_for skyline.xlsx 
view request
Isotopic label not recognized?
(6 responses) michael plank 2018-04-27
Dear Skyline team,
it would be great if you could help me with the following issue:
(I`m on Skyline

I ran a DDA experiment with a mix of heavy and medium labelled samples (no light sample was mixed in) and I want to define an inclusion list from this.
I set the modifications as shown in scr1 and scr2 attached. The mass of the lysine label in the medium sample is 4.0251 (according to UniMod and MaxQuant). I created a library from the MaxQuant msms.txt on these samples.
When I look at the library, Skyline complains that the modification 4.02291 couldn`t be interpreted (scr3). As I think this is related to the issue, my first question is: where does skyline get this modification from? I did include a dependent peptide search in MaxQuant, but in the msms.txt I only get the modifications I defined in the search or 'Unmodified'.
In the library there seems to be a high number of peptides with lysine 4.02291 (which don`t have the peptide icon), but none with the expected 4.0251 (scr4).
When I then load the peptides into the target list, there are light and heavy peptides containing lysines, while for peptides with arginine there are light, medium and heavy (scr5).
In msms.txt there are IDs also for medium labelled peptides with lysine.

Thank you,
 scr1.PNG  scr2.PNG  scr3.PNG  scr4.PNG  scr5.PNG 
view request
Double files appear in skyline dialog
(1 response) philip remes 2018-04-27
I have a data folder with 664 .raw files, which is 40.4 GB. On skyline daily, often when trying to import files, it takes about a minute for files to show up in the file picker dialog, and when they do, each file shows up twice. When the dialog is then canceled and you reimport, it is fast to populate the file names, and each shows up once.
view request
Associate proteins after peptide refinement
(1 response) Priya Ghodasara 2018-04-26
Dear Skyline team,

I am a new skyline user and I am using skyline for processing SWATH data in Skyline version 4.1. I have a library file from which I create a list of peptides for targeted data extraction (View > Library > Add all and I untick association with proteins). After I finish extracting SWATH data, I re-associate the peptides with proteins (Edit>Refine>Associate protein) but it seems like it associates all the proteins present in the background proteome file (FASTA file) irrespective of curated peptides. I was expecting the proteins from curated peptides only. I am not sure how this function will work. It would be great if you can give your suggestions on how to associate curated peptide list to the specific proteins.

I would really appreciate your advice on it.

Thank you,
view request
small molecule transition list error
(4 responses) dspiciarich 2018-04-25
I've generated a small molecule library that I'm able to manually inport into skyline (see attachment #1). Everything seems to load correctly and I can export the transition list. However if I close the window and then try to re-import the transition list into the gui, I get an error (see 2nd attachment).

When I try to use skylinerunner I get the following error:

Error: Failed importing the file c:\skyline_test\exported_sml_molecule_translist.csv. Failed to find peptide column., line 1.

I think my .sky file is expecting proteomic data and that I need to tell it that it should expect small molecule data. Is this correct? How do you recommend proceeding?

These are actually peptides but that I want skyline to treat the transitions as small molecules because the transitions wouldn't be predicted by the molecular weight or the molecular formula.

Thank you.
 Screen Shot 2018-04-25 at 2.38.14 PM.png  Screen Shot 2018-04-25 at 11.44.59 AM.png 
view request
Skyline daily ran out of memory when doing MS stats group comparison
(6 responses) sas28 2018-04-18
 I hope you can give me some advice. My Skyline document contains 12 SWATH files from a 5600+ instrument (the skyd file is 35GB). The library contains 3500 proteins. When trying to run MSstats group comparison, I get the error message below (Skyline daily ran out of memory). I am wondering how to solve this. Would you have any suggestions? I am using the latest Skyline daily version. Do I have to use Skyline runner?

Many thanks,

pwiz.Skyline.Model.Tools.ToolExecutionException: The tool MSstats\Group Comparison had an error, it returned the message:
Array dimensions exceeded supported range. ---> System.OutOfMemoryException: Array dimensions exceeded supported range.
   at System.Windows.Forms.Control.MarshaledInvoke(Control caller, Delegate method, Object[] args, Boolean synchronous)
   at System.Windows.Forms.Control.Invoke(Delegate method, Object[] args)
   at pwiz.Skyline.Model.Tools.ToolDescription.CallArgsCollector(Control parent, String args, String reportCsv, String pathReportCsv, ProcessStartInfo startInfo) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Tools\ToolDescription.cs:line 530
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Model.Tools.ToolDescription.CallArgsCollector(Control parent, String args, String reportCsv, String pathReportCsv, ProcessStartInfo startInfo) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Tools\ToolDescription.cs:line 548
   at pwiz.Skyline.Model.Tools.ToolDescription.RunExecutableBackground(SrmDocument document, IToolMacroProvider toolMacroProvider, TextWriter textWriter, IProgressMonitor progressMonitor, Control parent) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Tools\ToolDescription.cs:line 393
   at pwiz.Skyline.Model.Tools.ToolDescription.<>c__DisplayClassa.<RunExecutable>b__9() in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Tools\ToolDescription.cs:line 335
view request
choosing the best trendline option for biological data
VJ 2018-04-25
This question is not directly related to Skyline. I've analyze the data and now just playing it with. I have not advanced mathematical skills to find a solution, hence thought to ask you all.

I see MS Excel has several trend-line options; linear, logarithmic, polynomial, exponential, and power functions. What is basis/logic for selecting these functions for biological data. For e.g. I'm interested in understanding the change of peak area of protein vs different time course; my data fitting with polynomial trend-line. how can I compare different samples in this option?

if Excel is not a good option, how can I do this in R?

view request
Problems (PRM workflow and isotope tags targeting cysteine residue)
(5 responses) ylam 2018-04-24
I have a question regarding Skyline and am wondering if you could help me out.
I used Skyline a couple of years ago, and it interpreted the data very well for one of our studies. Thanks for your help at that time!
1) I am now trying to use Skyline again for another study, and I am having several problems/questions.
I am using the PRM workflow and trying to import the Proteome Discoverer 1.4 msf file into the Skyline. When I input the sequence of the protein of interest with trypsin as an option (Import FASTA), it says the protein only has 4 peptides (see attached: SkylineQuestion.jpg). However, the protein clearly has more than 8 peptides, how come? The sequence is listed below:
>sp|P12931.3|SRC_HUMAN RecName: Full=Proto-oncogene tyrosine-protein kinase Src; AltName: Full=Proto-oncogene c-Src; AltName: Full=pp60c-src; Short=p60-Src

2) It looks like the Skyline was not able to incorporate all the peptide identification from Proteome Discoverer results, although I have set the cut-off score = 0, when I imported the results.

3) We are using a cysteine-reactive tag C(8)H(10)O(2) [138.06808 Da] and its isotope (deuterium) counterpart 2H(6)C(8)H(4)O(2) [144.105740 Da] for our experiments. I have set these two as variable modification and 2H(6)C(8)H(4)O(2) - C(8)H(10)O(2) = 6.03766 Da as "heavy isotope modification". But Skyline seems to also see 144.105740 Da + 6.03766 Da as "heavy" as well.

It has been a while since I used the Skyline, any help will be greatly appreciated!
Best wishes,
Ying Wai Lam
VGN Proteomics Facility
University of Vermont
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Error Updating Skyline
elizabeth waldron 2018-04-24
Hi there,

I am trying to install Skyline 4.1. I am able to download the exe Setup file, but when I try and run it I get an error. I have attached the txt file containing error messages. Your help would be welcome!

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

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

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How to manage the order of sample in SL?
(1 response) VJ 2018-04-22

I've ordered my samples as you mentioned; "The order of samples in the document can also be changed by selecting Edit > Manage
Results and selecting Up or Down to move the sample to the desired position"

However I only see the changes in; Peak Areas-Replicate comparison and Retention Times Comparisons, not in EIC panel.

How can I get the same order in the bottom panel (EIA) too?
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Agilent QTOF targeted method optimisation
(2 responses) reka haraszi 2018-04-19
I know it is not very common to do targeted on a QTOF but... I did a set of peptide transitions with the help of the latest version of Skyline and exported the transition list for Agilent QTOF, see attached as glutenCTR2QTOF.csv. What I do not understand is what are the 0.01 m/z increments in the product ion mass associated with an increase of 3 in CE? The optimisation setting I put was for transitions (not precursors). When I set it for precursor, it gave me only one CE value (attached as glutenCTR2QTOF_pre.csv). Thanks, Reka
 glutenCTR2QTOF_pre.csv  glutenCTR2QTOF.csv 
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spectral library creation
(2 responses) klemens froehlich 2018-04-19
Dear Skyline support team,
I am currently experiencing difficulties creating a spectral library. While the MaxQuant output MSMS.txt can be read without error messages from Skyline, no y+ are displayed when I explore the library (all other ions are displayed as identified by MQ, also y++). I experience this problem with multiple versions of Skyline (3.7 & 4.1) and multiple different input files from MQ.
The tutorial spectral libraries could be created with y+ displayed and intact, so I don't think I got some setting wrong.
Please find attached the MSMS.txt I'm using. I've looked and it seems die y+ ions are reported correctly.

Could the import be somehow the problem?

Thanks for any help! Best,
 mqpar.xml  msms.txt 
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LOD and LOQ determination method
(3 responses) Wael 2018-04-17
Dear Skyline team,
as a skyline user I am very grateful for the newly implemented LOD calculation feature (blank SD-based) in Skyline-daily. Based on a paper from Steven A Carr group (BMC Bioinformatics. 2012; 13(Suppl 16): S9) it was shown that calculating LOD (LOD = μB +t(1-β) (σB + σS)/√n) by using blank-SD (σB) and low concentration sample-SD (σS) consistently produces more confident LOD values for most practical purposes in comparison to blank-SD methods. We have also observed the same in our lab.

Will it be possible to integrate different LOD calculation method in future in Skyline ?

Best regards
Wael Naboulsi
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Unique Modifications
(1 response) kerry hassell 2018-04-19
Hello, I can not seem to get the right mass for Goserelin in Skyline:

Pyr is a saved modification, but can not get 1269 m/z.

Can you please help?

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Import TIMS TOF data
(1 response) benoit fatou 2018-04-19
I have a problem with the import of TIMS TOF raw data and I have a last version of Skyline.
Could someone help me solving this issue?
Thank you very much,
Best regards,
 Error - TIMS TOF Import results.JPG 
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Can Thermo LTQ Orbitrap Elite run DIA​?
(2 responses) charlie yang 2018-04-17
Hi There,

Could anybody run DIA on LTQ Orbitrap Elite?

Thanks in advance!

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Small Molecule Peak integration issue with Thermo .raw QE+ data
(1 response) prasadpb 2018-04-17
Dear Skyline team,

I am facing peak picking and integration issue with the skyline with small molecule LC-MS data acquired on QE+ system.
I have attached images of 13C Creatinine peak integration done on Skyline (small molecule feature) vs Thermo Xcalibur software.
With the same files, I found there is a huge difference in pick detection and integration.
Can you please guide regarding what I am doing wrong?
Is there any steps I am missing or how can refine the peak detection method better.

Thanks and Regards,

Prasad Phapale
EMBL, Heidelberg, Germany
 Skyline peak integration.png  Xcalibur peak integration.png 
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Win32 Error: 3 when importing .raw file
(4 responses) kathrin wenger 2018-04-16

i always get this error message when I try to import result files (.raw) to my target list.
Any suggestions what I do wrong?


At 15:05:
Failed importing results file '\\\users\bib\users1\wengerk\Desktop\20180411_tMSMS_Z2u3_KW_undepl_101.raw'.
Win32 Error: 3
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Registration for the targeted proteomics course in Seattle
marleen enthoven 2018-04-16
Hi, I tried to Register several time for the Targeted Proteomics Course in Seatlle but the form keeps asking to add more text in the field Interest, although I already added over 50 words.
As the Registration Deadline is tomorrow, could you please help me with this asap.
The Registration is for Dr. Monika Edler, CSL Behring AG, Wankdorfstrasse 10, 3014 Bern, Switzerland.

Thanks a lot
Marleen Enthoven, assistant to Research
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Error in importing the transition list for small molecules
(7 responses) yih2 2018-04-13

I have encountered a problem when importing the transition list for small molecules. Some of the parent ion and their fragments can't be imported correctly (for example, CL96-169 and CL96-147 in the attached file). Please let me know if you need any additional information.



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

I would appreciate any input,
 MS1 scan error.JPG 
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Transition List
(2 responses) dspiciarich 2018-04-13
I have a number of transition lists made on an Agilent QQQ that I would like to use in skyline for quantification but many of my peptides have complex post translational modifications

For Example:

PEPT[large glycan] IDE PrecursorMz FragmentMz

The fragments may include the glycan portion and not the peptide portion. I ideally would like to do this without putting in the amino acid sequence but just a name and a precursor and fragment m/z mass. Is this possible?

Last question, I have the precursor mass and the fragment mass and the glycan mass but sometimes would like to use the average mass, whereas other times want to use the monoisotopic? Is this possible?

Happy Weekend!

Thank you,
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Peak Area Ratio missing (absolute quantification, small molecule application)
(2 responses) MP 2018-04-12
Dear Skyline community,

I have performed a targeted analysis of a small molecule, including 13C internal standard.

After importing the results into skyline, transitions and peak areas for the small molecule analyte and 13C standard are properly assigned and calculated (MRM, centroid, Waters TQ).

However, the Peak Area ratios of "Analyte" / "13C internal standard" are not shown and give an error (missing values) in the calibration curve. Both transitions, analyte and internal standard (=heavy)) are assigned to the same small molecule (I followed the instruction from the absolute quantification application note, for peptides it works well)

Any help is highly appreciated.

Best regards.
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EIC of product ions with multiple neutral loss
(1 response) lorrain 2018-04-11

I encounter this problem in both PRM analysis and MRM method setup when i was trying to look for a product ion which can have more than one neutral loss (for example, a y7 ion with two phosphorylation sites). The peptide and transition settings can predict the product ion with either one of the neutral loss (y7-98), but not both (y7-196). I was just wondering is there a way that we can have the skyline do that, or we can manually put in a product ion with both neutral loss (-196) in the list (especially for PRM analysis).

Thanks very much in advance!

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How to develop MRM method for interested proteins
(4 responses) VJ 2018-04-11
I'm new to MRM and going to develop a method for organelle markers.

I would be so much grateful if you could send me tutorials for;

-How to develop MRM method for interested proteins; with spectral libraries and without libraries (with theoretical digestions)

- Normalization and scaling the data.

Thanks for considering my request. I’m looking forward to your reply.
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Skyline won't import my .wiff results files; claims they "contain no usable data"
(5 responses) jmblakehedges 2018-04-06
Hi Skyline team,

I'm trying to import some data from my ABSciex 4000 QTRAP into Skyline. This is from a small molecule experiment using the QTRAP in Q1 Multiple ions (Q1 MI) mode. When I go to import my results, Skyline recognizes the .wiff files for my standards as results files, but when I try to actually import the files (File-->Import-->Results-->Add single injection reps...), I get the following error:

At 11:27 AM:
Failed importing results file 'C:\Users\jblakehedges\Documents\Proteomics\Small molecule test\Data Files\12.5uMpyrone-0.5uL-01.wiff'.
The sample 12.5uMpyrone-0.5uL-01 contains no usable data.

Inner exceptions:
Exception type: System.IO.InvalidDataException
Error message: The sample 12.5uMpyrone-0.5uL-01 contains no usable data.
The sample 12.5uMpyrone-0.5uL-01 contains no usable data.
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in c:\proj\skyline_4_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 277

I'm attaching the Skyline file that I'm trying to import into and also the data files I've tried and failed to import. This was performed both on my personal laptop (64-bit, Skyline v.4.1) and on the instrument computer (32-bit, Skyline v. 3.7), and both times I encountered the same error. Please help!


 20180406_lactones stds 
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Installation error
(2 responses) kblack 2018-04-10
Having trouble installing on Windows 10... Skyline tries to install .NET3.5 but windows 10 comes with .NET4.6 so will not allow....
 2018.04.10 skyline.PNG 
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Error in QuaSAR- Error in Calculation
(5 responses) patricia fernandez puente 2018-04-09
Today I installed the QuaSAR software to calculate the LOD and LOQ of my experiment in Skyline.
My experiment consists in the quantification of several proteins with the internal standard method.
To make the calibration curve I put, in the same quantity of total digested serum proteins, increasing quantity of heavy peptides.
When I performed the QuaSAR run with my samples this is the error I recived back:
"C:\Program Files\R\R-3.0.1\bin\R.exe" -f "C:\Users\usuario\AppData\Local\Apps\2.0\BRZVJE6R.N0V\6X83QJAC.83W\skyl..tion_e4141a2a22107248_0004.0001_c221031cbe0943a3\Tools\QuaSAR-1_33\QuaSAR-Skyline.R" --slave --no-save --args "C:\Users\usuario\AppData\Local\Apps\2.0\BRZVJE6R.N0V\6X83QJAC.83W\skyl..tion_e4141a2a22107248_0004.0001_c221031cbe0943a3\Tools\QuaSAR-1_33\QuaSAR.R" "C:\Users\usuario\AppData\Local\Apps\2.0\BRZVJE6R.N0V\6X83QJAC.83W\skyl..tion_e4141a2a22107248_0004.0001_c221031cbe0943a3\Tools\QuaSAR-1_33\common.R" "C:\Users\usuario\AppData\Local\Temp\QuaSAR_QuaSAR_Input.csv" NULL "Calibration Curve_09042018" "heavy Area" 1 "light Area" fmol/ul 1 1 -1 1 1 1 0 150 150 1 0.2 0 NULL "Calibration Curve_09042018" 0
[1] $Id: QuaSAR-Skyline.R 149 2014-10-23 13:56:13Z manidr $
[1] $Id: QuaSAR.R 149 2014-10-23 13:56:13Z manidr $
Attaching package: 'boot'
The following object is masked from 'package:lattice':
The following object is masked from 'package:gtools':
    inv.logit, logit
KernSmooth 2.23 loaded
Copyright M. P. Wand 1997-2009
Attaching package: 'gplots'
The following object is masked from 'package:stats':
[1] Processing arguments ...
[1] >>> Processing part 1 of 1
[1] Initializing ...
Error in calculate(data.subset, concentrationFile, output.prefix = outputPrefix, :
  Duplicate transitions found -- try including neutral loss
Calls: tryCatch -> tryCatchList -> parse.cmdline -> calculate
Execution halted

The folder Calibration Curve_09042018 is empty.

What should I do to fix the problem?
And, is available a workbook with all the possible troubleshoot problems in QuaSAR and the possible ways of solving the problems?

Thank you for your help.
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Skyline Results grid - peptide results
(4 responses) Tomas Vaisar 2018-04-10
Would anybody know if it is possible to get a Skyline quantification report where would be for a replicate included "calculated concentration", "ratio to standard", as well as Total fragment ion area for measured peptide and Total fragment ion area for the Standard.
In the Results grid I can get a view which gives me the former two, but does not allow to include also the actual peak area responses used in the calculation of the Ratio to standard and the Calculated Concentration.

Is there a different view?


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