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.

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Showing: limited to 100 requests
Prosit in skyline
(2 responses) Tobi 2019-12-05

Dear all,

thank you very much for integrating Prosit into Skyline. I noticed that the dotp in the new library match mirror view does not treat y ions from deferentially labeled peptides as equivalent. Is it possible to extract the Comparison dotp via results grid for all peptides at once?


 Prosit in Skyline.png 
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Denovo peptide import - No enzyme or protein ID
(1 response) john muchena 2019-12-06

Dear All,

Is there a way to import denovo peptides with no enzyme or protein ID to skyline? Useful in quantifying small peptides (2-4 AA).

Thank you,


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(48 responses) Brett Phinney 2019-11-07

Hey Skyline team, I’m loading some diaPASEF into skyline. It’s super fast!!!

One question. How should I make the library? What formats/ search engines are you using? Does the library use the ion mobility?

I’ll be happy to share some data with everyone



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Mass Error and Mirror Plots
(2 responses) sgoulding 2019-12-04

Hi, Skyline team.

I find the mirror plots very helpful when comparing synthetic to endogenous spectra but would love to see the mass error (in PPM relative to theoretical m/z) above the fragment ions. Is this possible? If not, would you please add this feature?

Thanks in advance,

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Error building spectral library from MaxQuant msms.txt
(1 response) Alon Savidor 2019-12-04

Following up on David's comment below, in the latest Skyline release there seems to be a bug with library building from MaxQuant's msms.txt file as well. It seems like the scan number which is used in the library is actually the one after the identification scan as it appears in the msms.txt file (Skyline library scan=msms.txt scan+1).
Thanks for your help

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Error building spectral library from TPP/PeptideProphet pep.xml
(14 responses) david schmidt 2019-11-06
I'm trying to import search results produced by X!Tandem and further analysed by PeptideProphet using the TPP, in order to build a spectral library in Skyline. The original data was acquired using a DDA approach on a waters QTOF, centroided and lockmass-corrected in MassLynx and then converted to MzML using MSConvert. Importing the MzML as results works fine. However, when trying to build a spectral library from either the interact.pep.xml or the tandem.pep.xml file, I'm getting the error: No spectra were found in the new library (see attached).

I suspect that this may be due to the link within the search results that point to the spectra in the MzML file. A pretty similar problem was described before, but I couldn't figure out what the solution to that was:

Thank you very much for your help,

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Error building spectral libraries from Comet/percolator output
(15 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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Export protein abundance from MRM data
(1 response) benoit fatou 2019-12-03

Dear Skyline Team,

I was wondering if there is a way to export the protein abundance in Skyline based on the average of the peptide areas.

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Import QuanDirect (aka IS-PRM) data to Skyline
(3 responses) Markus 2019-11-26

Hi Skyline team,

we are experimenting with the QuanDirect (aka IS-PRM) method on the Orbitrap Lumos using heavy spike-in peptides and everything seemed to have worked fine so far. When importing the data into Skyline (daily I run into the problem, that Skyline extracts all MS2 scans, i.e. also the ion-trap scans which are only targeted at the trigger y1 ion and which do not contain any other ion information. When combined with the MSMS Orbitrap scans that actually contain the fragment ions of interest this results in sawtooth-like peaks (see picture attached). How do I tell Skyline to only import/extract the hi-res Orbitrap PRM scans?

Many thanks,

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Upload MS files (.raw) to build a libary
(5 responses) melanie gut 2019-11-28


I am trying to upload measured results to build a libray. I measured my data with a Q Exactive HF 2. It seems that Skyline cant read my raw files. Am I doing something wrong?

Thank you very much for your support!

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Error reading MaxQuant msms.txt file for building spectral library
(3 responses) s26guan 2019-11-29

When I attempted to import "Peptide Search..." from MaxQuant search results, I got the attached error. What should I do? Thank you.

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EIC coelution problem
(6 responses) akulyyasov 2019-11-27

Dear Skyline team,
I am a new user and I have a problem with extracted ion chromatograms in Skyline. I run BSA sample on Q-TOF Impact II instrument (Bruker) using MRM method. When I analyzed raw file on DataAnalysis program I see ideal coeluting peaks corresponding to EIC from precursor ions 740 and 820.
Why I don't see the same coeluting peaks in Skyline? I used the tutorial recommended for the Bruker instrument.

Thanks in advance,

 DataAnalysis_BSA.png  Skyline_BSA.png  704449_AppTut_Skyline_Rev1.pdf 
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Not able to see
(1 response) Monica E 2019-09-15


I am trying to compare the peak areas of my different proteins among 16 different samples. I can now see the different peptides for each protein as well as the spectra corresponding to every replicate. However, I cannot see the "library" peak area, only the peak area for each replicate. I had a read through the forum and found a suggestion that said to "right click" on the replicate comparison panel and select to see the library peak area, however, this option does not come up when I "right click". Would you be able to help me? The library I am using in peptide settings is a reference pool sample of all the 16 replicates. I attach a screen shot as well in case it is any help.

Thanks in advance,
Monica Espinosa

 Skyline query.png 
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the conversion of spectronaut library in EncyclopeDIA
(7 responses) sunbergsoon 2019-11-26


recently I tried the EncyclopeDIA from your group, I want to use the library built by spectronaut.
However, I got nothing after conversion. So could you give me some advice about the format
and details on this ?

Thanks a lot.


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Poor System Utilization by Skyline
(1 response) chinmaya k 2019-11-27

Hello Skyline Team,

I am trying to perform DIA analysis with 2 DIA raw files against a quite large spectral library having 581197 precursors corresponding to 477997 peptides using Skyline 19.1 in our Windows workstation (Please find the attachment for details).

However, I find Skyline hardly utilizing CPU and RAM for any small changes with occupying all the available disk space (Currently the disk containing this skyline document has 1.8 TB space).

It would we great if there is an option in the skyline to set the number of processors/cores to be used, I have not seen any.

Thank You,

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protein quantification report
zhangfa 2019-11-28
Hi Skyline Team,
Can Skyline provide a built-in labelā€free protein quantification with control at protein level for DIA data? Or is there any external tool in the Skyline ecosystem ? If so, could you please provide any relevant tutorial?Thanks.
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Upload of PRM data to Panorama
(1 response) ingo.wohlgemuth 2019-11-27

Hello Skyline team,

I tried to Upload PRM results to Panorama and failed. Because I had identical analytes in different experiments, I erased the data in one of them and imported the new PRM data. This second file I cannot upload. Obviously, because I uploaded the file with the previous experiment (Although file name and PRM content are different).

The error message is:
Failed attempting to create sharing file C:\
An item with the same key has already been added.

Can I solve that somehow?

Thanks a lot!


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The version of Skyline compatible for Windows 8.1 Pro.
(1 response) lindagpaliyath 2019-11-25

I have tried to install version 19.1 of Skyline in my Windows 8.1 Pro laptop. I also tried Skyline daily. Both are not compatible and there is an error installing it. I have attached the screen shots of the error for your reference. Please do suggest the best version that is compatible with my OS.

 IMG-20191126-WA0010.jpg  IMG-20191126-WA0012.jpg 
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PRM - low precursor signal
(8 responses) lisa crawford 2019-11-25

I am working in establishing a scheduled PRM method of the Pierce PRTC standard mixture using a QEHFX.

I am noticing that there is an issue with the precursor for most of the peptides (do have signal for transitions). I’m assuming I have a global setting that is wrong?

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Failed Importing .raw Waters MSe Data
(4 responses) mcbagley 2019-11-07

Hello Skyline team,

I have spent the past while searching previous posts for my particular error and could not find it. I am trying to upload MSe proteomics data acquired on a Waters G2. When I try to import the .raw files I get the attached error. Unfortunately, these data files cannot be shared. The error occurs for any attempted import of these .raw files.

I'm using Window 10 Pro with 64-bit operating system. I'm using Skyline (64-bit)

I do apologize if this is a trivial fix.

Thank you!

 skyline data import error message.txt 
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Optimizing FAIMS on new Thermo instruments
(3 responses) Martijn van Duijn 2019-09-18


Our institution acquired a shining new Orbitrap Eclipse with a FAIMS source module. The FAIMS module need optimization for targeted analysis of our analytes, in order to obtain the best compensation voltage for transmission of the ion of interest.

It would be great if Skyline could facilitate this optimization step, similar to the optimization of collision energies that is already done. In fact, Skyline already seems to include a very similar optimization feature "Compensation Voltage', which seems to be intended for use with the SciEx Differential Mobility Separation. Can this feature be adapted to be used with Thermo FAIMS Compensation Voltages, or is this something that is intended for implementation in a future release of Skyline?


Martijn van Duijn

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Install issue
(2 responses) Brett Phinney 2018-01-30
Hey everyone, not sure if this is just my messed up win10 computer but I tried to install

And win10 would not let me install it even as the admin.

This seemed to fix it

just an FYI if someone else has this issue


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FAIMS unit on Thermo Lumos
(1 response) erik.soderblom 2019-11-20

Hi Skyline Team! I wanted to "bump" a thread entitled “Optimizing FAIMS on new Thermo instruments” from Martijn van Duijn back in September. We are now in the same boat (acquired a shiny new FAIMS device for our Lumos!) and at this point we just want to incorporate the data (say, 3 different CVs across 7 different peptides) into Skyline from our normal system suitability PRM acquisitions. I see where compensation voltage is a selectable unit under the ion mobility settings, but it doesn't look like Skyline is able to extract data for a single ("best") CV in the chromatogram view. Is this possible with current Skyline Daily ( yet?

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Generating library manual from PRM runs
(1 response) tsveth 2019-11-19

Hi everybody,

During development of my HL library sometimes software (MQ, PD, etc) does not detect peptides even though they can clearly be found in the spectra. Is it possible to load my PRM runs into Skyline and manually select the spectra that should be used for the library by using the Skyline chromatograms?

Thank you!

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Problems Installing Skyline
(2 responses) alex apffel 2019-11-19
I'm installing Skyline 64bit on a new computer within the Agilent Technologies Firewall. I have done this before on earlier versions without mishap. Having disable our normal security parameters, I open setup as an adminstrator and get this message shown in figure 1. Clicking on the Link in this window to skyline sends me to my browser home page. Clicking on the link to University of Washington Link sends me to the screen shown in figure 2. Accept the certificate and retrying yields the same result. Any ideas? Alex
 Fig1.png  Fig2.png 
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Method Development Workflow for Small Molecules
(3 responses) Richard Lam 2019-11-13


Do you have tutorials on optimizing MS/MS parameters for small molecules?
Note: I only have the precursor m/z info, fragments ions are unknown. It seems like the small molecule tutorials available in Skyline website are only for for both precursor and fragment ions are pre-determined already.



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Apply peak to subsequent error
(2 responses) Jason Held 2019-11-15

I'm assuming that the 'subsequent' in 'apply peak to subsequent' refers the result files lower in the file list. Is that true?

If either case, when I use it I get an error:

Top bit of the error window says "Failed to apply peak. Specific argument was out of the range of valid values. Parameter name: count:". More info gives this info:

System.Reflection.TargetInvocationException: Specified argument was out of the range of valid values.
Parameter name: count ---> System.ArgumentOutOfRangeException: Specified argument was out of the range of valid values.
Parameter name: count
at System.Linq.Enumerable.Range(Int32 start, Int32 count)
at pwiz.Common.PeakFinding.FoundPeak.SetBoundaries(Int32 startIndex, Int32 endIndex) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\PeakFinding\FoundPeak.cs:line 97
at pwiz.Common.PeakFinding.PeakFinder.GetPeak(Int32 startIndex, Int32 endIndex) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\PeakFinding\PeakFinder.cs:line 52
at pwiz.Skyline.Model.Results.ChromatogramInfo.CalcPeak(Int32 startIndex, Int32 endIndex, FlagValues flags) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromHeaderInfo.cs:line 2579
at pwiz.Skyline.Model.TransitionGroupDocNode.ChangePeak(SrmSettings settings, ChromatogramGroupInfo chromGroupInfo, Double mzMatchTolerance, Int32 indexSet, ChromFileInfoId fileId, OptimizableRegression regression, Transition transition, Nullable1 startTime, Nullable1 endTime, PeakIdentification identified, UserSet userSet, Boolean preserveMissingPeaks) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\TransitionGroupDocNode.cs:line 2710
at pwiz.Skyline.Model.SrmDocument.<>c__DisplayClass162_0.<ChangePeak>b__0(TransitionGroupDocNode node, ChromatogramGroupInfo info, Double tol, Int32 iSet, ChromFileInfoId fileId, OptimizableRegression reg) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1721
at pwiz.Skyline.Model.SrmDocument.ChangePeak(IdentityPath groupPath, String nameSet, MsDataFileUri filePath, Boolean loadPoints, ChangeNodePeak change) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1774
at pwiz.Skyline.Model.SrmDocument.ChangePeak(IdentityPath groupPath, String nameSet, MsDataFileUri filePath, Transition transition, Nullable1 startTime, Nullable1 endTime, UserSet userSet, Nullable1 identified, Boolean preserveMissingPeaks) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1719 at pwiz.Skyline.Model.PeakMatcher.PeakMatch.ChangePeak(SrmDocument doc, SrmTreeNode nodePepTree, TransitionGroupDocNode nodeTranGroup, String nameSet, MsDataFileUri filePath) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\PeakMatcher.cs:line 418 at pwiz.Skyline.Model.PeakMatcher.ApplyPeak(SrmDocument doc, PeptideTreeNode nodePepTree, TransitionGroupDocNode& nodeTranGroup, Int32 resultsIndex, ChromFileInfoId resultsFile, Boolean subsequent, ILongWaitBroker longWaitBroker) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\PeakMatcher.cs:line 150 at pwiz.Skyline.SkylineWindow.<>c__DisplayClass1198_2.<ApplyPeak>b__0(ILongWaitBroker monitor) in C:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineGraphs.cs:line 2111 at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action1 performWork) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 232
--- End of inner exception stack trace ---
at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Util\Util.cs:line 1909
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 180
at pwiz.Skyline.SkylineWindow.ApplyPeak(Boolean subsequent) in C:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineGraphs.cs:line 2111

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How to get the peak area for the precursor in a PRM run?
(2 responses) ekramh1982 2019-11-14

I have a PRM run with only one precursor (file attached). At the end of run I would ideally like to have the peak areas for the precursors. In the attached images the areas that are plotted are of the product ions (View->Peak Areas->Replicate Comparison). How do I get the area for the precursor in different runs. Also, in the area plot in red colors, what are those numbers (126, 129...) at the baseline of the peaks? And is there any way to print out the numbers or plot the numbers? If someone can point to a webinar that shows this, that will be great. Thank you.

 Bz_Test_1.PNG  Bz_Test_2.PNG  Bz_Test_3.PNG  Bz_PRM_IsolationList.csv 
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Is the light version of the peptide present?
(7 responses) sa825 2019-11-11


I imported some heavy labelled PRM results into skyline and for the light version of a peptide I am interested in, only 2 fragment ions were detected.

In the heavy version of the same peptide, all 3 fragment ions were detected.

Can I assume from this that there is close to 100% labelling, in the sense that the light peptide is no longer present in the sample? And its only the heavy version left in the sample?



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Multiple working groups on Panorama
(3 responses) Sarah Parker 2019-11-13

Hello! I am still a user in my Post Doctoral lab, but now I have my own lab and am an administrator for a new Panorama project that will house my independent projects. When I try to upload to Panorama from Skyline, I only see my old lab's folder and not my new folder recently made of which I am the administrator.

I've tried resetting Panorama log in within the Tools > Properties window, but nothing is working. Can you please advise how I can make my Skyline program talk to my new Panorama folder?



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Background proteome not digesting
(1 response) gmurray 2019-11-13

Hi there,

Recently installed Skyline on Win10. Trying to create a background proteome with no luck. I can successfully create the .protdb file containing 10,000 proteins from a single FASTA file; however, when I select my enzyme it fails to digest this background.

Any help would be appreciated.

Many thanks,


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MSstats download error
(1 response) supitcha pan 2019-11-12

Hi, everyone
I've got the problem about MSstats download.
Once I go to Tools> Tool store
The error was occurred as "Error connecting to the Tool Store: The remote server returned an error (407) Proxy Aythentication Required.

Is there anyone know the solution, please

Thank you

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Missing expected Retention Times
(3 responses) SW Test 2019-11-11

Hi Skyline Support,

We observe that if Skyline detects maxima at different retention times for the different charge states, it appears to use an Average value for the method for all the charge states. This results into incorrect Scheduled retention times in the MRM method generated by Skyline.

Please advise if this is a feature limitation for the current version? ...

Best Regards

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Setting different analyte concentrations for different peptides
(2 responses) lee julie 2019-11-12

Hi, I am trying to define the analyte concentration for samples defined as QCs in the Document Grid under replicates. I am unable to define separate analyte concentrations for different peptides - it applies the last defined concentration to all peptides. Any advice on how to correctly approach this?

Thanks in advance

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Using Panorama .clib File to generate scheduled MRM methods
(1 response) hogan 2019-11-06


I am wondering why you cannot generate a scheduled MRM transition list using just a .clib file from a Panorama Chromatography library. I have uploaded multiple proteins from multiple runs/documents into the library and can export one concatenated clib file that I thought would allow me to generate a scheduled method with all of the validated targets. I pull the .clib file into a new document and when I attempt to generate a scheduled method it throws an error that I need scheduling information from a data file to generate a scheduled method/transition list.

The clib file contains the RT, Precursor, and Y ion information for all the peptides in the library. Why can it not be used to generate a scheduled transition list without importing data.



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Absolute Quantification in Skyline for (Peptide/Protein)
(6 responses) iej7 2019-10-18

I'm working with skyline (v19.1) to do some peptide based absolute quantification. All experiments are from MRM files (Instrument: 6500 QTRAP). As a general example, let's say the skyline file contains 2 proteins that are in different concentrations in my cal curve :

Protein A
-Peptide ABCDE
=Peptide FGHIJ

Protein B
-Peptide KLMN
-Peptide OPQR

I am very likely missing something basic, but don't see how to specify unique concentrations for the calibrators if Proteins A and B are in my cal curve at different concentrations. It seems like I can only specify analyte concentrations for 1 protein only and those values are repeated for all other proteins in the skyline file (i.e. Calibrator concentrations for Protein A will also be the concentrations for B).

How do I specify different concentrations for the proteins in my skyline file so that cal curve data can be evaluated appropriately?

I'm sure I'm overlooking something simple. If this is true, just point me to the right tutorial.

As always, thank you.

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Meaning of 'Min peak found ratio' (refine->advanced settings)
(3 responses) Emma Jappe 2019-11-08

I am analysing DIA data and have a spectral library that I am searching my DIA data against. Some of the transitions are of very poor quality (indicated with a red dot) and I would like to remove these; however, I have a lot of data and it would take a few days to do this manually. I have been looking at some of the filtering options in edit->refine->advanced and in the results tab, I have set a cut-off on the 'Min peak found ratio' to 0.8. Can you describe to me the meaning of this setting as well as the other settings (max transition peak rank etc).
Thanks a lot!

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Reverse calibration curves for determining LOD and LOQ in PRM assays resulted in some kind of paradox
(3 responses) sofia farkona 2019-11-08
Hello all. I have a question which is not limited to Skyline software. However I would appreciate it if you could have a look. I have developed PRM assays targeting of some proteins of interest. The biological sample into which I apply the PRM assays is bronchoalveolar lavage. I have constructed REVERSE calibration curves to determine LOD and LOQ because the majority of the endogenous peptides I look at are present in most of the samples. Basically the heavy peptides are spiked in, in different dilution in the same BAL sample (or in a pool of BAL samples). In that way our calibrator sample have a constant concentration of the light or endogenous peptide while the heavy counterparts are in different concentrations. What we plot is the H/L ratio over the theoretical concentration of heavy peptide spiked in the sample. The paradox is this: While in this way I manage to "see" the heavy up to a concentration of for example 0.05 fmol/uL (or up to an amount of for example 22 fmol) when I apply my PRM to monitor the light peptides in other BAL samples, following the calculations, it seems that the light peptides exist in lower concentrations than this 0.05 fmol/uL (or in a lower amount than this 22 fmol).

This has confused me very much and I don't even know how to look for this in the literature.

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Importing iRT values
(2 responses) Erik 2019-11-07

Hi Skyline team,

For importing srm/mrm assays nowadays it becomes more regular that together with Q1/Q3 coordinates also the iRT is provided.
Is it possible to import a list of (oxi/phospho modified) peptides with their iRT?
I know you can import an iRT peptide database to an iRT calculator but this is only possible when you have measured the peptides yourself and export them as an iRT database .
So ideally when you first want to run an SRM assay with >100 peptides you can already let skyline predict the RTs based on iRT and export a method to measure the data with wide RT windows. It would be nice to be able to import an excel list with two columns [modsequence] [iRT] like you can also do with Q1/Q3 coordinates.

Hopefully you have a solution so I do not have to run many unscheduled runs first to let skyline know the iRTs of my peptides of interest!

Kind regards,

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Filtering data using rdotp values
(1 response) Neves LX 2019-11-06

Hi Dear

I'm wondering if it is possible to get rid of peptides that had not been consistently detected in both light and heavy channels (or its noisy in some) by filtering the data using rdotp values. In addition, has the signal to noise/background been implemented in Skyline?

Best regards,

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Using more than one calibration curve in a single skyline document
(3 responses) hogan 2019-10-30


I have been using skyline smoothly to run a quantitative small molecule assay with a calibration curve and check standards and the whole nine yards for a few months now and it is great!!! Thank you all so much for your hard work in bringing small molecule functionality into this great platform!

Now the issue.
Skyline works fine for 1 analyte with 1 standard curve. I have recently tried to add a second analyte with a separate calibration curve to the same skyline document and I am only able to input one value in the "Analyte Concentration" column of the document grid for each replicate that is set as a "Standard". (I have multiple analytes in my calibration curve and their concentrations are different in the CAL levels). I can only load up one analyte curve worth of values and then they repeat over and over again in the grid even if the "Molecule Name" or "Precursor" are set to different values.

Is it possible for skyline to have multiple calibration curves set in the same document? Can you add a custom sample type like "Standard2", "Standard3", etc. and assign separate calibration values there?

My current workaround is to pull the data files into two separate skyline documents, with different analytes. I then import the annotations separately in each one and combine the calculated concentration data afterward using the replicate name as the index to join them on. Please let me know if there is another way to do this or if there is something in the works!

Again. Thanks so much for your work!


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Is there a way to elimate false postives in Small Molecule mode?
(1 response) Allison Haase 2019-11-04

Hi All,

Sorry for the bother, but I was wondering if there any way to set a threshold for peak intensity in small molecule mode in Skylines.

My group uses Skyline for a GC-MS data set that uses a surrogate standard for quantification. This method has a pretty noisy baseline, especially in our blanks and double blanks, as the lack of filtering causes a lot of product ions to be present.

The issue we have is with the double blanks. Due to the noisy baseline, Skyline picks out a very small peak for the internal standard and for the other molecules of interest. Due to the calibration curve being normalized to the surrogate standard, this causes incredibly high concentration values to be reported for our double blanks. Currently we deal with this problem by deleting the Surrogate Standard peaks for double blanks, but we wish to automate this process and to make sure that peaks are not picked out when they are not actually present. Is there a way to set an intensity threshold for Skyline to pick peaks?


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Total area normalized
(2 responses) aqassab 2019-11-04

Dear Skyline team,
I have two questions:

  1. I have a set of data created by skyline that needs to be normalized. In my exported transition list, I have included total area normalized and I use normalized to global standard in my peptide setting. I am wondering how this normalization method was performed. What is the "mathematical formula" used to calculate total area normalized. Please find a screenshot showing the total area normalized for one of the peptides in the attachment.
  2. I have also attached another screenshot showing one of the heavy peptides that is peaked twice and are located adjacent to each other, with similar transitions, peak areas but at different retention times. Could this be a dimerization issue?
    Thanks in Advance!
 Skyline exported data.PNG  image.png 
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no product ion chromatograms found
(6 responses) ddeleoni 2019-10-31

After importing files, I am not able to see the product ion chromatograms. Also noticed that the file size for the skyline chromatogram file is much larger than the skyline document file, when it should be the other way around.

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Problem Importing pep.xml File from PeaksX Search of Waters MSe Data
(3 responses) dwwilk02 2019-10-29

I've gotten the error message listed below in Skyline when I try to import a pep.xml file from a PeaksX search of waters MSe data. The MSe data was converted to mzML prior to the search in PeaksX. I attached the pep.xml file that was generated by PeaksX. Thanks for any suggestions. The error message is:

ERROR: peptides.pep.xml(line 3463): [Serializer_mzXML::translateSourceFileTypeToNativeIdFormat] unknown file type

Command-line: C:\Users\dwwilk02.AD\AppData\Local\Apps\2.0\VLVOVZ8Y.QJE\9QMYDTGV.WT6\skyl..tion_e4141a2a22107248_0013.0001_f6e2bff174ff9e23\BlibBuild -s -A -H -o -c 0.95 -i CytokineQuanSkylineTemplateTest191029 -S "C:\Users\dwwilk02.AD\AppData\Local\Temp\tmp2B91.tmp" "C:\Users\dwwilk02.AD\Desktop\Temp Data\Cytokine Quantification\CytokineQuanSkylineTemplateTest191029.redundant.blib"
Working directory: C:\Users\dwwilk02.AD\Desktop\Synapt_MSe_Test_191001_PEAKS_91
OK More Info
System.IO.IOException: ERROR: peptides.pep.xml(line 3463): [Serializer_mzXML::translateSourceFileTypeToNativeIdFormat] unknown file type

Command-line: C:\Users\dwwilk02.AD\AppData\Local\Apps\2.0\VLVOVZ8Y.QJE\9QMYDTGV.WT6\skyl..tion_e4141a2a22107248_0013.0001_f6e2bff174ff9e23\BlibBuild -s -A -H -o -c 0.95 -i CytokineQuanSkylineTemplateTest191029 -S "C:\Users\dwwilk02.AD\AppData\Local\Temp\tmp2B91.tmp" "C:\Users\dwwilk02.AD\Desktop\Temp Data\Cytokine Quantification\CytokineQuanSkylineTemplateTest191029.redundant.blib"
Working directory: C:\Users\dwwilk02.AD\Desktop\Synapt_MSe_Test_191001_PEAKS_91
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_19_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in C:\proj\skyline_19_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 186
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 144
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Standard deviation in quant experiments
(5 responses) julius fuersch 2019-10-16

Hi guys,

I have a very general question regarding error distribtion in quant proteomics experiments. Often the standard deviation and therefore the p-value is calculated using log values of the extracted ion chromatogramm areas. By using the log values instead of the original values you will get a completely different distribution and a much lower relative standard error! Why do people use the log areas instead of the original values? In my very small understanding of statistics, this artifically improves the test statistics and makes the results better than they appear! I would be very happy about an explanation because we are using an in-house developed cross-link quant software which also does it in that way and I would really like to understand the background!

Thanks a lot in advance


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Calculation standard error
julius fuersch 2019-10-29

Hi Nick,

I posted a last question in our recent conversation regarding error calculation based on log or non log values? Did you see this? Do you have an answer to this?
Thanks a lot
Kind regards

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Selecting neutral loss transitions
(2 responses) michael plank 2019-10-28

Hi Skyline team,

is there a way to specify the extraction of y-ions with a phosphate loss? I checked that the 'Phospho (ST)' modification on the peptide of interest has the 97.9769 neutral loss specified, but neutral loss transitions are not defined automatically.

(The context of this question is that I observed a y-98 ion as a site-determining ion for a phospho-peptide that is in my library. Now I want to see if I can detect site-determining ions for isoforms that were not identified, i.e. are not in the library.)

Thank you,

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Question regarding DIA Isolation Window Scheme output for Thermo Fusion DIA m/z list
(4 responses) dhardie 2019-10-16

Dear Skyline Team;

I have a question regarding the DIA Isolation Window Scheme output when using Skyline Daily 64-Bit the output table contains "Start and End m/z" with the ability to show "Margin" and "CE Range" and has the "optimized window placement" feature but there is no "Centre m/z" similar to the EncyclopeDIA Scheme Wizard? Older versions of Skyline and presentations show "Generate Target " toggle that would show a centered target m/z in the output list that could be exported as a csv into the Thermo Fusion MS2 targeted list. The current output is just start and end m/z. Do you suggest manually calculating the "centre mass" for the DIA acquisition as a work-around.

Also I have noticed the masses generated when using margin widths of 0.2 if using Skyline and EncyclopeDIA window generators give different values. I have seen Searle's bitbucket documents describing Skyline's settings for DIA windowing acquisition settings.

Is there a formal document explaining the windowing scheme settings MSX,Overlap, Overlap MSX, Fast Overlap Experimental and what these settings are? I see many presentations for Q-Exactives but not much for Lumos or Fusion.

Thanks for entertaining my questions


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iRT error when adding Protein Prospector generated blib
(5 responses) anatoly.urisman 2019-10-16

Dear Skyline team,
I am getting an error while adding an “on-column” spectral library (blib generated directly from Protein Prospector) to an iRT calculator (Pierce iRT set supported by Skyline). The error is “1 run was not converted due to insufficient correlation.”

I’ve uploaded a dummy Skyline document to the Upload folder. It contains two libraries, both of which are generated from the same DDA data, just two different searches (one focused on identifying the iRT peptides and the other on other peptides in the samples).

Could you take a look at the error and let us know if it is something that needs to be fixed on the Protein Prospector end?

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Unable to auto-calculate regression
(5 responses) michael plank 2019-10-22


I`m trying to set up a targeted PRM method by applying an RT-predictor on a sample where I only measured the iRT peptides (Biognosys iRT-11) in the calculator.
When I go to Export: Isolation list and try to select 'scheduled', I get:
"Retention time predictor is unable to auto-calculate a regression. Check to make sure the document contains times for all of the required standard peptides."
All 11 iRT peptides are detected with good signal and when I look at only the standards in View> Regression> Score to Run, the regression looks fine.

The complete file is attached.

Thank you,
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Ignoring the PDA data trace (FUNC003)
(3 responses) stsyp 2019-10-22

Hey Skyline

I'm using skyline to analyze SmallMolecules. Back in the days I had QC data files that only consisted out of two traces (FUNC001 and FUNC002) whereof the first one is the data of the sample and the second one the data of the lockmass. For a while I added a third trace into the data acquisition method (FUNC003) that consisted of the data from a PDA detector. These data traces are huge but didn't botter skyline to function as it did before (it was just very very slow). My workaround was opening that FUNC003 trace in notepad and deleting a bunch of data points in the middle before I processed the data in skyline --> slow in the beginning, fast in the middle and slow in the end).
I've been running without PDA for over a year now, but since short I need it again and my workaround doesn't seem to work at all anymore --> skyline stops working (even when I don't delete a bunch of data points in the middle).

Are there settings that I can change so the FUNC003 trace is ignored like it was only a file with two FUNC files?
In attached I've already shared a .raw file to clarify my problem.

Steven Vds

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(2 responses) Tobi 2019-10-22

Dear Skyline Team,

filtering for 1% FDR by mProphet model is easy in Skyline by filtering for Detetction Q value <= 0.01. However, we have some issues with that and would like to export data of target and decoy peptides, filter the data ourselves and then perform the 1 % FDR cutoff.

What would be the most feasable way to perform the 1% FDR filtering (Q value calculation) outside of skyline with only z-scores available? Is it a simple formular or more complex? In case it requires complex code, could you please link it? (Noncoder here and I already checked the tutorials and cited paper but did not find q value mentioned seperately)

A short comment is highly appreciated.

With best regards,

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Failed importing results file
(9 responses) sandberg 2019-01-22

Dear Skyline Team,

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

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

I also attached the problematic wiff2 file.

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Wrong scans extracted when building spectral library from MaxQuant results
(2 responses) danielz 2019-10-22

Hi Skyline Team,

Tools used: Skyline and MaxQuant 1.6.5

I realized (after hours of questioning my sanity :-) ) that there is a bug when using this new feature where Skyline takes the MaxQuant result and goes back to the raw file to extract the raw scan (great feature, by the way).

When one uses the MaxQuant msms.txt as is, the scans that are extracted by Skyline look nothing like they should look like, because the wrong scan is extracted. I tested this on multiple instances, however not MaxQuant versions (will follow). What happens is, that Skyline always takes the scan subsequent to the actual scan-number (ScanNr + 1) it should extract. Probably an indexing error or the difference between the scan number and the internal counting when accessing the raw file?

A working quick-fix for this issue is to edit the scan number column of the msms.txt in Excel and subtract 1 (ScanNr - 1) from the respective scan number. Skyline will then extract the correct scan.

Can you reproduce this or is my MQ version bogus? I will try an older one tonight.

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How do I limit the RT window when use 'Apply Peak to All' function
(3 responses) qzhang31 2019-10-17

Hi there,
I am running MS1 filtering for a bunch of peptides, I have a standard with high concentration and every peptide's peak is properly picked. Then I am loading some data with much lower concentration of those peptides and I find that skyline will pick wrong peaks at a very different RT.

How do I tell skyline to just look at 1 min RT window within the peak in file A for all the other files?


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Real-time transfer of data to Skyline
(2 responses) benoit fatou 2019-10-18

Hi Skyline Support,

I was wondering if there is a possible way to perform an automatic transfer of data to Skyline just after the acquisition.
That would be very helpful especially for large number of samples.

Thank you very much for your help,

Benoit FATOU

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PRM Workflow with Spectral Matching
(1 response) sa825 2019-10-18

Good afternoon,

I have watched Webinars 3 and 4 and found it very helpful but I have a question regarding spectral matching.
I conduct PRM on the Thermo Q Exacitive and this is my current workflow:

  • I choose the precursor and product ions of interest and export an isolation list for the Q Exacitive
  • I get the Raw Data file from the Thermo and run it through PD (with Percolator) which produces a PD result file
  • I then import this PD result file as a PRM search (File -> Import -> Peptide Search) and match it with the same PRM Raw file by following the instructions in the Import Wizard
  • This produces what I would call really good spectra. The chosen product ions are clearly detected with Peptide ID annotations.

However, when I import the raw result file ( File -> Import -> Results), I don't get the same level of detection. In most cases, none of the product ions detected when I do the workflow above are seen in this result file approach.

Can you please explain why there is this discrepancy? And if my current workflow is correct?



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Not possible to extract chromatograms from MGF files
(3 responses) marc isaksson 2019-10-17


I tried to use Skyline(v.19.1) for MS1 filtering + spectra library building, by importing a mzIdentML file from PeptideShaker (v.1.16.42). While the spectral libraries were built, I got stuck in the peptide search import at the Extract Chromatograms part. The MGF files referred to in the mzIdentML file were missing. SEE ATTACHED.

When I tried to help Skyline locate them by clicking on the Find button, and navigating to the MGF file location, nothing was displayed. Apparently, Skyline does not support the MGF spectra files referred to in the mzIdentML file.

Will MGF files be supported for chromatogram extraction in a later release? Then I could do MS1 filtering on the PeptideShaker result, something which I think would be a nice feature.



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issue_exporting sepctral lib.
(4 responses) Wael 2019-10-15

Dear Skyline Team,
I am getting an empty .blib file after exporting spectral lib from skyline. can you please help me?

I have generated the lib from PD2 msf file and everything was good, but when I try to export the lib. I am getting a 20kb empty file. I have tried to generate the lib again but that did not help. I have also tried to open the skyline file using skyline-daily and export the lib. but I am getting the same emtpy folder.

Best regards


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The error with cdb file
(3 responses) shin 2019-10-09

Hello skyline team,
I'm trying to analysis proteome data with Mascot dat file.
Previously I succeeded to analyze other data.
However It doesn't work in this time.
Investigation of the reason of error clarified some files made "dat.cdb" file.
What is dat.cdb file and how do we analyze them?

Thanks for your assistance and effort.


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Missing MS2 from Mass Inclusion List.
(5 responses) mwmann 2019-10-10

Hi, much like everyone else here, I should probably mention how incredibly useful Skyline has been for me. I've been using it for the last 2.5 years, starting as a learning tool for MS and now using it for my own projects as a graduate student. That being said, I would probably still consider myself a beginner with this software. Right now I'm using Skyline to help me quantify changes in histone ptms, and it's not an understatement to say that I couldn't do it without this software. However, I'm running into a weird issue related to MS2 spectra not being recognized in Skyline.

I'm running Skyline Daily on Windows 10.

I'll start by detailing my workflow:

  1. I run my samples on a bruker QTOF using a DDA workflow. I've manually added certain masses to a mass preference list so that some ions would fragment at all points during their elution. This is to facilitate quantification of co-eluting isobaric ions. (I'll admit, it's possible that this step isn't working as intended, but it wouldn't explain all the behavior I'm seeing).
  2. I convert my files to centroided .mzml files and use Metamorpheus to calibrate and run a search for modified peptides. I import the resulting .mzid files into skyline as a spectral library with a cutoff of 0.99. My peptide settings allow a large number of PTMs (10), but restrict peptides to those found in the library.
  3. I import a fasta of selected histones (just H3 for now), and remove repeated peptides.
  4. I import my .mzml files and use the peptide identification times on each spectra to select peaks.

All of this seems to work well. However, when I look at MS2 spectra, I notice that I have relatively few MS2 peaks, even when peptide ids are shown on the spectra. I've attached an image demonstrating this. I can click on the IDs and see my fragment ions changing, but Skyline doesn't seem to have any MS2 at those timepoints. On the image, you can see the highest scoring ion as a straight line from practically zero abundance to its max, That same ion in the ID spectra increases, but not in a straight line manner.

Noting another poster had a similar issue which was resolved by changing the MS/MS filtering to a DDA strategy, I attempted that without success.

Any help would be appreciated. I've uploaded a minimal Skyline project that replicates this as a .zip to Uploads Page (File Name: M.Mann I imagine you'll need the raw files too, so I've uploaded those as well (File Name: M.Mann Raw

Thanks regardless! Hopefully this can be resolved easily.

Morgan Mann
University of Wisconsin-Madison
Brasier Lab

 M.Mann Bug.png 
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MSstats analysis using ratio light:heavy
(1 response) Taina Marques 2019-10-11


I was wondering if is possible to use the ratio light:heavy to perform group comparison analysis using the MSstats in skyline?
As far I could see, skyline only does with the area without any ratio light:heavy. Is there a way that I can switch to ratio?

Thank you!

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Small Molecule Quan using light:heavy ratio without deuterated (heavy) version of each analyte (light)
(1 response) kbrady 2019-10-08


The assay I am working with quantifies 5 metabolites of methotrexate (MTXPG1, MTXPG2, etc), but rather than using the deuterated version of each compound as internal standard for each metabolite, the method cheats a bit and quantifies MTXPG1 and MTXPG2 as the ratio of MTXPG1:MTXPG1-d3 and MTXPG2:MTXPG1-d3, respectively. MTXPG3-5 are quantified as the ratio of MTXPG3:MTXPG3-d3, MTXPG4:MTXPG3-d3, and MTXPG5:MTXPG3-d3, respectively. (Yes I realize this is less than ideal, but I did not make the method :) )

Following your advice here (, I formatted my transition list as shown in the attached translist.csv ('True Identity' and 'True Identity Purpose' columns are just for clairity, I'm not pasting those columns). Can skyline handle an experiement set up like this?

Also attached is the document grid generated by Skyline through Edit -> Insert -> Transition List. You can see Skyline has not kept the m/z as provided in the transition list, and has instead constrained 'heavy' MTXPG2, MTXPG4, and MTXPG5 to be similar in mass to the 'light' precursors. I am guessing you have some internal check along the lines 'A heavy transition must be heavier than the light transition it is associated with'? Is there a way around this? I would like to use light:heavy ratio for my calibration curve, but this currently only works for MTXPG1 and MTXPG3 (which are actually matched with a deuterated compound, unlike MTXPG2, MTXPG4, and MTXPG5).

 translist.csv  doc grid.csv 
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Differnt product ions not shown together in Skyline
(2 responses) FlorianBonn 2019-10-10

I have a strange problem with Skyline I want to quantify a large peptide in a SRM experiment with a triple quad. The precursor is m/z ~700 with +4. We measured 3 product ions: b3 (m/z 350 +1), y19 (m/z 1050 +2) and y21 (m/z 760 +3) with a Qtrap and loaded the data in Skyline.
If I select all three Transitions it shows me data for y19 and y21, but for b3 only a zero signal. If I remove either y19 or y21 from the list, only data for b3 is there and for y19 or y21 only the zero signal is there. If I again add y19/y21 to the transition list, the b3 trace is gone and the two from the large ions are both displayed. Can anyone help me out to get all three transitions displayed at once or at least b3 and y19 together?
Sorry if this questions was answered, but search terms I tried were either to specific (nothing was found) or to general.

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Cannot import iProphet pepXML file to build a spectral library
(5 responses) marc isaksson 2019-09-24


I tried to generate a library in Skyline from an iProphet pepXML file, but failed in the last step.

Long story short:

I tried to create it the library by first starting a blank document. Then I went to Settings>Peptide Settings and clicked on the Library tab.
I entered all info regarding file names, output path for library, cut-off score and what iRT peptides I used.

In the next step, I added the iProphet pepXML file, and then clicked on the Finish Button. After a few seconds, the software threw an error. It seems like Skyline/BlibBuild cannot find an attribute called SpectrumNativeID in the iProphet pepXML?


FYI: I made sure to copy the iProphet.pep.xml file into the same directory(Working directory: D:\Data_TPP\RAW_files) as the mzML files used for the database searching (combined both Comet and X!Tandem searches into a single iProphet file).

I used the TPP v.5.0.2, with X!Tandem (old: Jackhammer 2013.06.15.1) and Comet (v.2018.01 rev.4).

If you could help me solve this issue, I would be really happy.



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Problem installing Skyline Daily
(2 responses) joel federspiel 2019-10-09

Hello Skyline team,

I have one computer that is refusing to install Skyline Daily ( We have used daily on this computer routinely in the past, but at some point recently the software was removed. When I try to install it again, I get the attached error and am unable to complete the install. This computer is running Windows 7 Professional.

Any help would be greatly appreciated. I am happy to provide any other information that would be helpful.



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Skyline not recognizing Heavy Acetyl modification of lysine residues
(13 responses) dbade001 2019-09-20

I am trying to upload my maxquant files to skyline. It has worked for my other files but it seems to have an issue with the heavy acetyl modification on the lysines. I am uploading a pic of this. Have you seen this issue before?

Thanks for your assistance and effort :)


 skyline error heavy acetyl.png 
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Skyline cannot import iProphet pepXML files to build a spectral library - if X!Tandem was used as a search engine
(1 response) marc isaksson 2019-10-07


I have noticed that Skyline (v.19.1) cannot import a iProphet pepXML file, if X!Tandem was one of the search engines, or the only search engine. I have tried to combine Comet searches and X!Tandem searches into an iProphet pepXML. When importing into Skyline, I get a error message saying(see attached for full error message):

ERROR: comet_tandem.ipro.pep.xml(line 555966): Missing required attribute 'spectrumNativeID'.

Command-line: C:\Users\marc\AppData\Local\Apps\2.0\W8H7VL7Y.7R9\HMJZHTQA.5XR\skyl..tion_e4141a2a22107248_0013.0001_f6e2bff174ff9e23\BlibBuild -s -A -H -o -c 0.999677 -i H9D_6F_190913_lib1 -S "C:\Users\marc\AppData\Local\Temp\tmp17BF.tmp" "D:\Data_Skyline\H9D_6F_190913\H9D_6F_190913_lib1.redundant.blib"
Working directory: D:\Data_TPP\RAW_files

Apparently, Skyline/Blibbuild is looking for the term SpectrumNativeID. Problem is that only Comet results have such a reference. X!Tandem does not produce any column or reference saying SpectrumNativeID. I was using X!Tandem version Jackhammer 2013.06.15.1, as part of the TPP 5.0.2 distribution.

When I created an iProphet pepXML file from Comet searches only (v.2018.01 rev.4), it was no problem to import the pepXML file into Skyline.

I would be really happy if search results from Comet and X!Tandem could be combined into iProphet and then imported into Skyline! As I understand, BlibBuild is supposed to import X!Tandem results, given that the file format is xtan.xml.



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

Dear Skyline Team,

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

I have my partial skyline processing file attached.



 pic 1 _ base-peak intensity chromatogram.PNG  pic 2.png  pic 3_raw chromatogram of standard peptide.png 
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How to normalize the peak area with b-gal peptide
(1 response) drrenugoel 2019-09-30

I would like to normalize the peak area with one peptide of b-gal which we had added in all teh samples

There is a variation in the peak area. How to normalize all other sample peptides by this peptide?


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Calibration Curve Errors for Light:Heavy Ratio
(1 response) bwidner 2019-10-04

Hello Again,

I have been using skyline for small molecules and prior to now I have made linear standard curves using the peak area. That has worked great. I just introduced isotopically labeled standards, and I am now having trouble getting the standard curves to appear for some molecules. Because it works for most molecules, I dont think it's a problem with my settings or Document Grid, etc. The problem is that the Calibration Curve plot will have data points on it, but it won't draw a line and lists NaN for Slope and R-Squared. I have tried everything I can think of to address this problem, and I can't solve it. It does seem to happen with the curve is fairly crappy.. When I just plot the peak area of the light compound for these, I get a curve as normal. Is it something to do with very low intensity heavy peaks?

I attached a photo of the problem, and I will upload the files to the file share thing momentarily.


 ectoine curve error.tif 
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Failed Importing Results... Failed to Centroid Scan
(4 responses) bwidner 2019-10-02

Hello Skyline Team!

I tried to import a batch of ~30 files and got the following error for three of them

At 2:35 PM:
Failed importing results file 'C:\Filepath\mtab_BCderiv_BC43_092619_69.raw'.
[SpectrumList_Thermo::spectrum()] Error retrieving spectrum "controllerType=0 controllerNumber=1 scan=3119": [RawFileThreadImpl::getMassList()] failed to centroid scan

I opened each file in XCalibur and looked at the scan indicated in the error message (1972 for the example given above). The scan is for an MS2, and it appears to be completely empty of data (see attachment). I guess this is why it could not be centroided! Is there any way around this problem so I can import the files, as it seems to be just one bad scan?


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Skyline-daily importing (extracting product ion data) from only light peptide even though heavy & light are both in RAW file
(3 responses) mlane 2019-10-02
Hi, Upon recently updating Skyline Daily version to, targeted MS raw data files (PRM data generated from Orbitrap Lumos) is not fully imported into Skyline documents. We went back to official Skyline release (most current v 19.1) with the same file, created new document and the problem was resolved. Also, on multiple PCs, we created new Skyline Daily version document template, and the problem repeated. The raw files are confidential, but if you need help with source files to recreate the issue, let us know and we will try to come up with a example that is not confidential. At minimum, can you send link or instructions to install previous version of Skyline Daily (we did not have issue in previous release)?
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Two-step normalization
roman sakson 2019-10-02

Dear all,

I have a more general question concerning data analysis. Is there any possibility to have a two-step data normalization workflow implemented in Skyline? We often use spike-in SILAC experiments and normalization to heavy very nicely removes technical variance from our sample preparation workflows etc. However, it would be great if we could then use these L/H ratios and still normalize them to some housekeeping peptides to address biological variance (I realize that this can be done via global or surrogate standards but I would very much like to have it in addition to heavy normalization and not instead of it). Is something like this possible in Skyline or should I rather try working with MSstats for this purpose?

Thanks for this great piece of software!


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Group comparison
(5 responses) Enzo 2015-08-11
Dear Brendan:

First, thanks for developing such a nice software for targeted proteomics.
Second, got some questions about the group comparison:
1. We are focusing on the protein level instead of peptide level, so how did Skyline define the intensity for the protein? Does it simply add up all the intensity of peptides from same protein?
2. Take the first protein from the attachment for example, in the "fold change result" what does 38.66(99%,CL:24.83 to 60.21) mean? I assume 38.66 is the mean of the fold change from 3 repeats (I have done the 3 biological repeats) and 99% means 99% confident level.
3. Can I get the ratio for the 3 different repeats instead of median?

Thanks a lot for your help.
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diaPASEF data analysis
(1 response) thsofia17681 2019-10-01

Hi I would like to ask you if you have a basic tutorial on how to import diaPASEF data and how to see the MS2 raw spectra of the files.
Please note I am pretty rookie on Skyline. I have taken courses but never used it extensively.

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

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


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problems during install
(3 responses) zhengki 2019-09-29

Please help me with this problem in installing.

Thanks very much!!!

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remove peptides that are not present in all replicates
(2 responses) contet 2019-09-27
I am working with Skyline files from a DIA experiment in which samples were processed in 2 batches, which each contain replicates from all experimental conditions and have their own reference for normalization. I want to merge the Skyline files from the 2 batches to analyze the data in MSstats. What I have done so far is open the Skyline file of batch 1 then File>Import>Document to import the file of batch 2. I selected Add new replicates and Merge matching peptides, such that peptides that have been detected in both batches show up under the same protein ID. My problem is that some peptides were detected in only 1 of the 2 batches, which causes problems with MSstats (the error message says that "the input dataset has incomplete rows"). I only want to keep peptides that have been detected in both batches. How can I remove peptides that are not present in all replicates (e.g., in the attached screenshot, keep the aqua peptide but exclude the green one)?
Thanks for your help!
 Screenshot (40).png 
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Raw files moved
(1 response) ingo.wohlgemuth 2019-09-28


thanks for the great software.
I analyzed a larger DDA dataset with MS1filtering. Unfortunately, the external disk with the RAW files crashed. I still have the skyline file on my computer and a backup of the maxquant search plus raw files of the corresponding analysis. However, Skyline expects the original location of the raw files on the broken external memory.

  1. Is it possible to reimport the data in the skyline files from a new folder?
    or 2. is there another way to reconnect skyline analysis and raw data (plus maxquant search)?

Thanks in advance!


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IS it possible to do PRM data analysis using skyline?
(9 responses) ekramh1982 2019-09-26

The format (column headings) for the inclusion list of PRM in Q Exactive plus is different from the example provided in "" file. And creating a file similar to the values gets this problem: when I import the raw files it says "no usable data". How do I quantify the PRM files from Q Exactive Plus using skyline? The message is as follows:

At 11:52 PM:
Failed importing results file 'D:\Ekram\2019\Sept_20\Swab_Recovery_PRM\Pos_SwabRecovery_PRM\Pos_PRM_P40_Tang_100uM_stock_A10.raw'.
The sample Pos_PRM_P40_Tang_100uM_stock_A10 contains no usable data.

But I can open that file in quail browser:

How do I solve this? Thank you.

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how to upload chromatogram
(1 response) drrenugoel 2019-09-27

Kindly see the snapshot. I have built the library. Now I want to import my result chromatogram. However, it is showing library raw files. How to import result chromatogram. these are HR-MRM runs files.

 upload _chromatogram.jpg 
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Facing problem in instaling
(1 response) mchsahu 2019-09-27

Dear Sir/Madam,
I tried many times to install skyline daily but unable to do and getting the attached error. Will you kindly plz help me regarding this.

Thanks and regards

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PEAKS export for library building
(1 response) mauro galli 2019-09-26

I am a new user and I have a question about the Cut-off score setting in the library building for a DIA analysis.

I processed the data on PEAKS applying 1% FDR and I exported the file for Skyline. In the ion library building process in Skyline, I set the Cut-off score equal to 0, not to apply a double filter on my peptides (I don't want to cut 1% of the file where I already cut 1%). Do you think it could be a problem?

Thanks in advance!

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No transitions visible since latest Skyline daily update
(6 responses) lena reimann 2019-09-25

Dear Skyline team,

I updated our Skyline daily version to yesterday. Since this time point, we are no longer able to see any transitions in our AutoQC pipeline or following manual data import into Skyline. If I check for the transitions manually with Xcalibur or use older Skyline daily versions e.g. all the transitions are visible.
Is there anything I can do to recover the visibility/import of the transitions? At the moment they are also not imported into PanoramaWeb, so we are lacking some valuable information about our instument performance.

Thanks for your help and providing this great software,

 Skylinedaily_19-1-1-248.JPG  Skylinedaily_19-0-0-190.JPG 
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LOD determination
(1 response) djlj1 2019-09-25


I have been trying to determine LOD and LOQ for a series of calibration lines. If I use a bilinear model I can get both LOD and LOQ. I only get LOQ if I choose a linear model. Am I choosing a wrong setting please.

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Skyline won't import PD result files
(1 response) sa825 2019-09-24

Good afternoon,

I am trying to import some DDA files that have been processed in Proteome Discoverer (PD) using the Import --> Peptide Search format.
However, when I do this, I get the following error: "This file does not contain q-values. You can set a cut-off score of 0 in order to build a library from it, but this may cause your library to include a lot of false-positives."

If I reduce the cut off score to 0, it works but then as it says, such results are unreliable.

I have attached a screenshot of the error.



 Skyline 1.png 
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Easier visual for Heavy and Light in
(1 response) waltteri hosia 2019-09-24


Is there an easier way distinguish heavy and light versions of the same peptide in document grid? Looks like that total area is the only column that distinguishes them. At least with my settings, (double calibration curve, normalize to heavy), skyline doubles the rows with same calculated values for both, (which is slightly odd, heavy normalizing heavy should give 1, or?) In other words for those peptides that I have heavy peptide to use as ISTD I see in normalized grid 4 calibration sequences, from two sequences actually injected. But two of them have identical values, although total area reveals they are from the heavy ISTD. Modifying the "normalized" grid from "peptide modified sequence" does not quite work either. Choosing "show monoisotopic mass" shows the plain sequence for both light and heavy.

BR, Waltteri

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Heavy peptide as global or surrogate standard
(4 responses) waltteri hosia 2019-09-20

Hi, Skyline keeps coming better with every update, thank you for keep improving it.

Question. Is there a way to set the heavy peptide as a global or surrogate standard? I have an quant experiment with few proteins and I only have one heavy peptide per protein. I see though that sensitivity would be better with other peptide than the one that I have the heavy for. The heavy is in as one fixed concentration.

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AutoQC- No existing files found
(3 responses) davis 2019-09-20

Hi Skyline team!

I am a new user to AUTOQC. AutoQC is unable to import/find my existing files although the appropriate data folder is provided in the Folder to watch: field in the AutoQC Configuration settings. Please help me.

Current log message.

9/20/2019 8:25:27 AM] Getting the acquisition date on the newest file imported into the Skyline document.
[9/20/2019 8:25:27 AM] Running SkylineRunner.exe with args:
[9/20/2019 8:25:27 AM] --in="D:\Masslynx_projects\20180830_Metabolomics_Lipidomics.PRO\Skyline Results\AutoQC\" --report-conflict-resolution=overwrite --report-add="C:\Users\Administrator\AppData\Local\Apps\2.0\MY2EMOE9.LK6\QNADK77V.1XC\auto..tion_e4141a2a22107248_0001.0001_6f67fb96aa7015e7\FileAcquisitionTime.skyr" --report-name="AcquisitionTimes" --report-file="D:\Masslynx_projects\20180830_Metabolomics_Lipidomics.PRO\Skyline Results\AutoQC\AcquisitionTimes.csv"
[9/20/2019 8:25:30 AM] The name 'AcquisitionTimes' already exists.
[9/20/2019 8:25:30 AM] Resolving conflicts by overwriting.
[9/20/2019 8:25:30 AM] Success! Imported Reports from FileAcquisitionTime
[9/20/2019 8:25:30 AM] Opening file...
[9/20/2019 8:25:30 AM] File opened.
[9/20/2019 8:25:30 AM] Exporting report AcquisitionTimes...
[9/20/2019 8:25:30 AM] Report AcquisitionTimes exported successfully to D:\Masslynx_projects\20180830_Metabolomics_Lipidomics.PRO\Skyline Results\AutoQC\AcquisitionTimes.csv.
[9/20/2019 8:25:30 AM] SkylineRunner.exe exited successfully.
[9/20/2019 8:25:30 AM] The most recent acquisition date in the Skyline document is 9/10/2018 5:46:08 AM
[9/20/2019 8:25:30 AM] Initializing Panorama pinger...
[9/20/2019 8:25:30 AM] Starting configuration...
[9/20/2019 8:25:30 AM] Importing existing files...
[9/20/2019 8:25:31 AM] No existing files found.

Best! Sonnet

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DDA --> PRM not working
(9 responses) sa825 2019-09-19

Good afternoon,

I am trying to conduct PRM on a Thermo Q Exacitive for a list of proteins/peptides. They are heavy labelled so I am really interested in picking up the heavy peptides.

To do this, I first conducted DDA using the sample of interest on the Thermo and detected the many of the proteins/peptides of interest (Skyline shows that their precursors M, M+1 and M+2 are present)

However, when I run a Standard PRM (Exported a single method, standard isolation list) on the same instrument, using the same sample, only ~2 product ions are detected.

Can you please explain to me why this is the case? Maybe its a setting I have missed out?

I have attached a zip file containing the situation on the File sharing Page and titled it: "DDA -> PRM Not working"

Thanks and I look forward to your reply,


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

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

 gc-ms analysis 
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Spectral library problem from MaxQuant search (no isotopic labels)
(3 responses) Steve S 2019-09-13

Dear Skyline Support,

I created a spectral library in Skyline from a large MaxQuant search. There were no errors. I can find the expected peptide IDs in the library.
However, when I choose "View Library Match", the wrong spectrum is shown. The raw-file name and scan time are correct, however I don't know where the spectrum is from- it is another MS2 spectrum, but it certainly is not the correct spectrum.
Do you have any suggestions for what could be causing this?

I read a few earlier problems related to heavy-labelled samples, however in this case the MaxQuant search is from an unlabelled sample (DDA data).

Thanks for your help.

All the best,


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Install Version 19.1.
(2 responses) sandra maass 2019-09-18

Dear All,

I'm trying to install the newest Version in 64-bit. Unfortunatley I'll get an installation error every time I try. Please find the install log attached.

Do you have any idea what I do wrong?

Thank you in advance

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Peak picking within a specified retention time range.
(10 responses) wangqingok 2019-09-11

I have manually selected peaks corresponding to my target peptides in several reference samples and I have figured out my peptides' relatively stable and reproducible retention times. And I have 200 more samples to analyze. I know I could export and import peak boundaries to my future analysis when I want to quantify these peptides, but I found that for some peptides where a significant amount of noise signals are very close-by the target peaks, the quantification results are far off and the rentention times are not that reproducibly where to specify a fixed window for peak boundaries will have a lot of false positive signals being integrated and counted into the abundance. Therefore, I do want to also have skyline pick the peaks, but only within those predefined retention time windows (~ 2 minute windows) that I could manually get. Is there a way of doing this? Thank you so much for your help!

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Export structural modification list
(1 response) io 2019-09-17

Dear all,
I would like to export the structural modification list of Skyline to send it to a colleague
He wants to study O-glycosylations but I am not sure of the modification we have to add.
Thanks a lot

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Sciex analyst .wiff files fails to import showing: Object reference not set to an instance of an object.
(2 responses) kamal 2019-09-02

Dear Skyline Team,

In my experiment, each replication’s retention time is 6 hour. However the *.wiff and *.wiff.scan files comes in 3 part for each run. For example, P100_Cold_30min (Cold stress for 30 min) comes in P100_Cold_30min-1_1, P100_Cold_30min-1_2, P100_Cold_30min-1_3

On the other hand, the *.group and *.group.xmlcomes in single file P100_Cold_30min-1 as the following screenshot.
The MGF files also come in a single part which I used to generate mascot DAT file as an alternative to the group.xml files for creating the spectral library.

I am using these *.group and *.group.xml or just mascot DAT files to create the spectral library for MS1 based DDA analysis. However, it seems like that when importing the final results the following problem is occurring-

At 5:42 PM:
Failed importing results file 'C:\Kamal RIKEN DATA\190614\P100_Cold_30min-1_3.wiff'.
Object reference not set to an instance of an object.

Inner exceptions:
Exception type: System.NullReferenceException
Error message: Object reference not set to an instance of an object.
Object reference not set to an instance of an object.
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.get_PercentComplete() in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 805
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.UpdatePercentComplete() in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 598
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.GetChromatogram(Int32 id, Target modifiedSequence, Color peptideColor, ChromExtra& extra, TimeIntensities& timeIntensities) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 586
at pwiz.Skyline.Model.Results.ChromData.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\ChromData.cs:line 77
at pwiz.Skyline.Model.Results.ChromDataSet.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 236
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Load(ChromDataProvider provider) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 144
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 460
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 263

I am in desperate need of help because of my graduation deadline is approaching soon.

Best regards,

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using external calibration curve?
(1 response) Cedric 2019-09-09


is it possible with Skyline to use the calibration curve acquired for a molecule X and apply it to another molecule Y? This will allow to do semi-quantitative analysis of molecules which do not have a specific calibration curve.

If it is not possible, do you have a way to export the calibration curve equation determined in Skyline? Like this, we could use it in excel to perform concentration calculation.

thanks for your support and your great software

Kind regards,


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(8 responses) Tobi 2019-08-06

Dear Skyline Team,

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

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

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

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

Best regards,
tobi  AvantGardeDIA.rar  Error MSG.txt 
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