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.

If your question is about an External Tool, please contact that tool's developers directly. Contact information can usually be found on Skyline's Tools | Tool Store... menu.  

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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.

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Showing: limited to 100 requests
TMT neutral losses
(4 responses) aj10 2024-04-11 14:47


I am trying to quantify neutral losses from TMT-tagged peptides (the reporter tag + CO). I'd like to include the neutral losses regardless of their presence in the spectra used to generate the spectral library. When adding each tag as a neutral loss to the N-terminus modification, I set the losses to be included by default. However, these transitions are not added by default to my spectral library. I can add them manually using the Pick Children option, but I've got more than 10k peptides.

Second question. If we can resolve that, I only want the neutral losses from the precursor (i.e. not from every possible b ion). Is there a way to specify that and apply it to all peptides?

I'm running the Skyline daily build downloaded last Friday.

Thank you for your help!

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Ratio to surrogate ...
(3 responses) a rocher 2023-09-06 02:25


I quantify small molecules on Skyline. I wanted to use metabolite B as an internal standard for metabolite A, so I defined marked metabolite B as a standard surrogate in the tree structure. Unfortunately, in the Normalization Method column of the Document Grid, the name of the metabolite is replaced by its InChIKey, which makes it very difficult to read (see attached file).

How can we make the "Ratio to surrogate..." option show the metabolite name (as defined in the tree structure) and not an identifier?


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.wiff file chromatography import issue
(1 response) Jiny 2024-04-11 15:01

When I import my results for one file it shows the chromatography import all grey then I have no data, just blank screens with chromatogram unavailable. This is happening on only one set of data, I have sample sets that ran directly before and after the file that are importing just fine. I've been combing through the help tickets with no luck so far. Any help would be appreciated.

 20240411 grey chromatograms for skyline support.png 
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Unable to execute the setup file to install Skyline 23.1
(2 responses) khoil 2024-04-11 11:06

Hello Skyline team,

I am trying to update my skyline from version 22.2.0255 to 23.1. Unfortunately, whenever I try to open the setup.exe file I would consistently get this error message: "Application cannot be started. Contact the application vendor." I have tried uninstalling the previous version and also tried running it as an Administrator, but I am still getting this same error. Do you have a solution to this issue?

Thank you

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Avant Garde R Package Installation Failure
(3 responses) sgoulding 2023-10-05 14:05

Hi, Skyline Team.

I'm running into issues installing the Avant Garde tool in Skyline. I've tried to install it through both the Tool Store and as an External Tool, but I keep running into issues with specific R packages not installing. The two packages that won't install are stringr and ggplot2. This is happening on several different computers running the most current version of Skyline Daily.

Do you have any suggestions about how to remedy this? I tried installing the specified versions of ggplot2 (3.2.1) and stringr (1.3.0) outside of Skyline through R (v 4.0.3) but there were issues with the tool when I did that. I attached the error message I got related to the tool stoor failed installation of ggplot2 and stringr.


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AvantGarde tool install
(1 response) dkueltz 2024-04-11 08:21

Hi Skyline team,
I have been trying to install the AvantGarde tool but it always fails apparently because JAVA cannot be installed. I am not sure why this is but suspect that an older version of JAVA cannot be installed in addition to a newer version (which I use for the MSstats tool). Did you come across this before? I realize that this issue goes beyond Skyline but wanted to ask it anyway in case you have come across this before and have a solution.

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Display of transition chromatograms
dkueltz 2024-04-11 08:18

Hi Skyline team,
Is there a way to suppress the display of the axis labels for the transition EICs of each sample to enlarge the graph that is displayed? This would help a lot when having to display all samples simultaneously (tile view) for a large number of samples. Even with fontsize x-small the axis labels take up most of the space when the individual windows are small. It would be very nice to have a feature that allows suppression of the axis labels (i.e. in the properties window for EICs).

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(3 responses) naimamuntu 2024-04-10 23:44


I am looking at the quantification of the two peptides R.IEDEQALGSQLQK.K [1093, 1105] and R.ILNPVAIPEGQFIDSR.K [725, 740] in disease (GoH) and healthy hearts (NF). I have n=3 for GoH disease (2LV, GoH 5 LV, GoH 9 LV ), and I ran each sample in triplicate. The healthy group, n=5, NF_4LV, NF_13LV, NF_14LV, NF_18LV, NF_20LV and ran in triplicate.
I ran a multi-point calibration point for IEDEQALGSQLQK labeled 1 through 8 and ILNPVAIPEGQFIDSR labeled M6 through 8
Which transition to choose? How do we proceed to quantify these two peptides across disease and health?
I've attached my document as Skyline_Nitha_41024_

Thank you so much.

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Prosit server unavailable
michal zawadzki33223 2024-04-11 04:22


I am having an issue with connecting to Prosit server through Skyline (version - see the attached snip for details. I have used the test-netconnection command in Windows PowerShell as recommended by Brendan in one of the previous posts and it looks like my computer is able to connect to the server using port 8500. Any suggestions on how this could be fixed are appreciated.

Kind regards,


 Prosit server unavailable.PNG 
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Missing DIA acquisition
(1 response) guilherme pauperiolanfredi 2024-04-11 00:08

I’m having issues regarding the DIA acquisition on TimsTOF Pro, for some of my runs.

The chromatography of all runs are comparable and I can see it during the run, but when opening it on Skyline the acquisition is not there, not even noise.

In this screenshot, two runs are shown where I have complete signal acquisition and two others that only have the first few minutes and then there is no signal, besides having complete chromatography on the vendor software.

Any idea?

Thanks in advance.

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Ratio to surrogate standard using peak height?
(2 responses) per larsson 2024-04-09 02:12

Dear Skyline users,
I have a lipidomic MRM method where I use surrogate standards (one per lipid class). For one analyte I wish to use the ratio of the peak height instead of peak area, I cant find a way to set the peak height instead of area for the ratio calculation I would appreciate advice how to do this?

ratio=molecule_peak_height / surrogate_standard_peak_height

The reason i need to use peak height is that I have polarity switch in the scheduled MRM so I see peak apex but not the entire peak width, I preferer to use peak height to peak area for this particular lipid. I have searched the tutorials but not found how to do this.


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TMT quantification tutorials
(1 response) TK_1234 2024-04-09 07:42

Hi Skyline users, a new user here.

I was able to follow the tutorial on "Absolute Quantification.' Now, I'm interested in learning about TMT-based quantitative proteomics. My questions are the following: 1) Is there a skyline tutorial on TMT quantification? 2) If the TMT-labeling is applied only to the samples but not the heavy standard target peptide (used to generate calibration curve), what is the correct way to quantify the labeled samples?

Thank you so much!

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PRM run showing errors
(28 responses) cvadival 2024-04-03 12:32

In order to handle Skyline, I would truly need your assistance. Guide the way to the skyline run, please. While I run PRM, I get an error that says there is no chromatogram information available. My DDA run, however, is good and displays a nice chromatogram.

Comments from results "No precursor ion chromatograms found"


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Unassigned spectra
(3 responses) akhilabrai 2024-04-05 22:23

Hi team,

Can we export the unassigned spectra from our DIA data in skyline? Is there any option available?

Thank you.

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Missing transition
(4 responses) Joerg 2024-03-28 04:11

I have a problem with data extraction of our amino acid analysis done by FullMS and AIF on a QExactive. While most of the extracted transitions look fine, one transition is missing for isoleucine both in the internal standard and the analyte (the one that distinguishes isoleucine from leucine). I have no idea why the signal is missing (not even baseline), the signal seems to be present in the spectra, though. A file with a single run is attached. Any help is greatly appreciated!
Best Regards,
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The tutorials in the official website and the tutorials in the software should be consistent, it's too messy now!
839868794 2024-04-06 02:44

I am just learning to use it and when I wanted to learn about MS1 filtering, I found 2 ways to get the tutorial.

  1. I went to, the download link opened a new tab called MS1Filtering-20_1_zh-CHS, the download file was actually MS1Filtering-22_2_zh-CHS.pdf.
  2. In the Skyline, I went to the homepage > tutorials, after clicking on MS1 filtering, I noticed that the automatically created folder is MS1Filtering-22_2, but the actual downloaded is MS1Filtering-21_1_zh-CHS.pdf.

I'm not sure if this is isolated or if you guys forgot to update something after going through the upgrade.

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TQ-XS Data not loadable in Skyline-daily (64-bit) (b5b0d400b)
(3 responses) Marvin 2024-04-04 00:14

Dear all,
As I tried to load my measured data into skyline, the error:

  • are just spaces for intern names without special characters.

At 09:08:
Failed importing results file 'C:\Users***\MS24-0188_AltDB_074.raw'.
[pwiz::CLI::msdata::ReaderList::read] Unhandled exception: Generic Error
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\mbr\Desktop\Neuer Ordner\MS24-0188_AltDB_074.raw'.
[pwiz::CLI::msdata::ReaderList::read] Unhandled exception: Generic Error ---> System.Exception: [pwiz::CLI::msdata::ReaderList::read] Unhandled exception: Generic Error
bei filename, MSData result, Int32 runIndex, ReaderConfig config)
bei pwiz.ProteowizardWrapper.MsDataFileImpl..ctor(String path, Int32 sampleIndex, LockMassParameters lockmassParameters, Boolean simAsSpectra, Boolean srmAsSpectra, Boolean acceptZeroLengthSpectra, Boolean requireVendorCentroidedMS1, Boolean requireVendorCentroidedMS2, Boolean ignoreZeroIntensityPoints, Int32 preferOnlyMsLevel, Boolean combineIonMobilitySpectra, Boolean trimNativeId) in C:\proj\pwiz\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:Zeile 200.
bei pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:Zeile 295.
bei pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:Zeile 191.
--- Ende der internen Ausnahmestapelüberwachung ---


Do you have any suggestions how to tackle this problem?

We tried an older version of skyline, but still couldn´t open some of the files...

Best wishes,
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(16 responses) ekilic 2024-02-29 13:50

Dear Skyline Support Team,

I wanted to bring to your attention an issue we encountered while working with the BoxCar method for metabolomics on IQ-X system using the small molecule application mode. For your convenience, I've attached a PowerPoint file to this email detailing how we configured the method along with the raw file. In summary, the method consists of 2 spectra and 5 boxes per spectra with polarity switching.

Upon attempting to import the raw file to Skyline, we observed that the data in the second spectrum was missing. To investigate, we checked the Extracted Ion Chromatogram (EIC) of the absent compounds in the raw file using the vendor software (Quan Browser, Thermo). It came to our notice that the data existed there but wasn't being extracted into Skyline.

In our effort to resolve this, we attempted converting the data format to mzML as well as mzXmL, following a suggestion found in one of the threads on the website. We also explored various parameters in the Transition Settings, but unfortunately, we were unable to extract the data from the second spectrum.

Any insights or assistance you can provide on this matter would be greatly appreciated.

Thank you,

Ece and Soren

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Issue Report: AutoQC Execution Failure
(2 responses) lhy3221 2024-04-02 21:24


I am writing to inquire about an issue encountered during the execution of AutoQC same as

I received a message stating that
"The autoqc loader encountered an unexpected error."

Despite attempting to uninstall and reinstall all programs (Skyline, AutoQC), or install on another PC, the problem persisted.
Additionally, even when using the unplugged version of AutoQC, the same error persists.

Could you please provide guidance or advice on how to resolve this issue? Alternatively, if you require any further information, I am more than willing to provide it.

Thank you for your assistance in resolving this issue.

Best regards,

 AutoQCProgram.log  error.jpg  user.config 
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Feature request: allow controlling display m/z range for library match
(2 responses) Juan C. Rojas E. 2024-03-29 06:04

Hi again,

I often use the library match spectrum from Skyline to generate peptide spectra matches for publication. However, I struggle to normalize the m/z visualization range from the multiple spectra as this is (I suspect) automatically controlled by the range of signals stored in the fragment ion spectrum. Although I can play around with scroll button of the mouse to get it to show the same range it is usually a lot of hassle.

So, I was wondering it if would be possible to add a display range for the x- and y-axis in the "Graph properties" or wherever you see it fit?

As always, thank you for the awesome work on this software.
Juan C.

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Small molecules - Individual regression weighting for each analytes?
(1 response) johannes kutzler 2023-10-14 10:54

Hi everyone,
I have just started using Skyline after finishing the fantastic online introductory and small molecules course (2 days each).
Working in the forensic toxicology area dealing with small molecule quantification, I gained experience with SCIEX Analyst and Agilent Masshunter.

So here is my question: Is it possible to assign the regression weighting for the calibration curve for each analyte individually (e.g. amphetamine 1/x, methamphetamine 1/(x*x), ketamine none)? This is a common thing in forensic toxicology and would be crucial for a switch e.g. from SCIEX Analyst to your amazing software.

Thank you for your help in advance

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Audit log
(3 responses) Zac 2024-04-01 10:34

Is the audit log only available for Skyline Daily, or is there a way to enable it in Skyline

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DDA data missing retention time range of specific data file
(2 responses) wangyan2 2024-04-01 12:57

When trying to quantify a set of data (6 total) after DDA using precursor. Skyline miss integrated certain files, which is easy to fix manually, but for one file Skyline won't give me the time window where the peptide eluted. See file attached. While the peptide is detected in all other files at 83 min, for one file the displayed RT window starts at 88 min. How can I fix this? 
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not possible to open old skyline files
(1 response) io 2024-04-02 03:51

Hi all,
I have had to update the Skyline software but now I cannot open old files.
This is the error
Thank you

 Skyline error.JPG 
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Inquiry Regarding Potential Software Limitations or Known Issues
(5 responses) Jordan Martinez 2024-04-01 08:21

Dear Skyline Support Team,

I am writing to inquire about the analytical software "Skyline," specifically in the context of its use in forensic mass spectrometry data analysis. As part of the defense team in a legal case where Skyline-generated data is being utilized as significant evidence, it is imperative that we fully understand the capabilities and potential limitations of your software.

Could you please provide detailed information on the following aspects of Skyline?

  1. Known Bugs or Issues: Are there any known bugs or issues within the current version of Skyline that could potentially affect the accuracy or reliability of mass spectrometry data analysis? If so, could you please provide specific details on these issues and under what circumstances they might influence the results?

  2. Data Interpretation: Are there specific scenarios or conditions under which Skyline might produce ambiguous or less reliable results? For instance, are there any known limitations in the algorithms or methodologies that could lead to questionable data interpretations?

  3. Historical Performance: Have there been any reported instances where Skyline's analysis was later found to be inaccurate or called into question? If yes, could you elaborate on those instances and the outcomes?

  4. Validation and Certification: Could you provide information on the validation processes that Skyline has undergone, especially concerning its application in forensic analysis? Are there any certifications or endorsements from recognized bodies that attest to its reliability and accuracy in such contexts?

  5. User-Related Errors: Are there common user errors or misinterpretations when using Skyline that could potentially lead to inaccurate results? If so, what measures or guidelines do you recommend to mitigate such risks?

Our objective is to ensure that the evidence being presented is scrutinized with the utmost diligence, ensuring a fair and just evaluation in the context of the legal proceedings. Understanding any potential for error or misinterpretation within the software is crucial in this endeavor.

We appreciate your assistance and look forward to your detailed response.

[Generated by ChatGPT]

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Cannot export thermo exploris method
(2 responses) lkittinun1 2024-03-30 03:37

I tried to export skyline DIA method to the instrument. However, it failed to find the following file as the error message:

An error occurred attempting to export.
Thermo instrument software may not be installed correctly. The library System\Programs\dependencies\tng\Thermo.TNG.MethodXMLFactory.dll could not be found.

System.IO.IOException: Thermo instrument software may not be installed correctly. The library System\Programs\dependencies\tng\Thermo.TNG.MethodXMLFactory.dll could not be found. ---> System.IO.IOException: Thermo instrument software may not be installed correctly. The library System\Programs\dependencies\tng\Thermo.TNG.MethodXMLFactory.dll could not be found.
   at pwiz.Skyline.Model.ThermoMassListExporter.EnsureLibraries() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Export.cs:line 1164
   at pwiz.Skyline.Model.ThermoSureQuantMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Export.cs:line 2088
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\pwiz\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\pwiz\pwiz_tools\Skyline\Util\Util.cs:line 1922
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\pwiz\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 202
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\pwiz\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\pwiz\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2352

I also tried to perform a workaround as per, but when I checked the registry, it was already a full path (as provided in the attachment). I also checked the destination folder, and the "Thermo.TNG.MethodXMLFactory.dll " file was placed correctly.


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Simultaneously advance tabs in multiple windows
(4 responses) philippmaternus 2023-04-27 11:47

Dear Skyline team,

Suppose I have 120 replicates, I can use View/Arrange graphs/Groups... to split them across e.g. 6 windows with each 20 tabs. Now I can browse through the replicates by clicking ctrl+up or ctrl+down, but I still need 120 clicks to browse through all replicates. Is there a way to simultaneosly switch to the next tab in all 6 windows? This would allow me to browse through all 120 replicates in just 20 clicks.


P.S. I am using Skyline v21 on Windows 10

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Calibration Curve
(3 responses) naimamuntu 2024-03-20 21:28


I am currently in the process of developing an MRM assay for 4 peptides, each with four pairs of light/ heavy. I am still learning, and I went through the tutorial. I have used a multiple-point calibration approach. I utilize light peptides as internal standard and heavy peptides that are isotopically labeled. I am wondering whether the concentration of the heavy peptide spike-in within samples must match the concentration of the constant heavy peptide used for the calibration curve. Also, what are the factors influencing the choice of concentration for the heavy peptides?

Thank you so much.

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MRM QQQ transition peaks not detected in Skyline
(2 responses) ylee 2024-03-26 14:34


I ran three different m/z from a compound as shown in the transition list attached on Agilent QQQ. I can see the peaks fine at retention time around 4min for two transitions and less than 1min for one transition from Agilent Quantitative Analysis software. However, when I load the same raw files onto Skyline, I do not see any peaks and the retention time window is shown only up to 1.2min for the chromatograms which does not cover where the peaks are supposed to be in. Even after changing the explicit retention time and window, the chromatograms stay the same and I do not see anything.
I have attached the transition list, raw files, and Skyline template.

Please let me know how I can go about resolving this problem.

Thank you.  Skyline_transitionlist.xlsx 
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Total ratio
(1 response) vmohanty 2024-03-27 12:06

Hi All,

I get a dotp and total ratio next to my precursor for the peptide of interest. I am aware that closer the value of dotp to 1, good is the matching.

However, I want to know how much shall be the TOTAL Ratio mentioned after that. Is there any particular value, i have to look for?

How much should be the spike-in heavy standards be injected as compared to endogeneous peptides?
For example, for my endogenous peptide transition, the intensity is 6e3 and for the corresponding heavy transition is 2e6 (screenshot attached).

Thank you.

 Capture.JPG  Heavy_pep.JPG  Endogeneous.JPG 
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Doubts about transition list
(3 responses) gabidap 2024-03-27 06:46

Hello, I trying insert a transition list with M+H-H2O as adduct, but i receive the error message " calls for removing more O atoms than are found in the molecule" . What can be wrong in my list?
Many thanks

 Captura de tela 2024-03-27 103817.png 
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Chromatogram information unavailable
(3 responses) limsjy 2024-03-24 22:30

We build spectra libraries on skyline. For this current project, we combined 2 of the spectra libraries into 1 big library on skyline. After which we tried to import our 800 samples into skyline. I managed to import 600~ samples into skyline without any issues. However, the next day when i tried to open the skyline file, all of the chromatogram information was unavailable. I have attached the error message in this support ticket.

Please advise on how to solve this issue, thank you so much.

 Error message from skyline.PNG 
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Manual tuning of the spectral library
(1 response) hassan hijazi 2024-03-26 07:23

Dear Skyline team,

I want to have a curated spectral library from 3 replicates of DDA on samples spiked with iRTs.

After building the library, I found the following problems based on manual inspection:

1- some wrong picked MS1 peaks
2- wrong ID (2min far from the peak while having ID inside the elution width of the correct MS1 peak) assigned to correct MS1 peak.

With the aim to use them for the PRM assay, neither the iRT calculator nor the spectral library could be of further use if they contain incorrect information.

Those problems are expected as I am working on highly modified histone peptides where isobaric peptides co- or nearly elute with most of the product ions are shared.

Is there a way to modify the spectral library after it is built? For instance, replace a MSMS ID chosen by BiblioSpec with one from the redundant library or adjust the RT after manually adjusting the integration boundaries to the correct MS1 peak?

The only way I see for now to fix those problems is to build an assay library and then import it to Skyline.

Thank you for the help.

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Can not see Light/Endogeneous peptides
(10 responses) VM26 2024-03-20 18:28

Hi All,

I have been trying to develop a PRM assay for 1 protein for which I have 3 Heavy peptides. I have been able to develop a linear curve for LOD and LOQ using CSF (fixed amount) as a matrix with varying conc of Heavy peptides.
For Ratio, I used light as the internal that correct?

Using Light as the internal standard and heavy as Isotopically labeled, I could plot a linear graph for 1 peptide. However, I was unable to get a linear curve for 2 peptides of the same protein.
I can see the reason why as the endogenous peptides (Light peptides) might be way too low as compared to the Heavy peptide amount spiked that might be way too high for which the Ratio H/L becomes infinity.
To overcome this, I used a ratio CSF: Heavy peptide (in fmoles/ul) as 1:1.

However, I am unsure how to proceed with 1:1.
My concern is with body fluid in my case its CSF. I tried multiple way to do 1:1 ratio

First, i prepared different dilution of Heavy peptide standards from stock of 1mM such as 2500, 1000, 500,........0.25 fmol/ul.
I then took 5 ul from each standard and added to 5 ul of CSF and injected 1ul to column. My worry is the volume ratio 1:1 is right or not.

Secondly, I tried to prepare a 2500 fmol/ul from the heavy peptide stock. Took 5ul from 2500fmol/ul and added to 5 ul CSF. I prepared the dilution using this CSF+2500 Dilution as stock and diluted it down to 1000.......0.25fmol/ul. Here for every dilution, both CSF and Standard are equally getting diluted.

In Case 1; my every dilution vial has 5ul CSF (fixed volume so fixed amount of CSF) and 5ul from respective dilution tube (1000.....0.25fmol/ul)

In Case 2; my main mixed stock of 5ul CSF+ 5ul 2500fmol/ul is serving as highest stock and I am diluting using this as the main vial and 0.1% Formic acid as diluent.

Now, I am unsure which will work best as my endogenous peptides are way low but get overexpressed in the disease model of my interest.

CSF used here is the pool of 20 patients to avoid heterogeneity.

Can you suggest some solution.

Thank you.

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FAIMS CV optimization - Please Report "NA" in the Ion Mobility filtering settings for peptided with removed peak
(2 responses) Tobi 2024-03-20 03:52

Hi Skyline Team,

when using skyline to optimize FAIMS CV, that works in principle when having having data with multiple CVs and going via
Settings/Transition Settings/Ion Mobility/Ion Mobility Library/Add/Use Results

From there the optimized CV values can be copy&pasted and used elsewhere, I have not managed any other way to export the optimized CV values.

However, when some precursors or peptides had their peaks removed (chromatogram, righclick, remove peak), I would expect those to be listed with "NA" as optimized CV value, but they are simply missing from the ion mobility library window. This creates mismatches with the table of optimized CVs I would like to make.

The copy&paste is fine, but please keep the numbers of peptides and precursors consistent with the target list, that would be a great improvement.

Best, tobi

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2+ charge state not available for selection
(2 responses) sstoychev23513 2024-03-21 11:11

Hello Nick and Brendan,

Same question but on this forum.

I am building autoQC Skyline file and would like to select a 2+ precursor for one of the QC peptides but this is not available in the SKyline precursor selection three (see attached). Other peptides in this list have the 2+ option available as 2+ is also listed in the transitions settings filter tab.

Relevant files attached.


 20240320_Phosho_QC TIMS 
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here is the error code
(6 responses) jason.murphy 2024-03-20 11:45

I have both an Orbitrap eclipse and Ascend in my lab. I have acquired DIA data on both instruments, and I have successfully processed the Orbitrap Eclipse DIA data in Skyline. However when I try to process the Orbitrap Ascend DIA files Skyline throws an error and aborts the process. The software seems to have a problem recognizing the isolation windows in the .raw file for the Ascend. Even when I manually enter the isolation windows the processing still fails.
thanks for your help

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Calibration Curve
(1 response) idoia beloqui-ezquer 2024-03-21 02:53

Dear Skyline support,

I'm using the Skyline interface for small molecules, and I came across one question about the calibration curves.

I do not have individual standards, instead, I have standards mixtures.
More than one of the mixtures contains the same molecule, and therefore I can build the calibration curve for the same molecule using several of the standard mixtures.

I would like to compare the calibration curves built using different standard mixtures for the same molecule.
Is it possible to build more than one calibration curve for one molecule?

Currently, I'm using the "Exclude From Calibration" in the "Document Gird" to select which standards I would like to use for the calibration of each molecule. However, I do not know how can I build several calibration curves for one molecule.


view request
Importing MyriMatch search results into Skyline after parsing them through PeptideProphet
(9 responses) staab-weijnitz 2024-03-16 09:33

Dear Skyline support,

I have managed to do this before, but now I am again running into issues when importing MyriMatch search results into Skyline. I am trying to import these peptide search results to build a library in the "peptide settings -> library -> build" menu. However, Skyline does not recognize the files (MyriMatch results parsed through Peptide Prophet - this has, however, worked before). It would be great if you could please have a look at the attached example file here and let me know what is wrong with it or why Skyline can't load it.

In general, we have previously managed to use .mzXML and files like the attached one to build libraries with the DDA with MS1 filtering workflow.
It is my understanding that I load the raw files (.mzXML) using "Import Peptide Search, build spectral library, start from DDA raw (search and build library)" and the peptide search results (pep.XML) under "peptide settings -> library -> build"? Is this correct or am I completely off?

Skyline recognizes both the .mzXML files as well as thermo .raw files when I use the "Import Peptide Search, build spectral library, start from DDA raw (search and build library)" but after the percolator it always says "Search failed". Do you know why that is? I attach the corrsponding mzXML file, too.

I would really appreciate your help on this!

Many thanks,

view request
Setting up skyline for a single label
(1 response) rose gathungu 2024-03-19 23:04

I have a peptide with 2 Rs but i only have one Rs that is heavily labelled. When i set-up on skyline, the software is calculating heavy peptide masses with both Rs labelled. How do i set-up the software for a single heavy labelled R instead of both Rs?

view request
Standard curve based on heavy peptide
(1 response) negronil 2024-03-19 03:03

Dear Skyline support,
Is it possible to use the heavy peptide for both (i) the normalization by spiking-in, as usual and (ii) the quantification of the natural peptide based on the standard curve of heavy peptide ? The idea under the question is to buy only heavy and not light peptide.

view request
Intensity Threshold on MS2 fragments??
(5 responses) zrhopkins 2024-03-14 04:37

I was wondering if there is some in the background built-in intensity threshold on whether or not Skyline will show chromatograms for MS2 data?

We are using Skyline to quantify data collected in an Thermo Exploris 120. We have found that sometimes Skyline will not show MS2 chromatogram data for a fragment of a compound. However, when we review this data in Thermo software, there is clearly MS2 data generated for the fragment of interest.

I have attached an example. Showing 1) how in Skyline we don't see any fragment data for the 98 and 79 m/z and 2) If that same data is viewed in Thermo software I see the fragmentation event and a low intensity peak for the 98 and 79 m/z.

Maybe there is something that I'm missing, but any insight would be helpful!

 Skyline_Chromatogram.png  Thermo_MS2_Result.PNG 
view request
(2 responses) vmohanty 2024-03-14 09:53

Currently, I am plotting a reverse calibration curve using Skyline. I have used varying concentration of heavy peptide standards with fixed amount of my sample matrix.
My quantification parameters include following in PEPTIDE SETTINGS
Linear AND Linear through Zero (Ideally I should go for Linear?)
Ratio to Light
MS level: All
Max LOQ and LOD bias: 20%
Calculate LOD by
Blank + 2*SD

Could you let me know which settings are best to estimate the LOD and LOQ!
I went through few articles, some have used NONE and some 1/ x and 1/x*x. Pretty confused being a first timer. I am able to have a linear range for 7 out of 11 standards. Is that acceptable?
I am attaching the PowerPoint slides for referral.

view request
What are *.sky.view files and *.skyd and *.skyl. How can I open these files?
(1 response) Jenny Albanese 2024-03-12 19:05

What are *.sky.view files and *.skyd and *.skyl. How can I open these files? Thank you for you help .I received these files and do not know how to open them. Thank you, Jenny

view request
Trouble identifying glycan modified peptides and quantifying ions in DIA data
(3 responses) nkegley 2024-03-08 11:00

I am attempting to use Skyline to quantify ions from an existing DIA dataset I've generated using our labs orbitrap instrument. When using skyline to build a library using the related DDA dataset and extracting chromatograms using the DIA run, I get an error window about not being able to identify modifications (mostly glycans on Ser and Thr residues). Rebuilding glycan modifications in the "modifications" window of skyline doesn't seem to remedy this, and when I get the library built by skyline, all I generally see are peptides with fixed carbamidomethylation, no glycans.

I've used Byonic software to produce an mzident file for use in skyline, and there I can detect PSMs that include the glycan modifications (which makes sense considering I've already built an inclusion list for our instrument for those exact masses). Because of that, I assume I'm just going wrong somewhere in the skyline settings that results in ions from the glycan modified peptides being excluded from the library/chromatogram extraction.

Any help or advice would be appreciated!

view request
SRM/ MRM Method Development and Data Analysis with Skyline
(2 responses) naimamuntu 2024-03-11 11:17

Dear Skyline Support,
My name is Nitha, and I attended one of your sessions during Session 3 in October 2023. Firstly, I wanted to express my gratitude for the insightful session and the valuable information shared. For my project, I've been utilizing the SMR method development and analysis with Skyline.
I'm reaching out to inquire about any webinars or PowerPoint slides on SRM/ MRM Method Development and Data Analysis with Skyline.

Best Regards,

view request
"No Base Peak Chromatogram Found" when trying to integrate DDA data
(2 responses) jarrod a roach 2024-03-11 08:45

I'm trying to integrate small molecule DDA data from a Q-exactive plus in negative ionization mode. I made my transition list using MS1 and MS2 values that I know exist in my results, but when I import my results it does not recognize a base peak chromatogram. Does anyone have an idea as to why this might be happening? I've attached my skyline file. Thanks! 
view request
Skyline not compatible with Shimadzu LCMS-8060
(16 responses) William 2021-04-19 21:10

I am porting the method to our new purchased Shimadzu LCMS-8060. I exported transition list from Skyline (choose Shimadzu QQQ) to txt file. However, when importing compound list to Shimadzu, it is completely chaos. I then check the title of each rows and find they don't match. Here I attach the compound export file from Shimadzu QQQ. Pls update Skyline so that exported file can be recognised by Shimadzu.

view request
removing peaks' name
(1 response) mdorrani 2024-03-07 14:22

Hi, When you click on the "molecule" on top of the target (molecule) list, one can see all the peaks together which is pretty nice. However, I'd like to see a chromatogram without peak annotation. Is there any way I can remove the peaks' name from the chromatogram? (numbers in the screenshot). Thank you!

 Screenshot 2024-03-07 171717.jpg 
view request
PRM Target list creation from DDA data
(7 responses) Qssf 2024-03-05 18:41

Dear support team,
I use skyline as the method development tool for DDA and PRM quantitation of PTMs in protein conjugates. Byonic was used to generate mzid and mgf files from 3 DDA runs and I was able to create spectral library with no issue. I will need to create the targets from the library based on the customized PTMs etc. defined under peptide setting by loading the 3 DDA raw data files. On the Add Modifications page, it wouldn't let me add the new mod defined under peptide settings but I thought I could change that later so I left it as blank. I uploaded the fasta file for a bispecific conjugate with 2HC and 2LC but it finally gave me an error message as shown in the screenshot attached. I have never seen this type of error before (An error occurred during protein association). When I checked the library, it looked like unmodified and modified peptides were all present. I honestly don't remember I did anything differently this time and it was working well just a couple of months ago. Do you think this is related to the fasta file?
Thank you.

 error message.pptx 
view request
Chromatogram Information Unavailable Message
(2 responses) csb548 2024-03-05 08:51

Dear Support Team,

I am new to using Skyline and I am trying to create a workflow to enable rapid peptide identifications in my samples (the samples I am performing the test on have already been analyzed through Mascot, and the peptides I am targeting have been identified in all of the samples). My data is DDA-timsTOF HT data. When I try to import my data, I keep being met with a "chromatographic information unavailable" message. I'm not sure what it is I am doing wrong so any help would be appreciated!

I should also note, these samples have not undergone any digestion.

I have attached some Skyline files and some written instructions I was writing as I was making the document in Skyline to keep track of everything.


 Skyline human amel  Skyline human amel test.skyd  Skyline Test.docx 
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Predicted RT worsens after ?calibrating ("Use Results")
(6 responses) wphipps5 2024-02-29 15:54

I am trying to process DIA results (in for samples spiked with the Pierce iRT mix. After importing sample data files into a document containing only the PRTC mix peptides (14 peptides), I tried to recalibrate the retention time prediction (using the "Use Results" button under Peptide Settings in the prediction tab), but the predicted retention times all worsened...

For example, the Pierce SSA peptide elutes on our system at about ~12.6 min. The original predicted RT was 11.3 (appearing in the document after file import). I then tried pressing the "Use Results" button (in the calculator under Peptide settings) to refine further and the predicted RT jumped to 20.6, which would exclude the peptide based on a 10 minute extraction window. For the GIS peptide (actual RT = 14.3 min), the original "predicted" RT shown after importing the data was 14.7, whereas after pressing "Use Results" the predicted RT increased to 22.0. Clearly I am doing something wrong here...

The Skyline document is attached. The Text file also has a summary.

Sorry for all the idiotic questions.
Thank you
Bill  RT.txt 
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TSQ Atlis _ Chromatogram information unavailable
(5 responses) naimamuntu 2024-02-29 14:13

Good afternoon,
I'm trying to analyze the isotope-labeled Standard of the light and the heavy ratio of myosin heavy chain 7 processed with Thermo TSQ Altis. I am following webinar 13. However, I am getting the message "chromatogram information unavailable."
I can't solve the problem. Would you mind lending a hand? Your expertise would be greatly appreciated.
I've attached the method used and the raw files for the standards.
I really appreciate any help you can provide.
Loyola University Chicago

view request
Spectral library
(6 responses) andreas kalagasidis 2024-02-24 03:38

I am trying to create Spectral library for use with OpenMS analysis. I created Spectral library using DDA files. When I export this as a Report, it contains no fragment charge in the Skyline transition list due to the use of MS1 filtering flow.

Could this have a major impact on subsequent steps in the Open Swath workflow (I haven't been able to complete the workflow)?

Any suggestions?

view request
Chromatogram information unavailable
(10 responses) liuh1 2024-02-26 13:35

I use skyline-daily to process Agilent 6495C QQQ data. With several metabolites I always have problem to integrate the peak area. It shows the message: "Chromatogram information unavailable" I have followed the answer about the same problem, but it can't solve my problem.
Last week I worked for several times to get one file with less "Chromatogram information unavailable", but more metabolites show "Chromatogram information unavailable" after I added a few data file to the project.

I have checked the data file on Masshunter Qual, the metabolite shows great peak and right retention time.

What can I do next?


view request
error importing fragpipe output into skyline
(4 responses) jdemeter 2024-02-27 16:34


I have a similar problem to #63338: I am trying to import pep.xml files from fragpipe output for a group of DDA runs. I get the same error about not finding the scans. After doing the recommended modification of the pep.xml files, a new error occurs: please see the attached screenshot.

I am using Skyline-daily (64-bit) (a978938a7) - the previous version did not have this problem. Skyline (64-bit) is also able to read in the files without any problem.

can you please look into this?

Thank you,

view request
Failure opening Skyline-Daily file
(1 response) samartinez3 2024-02-28 09:50

Hello, I have been receiving an error when trying to open a file that has been previously analyzed. It gives me the options to click OK or More Info, More info gives the information below:

System.IO.IOException: Failure attempting to load the window layout file W:\FilePath
Rename or delete this file to restore the default layout.
Skyline may also need to be restarted. ---> System.ArgumentOutOfRangeException: Index was out of range. Must be non-negative and less than the size of the collection.
Parameter name: index
at System.ThrowHelper.ThrowArgumentOutOfRangeException(ExceptionArgument argument, ExceptionResource resource)
at System.Collections.Generic.List`1.get_Item(Int32 index)
at DigitalRune.Windows.Docking.DockPanelPersistor.LoadFromXml(DockPanel dockPanel, Stream stream, DeserializeDockableForm deserializeContent)
at pwiz.Skyline.SkylineWindow.LoadLayoutLocked(Stream layoutStream) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\SkylineGraphs.cs:line 526
at pwiz.Skyline.SkylineWindow.LoadLayout(Stream layoutStream) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\SkylineGraphs.cs:line 481
at pwiz.Skyline.SkylineWindow.UpdateGraphUI(SrmSettings settingsOld, Boolean docIdChanged) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\SkylineGraphs.cs:line 308
--- End of inner exception stack trace ---
at pwiz.Skyline.SkylineWindow.UpdateGraphUI(SrmSettings settingsOld, Boolean docIdChanged) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\SkylineGraphs.cs:line 317
at pwiz.Skyline.SkylineWindow.OnDocumentUIChanged(SrmDocument documentPrevious) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\Skyline.cs:line 588
at pwiz.Skyline.SkylineWindow.SetDocument(SrmDocument docNew, SrmDocument docOriginal) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\Skyline.cs:line 706
at pwiz.Skyline.SkylineWindow.RestoreDocument(SrmDocument docUndo) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\Skyline.cs:line 903
at pwiz.Skyline.SkylineWindow.SwitchDocument(SrmDocument document, String pathOnDisk) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\Skyline.cs:line 861
at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in D:\Users\nicksh\git_d\sky_applypeaktoall\pwiz_tools\Skyline\SkylineFiles.cs:line 397

I'm using a Skyline-daily with a special "apply to all" feature (from Nick). Please let me know what I need to do to properly open the files.

view request
Issues importing Small molecule transition list
(13 responses) m3g4n 2023-12-04 14:07

Hello! I'm having an issue where I'm trying to copy-paste a list of 30 compounds into skyline and it's breaking apart some of the entries (quant and qual are separate). I don't know how to add them together, or insert one into the other, etc. If anyone knows what I should be looking in depth into in terms of excel syntax that would be super helpful.

Thank you!

view request
(1 response) herath lakmini senavirathna 2024-02-27 12:01


I would like to know the difference between the total ion current area and the TIC chromatogram.
The total ion current area of each replicate can be obtained through skyline file---> export--> report--> peptide quantification.
TIC chromatogram can be obtained through Skyline file--> export-->chromatogram.
These two give different values and would like to know the difference.


view request
DIANN out in Skyline
(1 response) tania karasiewicz 2024-02-26 05:17


I have just started to use DIANN for some SWATH-DIA data. I am trying to visualise the data and do the stats in Skyline. I have managed to import the library and the peak boundaries of DIANN in skyline for visualisation of the different acquisitions.
I am having problem to do the stats, since I didn't run mprophet, is it possible to import the obtain q value from DIANN some how ?

Or in anycase if you have any tips for DIANN in Slyline, that would be great.

Have a nice day,


view request
Chromatogram information unavailable
(10 responses) Pa Nic 14 2024-02-23 13:02

I am working with a Q exactive in PRM mode. I created a transition list with my internal standard and thiol precursors. All compounds work well except my internal standard. I get a message saying Chromatogram information unavailable. I am sure about my retention time and fragmentation (I got them with a full ms d2). I don't understand that with the same parameters, I get a signal on Chromeleon for my internal standard. It is too bad cause I need my internal standard signal to continue working with Skyline, which is way more user-friendly...
Is there anything I could try

view request
Error Exporting to MassLynx Only with Scheduled Method
(7 responses) cbm11 2024-02-22 01:28
Good morning,
I have quite a large document with ~1000 peptides which I want to develop a method for, so I split them into a few 10 min runs with RepLiCal RT Predictor and am now using that to predict RT and schedule them all in one much longer run. The issue is that I am getting this error (which I assume might be a MassLynx problem?) but it only occurs when I try to export the method as "Scheduled", I am able to export the method with no issues if I just export it as standard unscheduled. I am also able to export other scheduled documents as expected, this one in particular using RT prediction just throws up the error. I was wondering if you could help me out with this problem please? Thank you!

(I am sharing the Skyline doc on fileshare as "RT Prediction Export Issues" too)


An error occurred attempting to export.
Error generating Quanpedia databank

Command-line: Method\Waters\BuildWatersMethod -w 1.5 -m "D:\Gez.PRO\ACQUDB\BCR 2 SRM Plus IStd.exp" "D:\ColleenM.PRO\ACQUDB\rgi0ks4h.fwn\transitions.txt"
Working directory: D:\ColleenM.PRO\ACQUDB
OK More Info
System.IO.IOException: Error generating Quanpedia databank

Command-line: Method\Waters\BuildWatersMethod -w 1.5 -m "D:\Gez.PRO\ACQUDB\BCR 2 SRM Plus IStd.exp" "D:\ColleenM.PRO\ACQUDB\rgi0ks4h.fwn\transitions.txt"
Working directory: D:\ColleenM.PRO\ACQUDB ---> System.IO.IOException: Error generating Quanpedia databank

Command-line: Method\Waters\BuildWatersMethod -w 1.5 -m "D:\Gez.PRO\ACQUDB\BCR 2 SRM Plus IStd.exp" "D:\ColleenM.PRO\ACQUDB\rgi0ks4h.fwn\transitions.txt"
Working directory: D:\ColleenM.PRO\ACQUDB
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass, Boolean forceTempfilesCleanup, Func`3 outputAndExitCodeAreGoodFunc, Boolean updateProgressPercentage) in C:\proj\skyline_23_1\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 181
   at pwiz.Skyline.Util.Extensions.UtilProcess.RunProcess(ProcessStartInfo psi, String stdin, String messagePrefix, IProgressMonitor progress, IProgressStatus& status) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Util\Extensions\UtilProcess.cs:line 45
   at pwiz.Skyline.Model.MethodExporter.ExportMethod(String exeName, List`1 argv, String fileName, String templateName, Dictionary`2 dictTranLists, IProgressMonitor progressMonitor) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Export.cs:line 4731
   at pwiz.Skyline.Model.WatersMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Export.cs:line 4523
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Util\Util.cs:line 1922
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 204
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\skyline_23_1\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2313
view request
Exporting scheduled MRM method to *.exp Masslynx
(8 responses) per larsson 2024-02-01 09:05

Dear Skyline support,
I run lipidomics using waters xevo tqxs. Managing the many MRMs in the Masslynx software is to time consuming (adjusting RTs manually) so I am trying to learn skyline. My main objective is to be able to write all MRM experiments into a *.csv file that can be used as a compound database. I can then use skyline to build the *.exp method for each application and update the RTs in excel.

I manage to insert transition list with the parameters needed in a way that seems correct into Skyline but I have problems to get all needed settings exported to the *.exp file.


  1. The problem is that when I export method I am not able to get the right polarity set for each MRM in skyline into the *.exp file, they will al be ES+ even if listed as ES- in my Skyline file.
  2. I have the retention time and retention time windows in Skyline but am unable to get export this to the *.exp file the start and stop time will be at the same value.

If I can find a solution to these problems I would be able to write and modify all my methods from templates in excel and export them very quickly as needed to Masslynx. Changing RT times and windows would be a very simple task in excel and making selections for MRMs for a new application would be very simple as well. To make this work I need to find the solution to export the scheduled RTs and the polarity for all MRMs from skyline to masslynx.

I am using Masslynx 4.2 SCN 1045 and Skyline and run this on the workstation used for controlling the instruments.

In case any one can help me I attach my csv file that I use to import into skyline, pasting into "insert transition list"
I attach my skyline file
I attach my .exp template for the export
I attach a raw file with results from the lipids in the template

Would be extremely nice to find a solution and I am think there should be lots of other users that would have use of this solution.
Excel->Skyline-> Masslynx for method setup would be great


 Skyline problem data.7z 
view request
Error when Upload MSFragPipe output file to skyline
(1 response) wchu3 2024-02-23 10:44

Good afternoon Skyline team

Recently, I tried to use Skyline to analyze the output files from FragPipe. I found a tutorial (Untargeted analysis of DIA datasets) about how to use Skyline to analyze the MSFragger data and it worked for the tutorial data but for my AAV data, it's always displaying the attaches error. Do you know what happened? Looking forward to hearing from you, thank you.

Best wishes,


 IMG_0092.jpg  IMG_0091.jpg  Tutorial-5-DIA-Fragpipe.pdf 
view request
Distinguishing between heavy carbon and heavy hydrogen
(7 responses) molly hopper 2024-02-22 11:24

I'm working on a small molecule tracing experiment where we've added a duterated molecutle (D5 Glutamate) as in internal standard in our study, and have a heavy labeled carbon tracer. We're trying to trace the heavy carbons, and in an unlabeled control, we've noticed a slight signal for [M5C13-H]. I am interpreting this as an issue with the masses being so close that skyline believes them to be the same peak. We have the resolution to see the difference between [M5H2-H] and [M5C13-H] on the instrument (Thermo Exploris 240). I'm hoping for some help with how we can resolve the peaks in skyline, as we're soon to start a study that uses C13N15 tracers.

Current Transition Settings:
MS1 Filtering, precursor mass analyzer: Orbitrap, resolving power: 240,000 at 200 m/z
Instrument: Method matcho tolerance 0.055 m/z.

I've tried changing both of these settings with no change to the integrations.

Windows 10, Skyline 64-bit


 Screenshot 2024-02-22 142143.png 
view request
error using "DIA raw" option when performing Import Peptide Search
(4 responses) wphipps5 2024-02-21 14:53

Hi there-- I am trying out this "Start from: DIA Raw" option during Import Peptide Search of our DIA results. It runs for several hours but then gets an error message. Here is how it ends:

"[2024/02/21 12:08:14] INFO: Issued command:
[2024/02/21 12:08:14] INFO: percolator --results-peptides D:\dia_mzml\231101_dia_mzml_rename\crux-output/ --decoy-results-peptides D:\dia_mzml\231101_dia_mzml_rename\crux-output/percolator.decoy.peptides.txt --results-psms D:\dia_mzml\231101_dia_mzml_rename\crux-output/ --decoy-results-psms D:\dia_mzml\231101_dia_mzml_rename\crux-output/percolator.decoy.psms.txt --verbose 2 --protein-decoy-pattern DECOY_ --seed 1 --subset-max-train 0 --trainFDR 0.01 --testFDR 0.05 --maxiter 10 --search-input auto --no-schema-validation --protein-enzyme trypsin --post-processing-tdc D:\dia_mzml\231117_dia_mzml_rename\
[2024/02/21 12:08:14] INFO: Started Wed Feb 21 12:08:14 2024
[2024/02/21 12:08:14] INFO: Hyperparameters: selectionFdr=0.01, Cpos=0, Cneg=0, maxNiter=10
[2024/02/21 12:08:14] INFO: Reading tab-delimited input from datafile D:\dia_mzml\231117_dia_mzml_rename\
[2024/02/21 12:08:14] INFO: Features:
[2024/02/21 12:08:14] INFO: retentiontime rank abs_ppm isotope_errors log10_evalue hyperscore delta_hyperscore matched_ion_num complementary_ions ion_series weighted_average_abs_fragment_ppm peptide_length ntt nmc Charge1 Charge2 Charge3 Charge4 Charge5 Charge6 Charge7
[2024/02/21 12:08:14] INFO: Found 28 PSMs
[2024/02/21 12:08:14] INFO: Concatenated search input detected and --post-processing-tdc flag set. Applying target-decoy competition on Percolator scores.
[2024/02/21 12:08:14] INFO: Train/test set contains 28 positives and 0 negatives, size ratio=inf and pi0=1
[2024/02/21 12:08:14] FATAL: An exception occurred: Error: no decoy PSMs were provided.
[2024/02/21 12:08:14]
[2024/02/21 14:20:21] Search canceled."

The full output is attached as well as a pdf of my settings. I can provide the mzML files and the fasta.


 settings.pdf  output.txt 
view request
Cannot Import Chromatograms from MassLynx
(5 responses) cbm11 2024-02-19 06:40

Good afternoon,
We are trying to import chromatograms from MassLynx to Skyline 23.1 and only just today have started receiving the below error:

"At 2:46 PM:
Could not load file or assembly 'pwiz_data_cli.dll' or one of its dependencies. The specified module could not be found.
System.IO.FileNotFoundException: Could not load file or assembly 'pwiz_data_cli.dll' or one of its dependencies. The specified module could not be found.
File name: 'pwiz_data_cli.dll'
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache()
at pwiz.Skyline.Model.Results.ChromatogramCache.Build(SrmDocument document, String documentFilePath, ChromatogramCache cacheRecalc, String cachePath, MsDataFileUri msDataFileUri, IProgressStatus status, ILoadMonitor loader, Action`2 complete) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Results\ChromatogramCache.cs:line 732:"

We have tried old skyline documents/raw data files that used to work and also tried uninstalling/reinstalling 23.1. Any idea what else could be happening? I've attached an example .raw file (zipped) and a skyline doc where this is happening

 DME trial patient 
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Explicit Retention Time from msp library for small molecules
(10 responses) meowcat 2024-02-13 02:56


we are using MSP libraries from in-house small molecule standards with specified retention time.
This is correctly loaded into the MSP (NIST) library. However, the retention times from the library aren't used as explicit retention times when adding the compound.

I made a github issue and coded a provisional "solution":

Aside from the fact that it would need to be improved, maybe this isn't needed at all:

  • Is there an existing way to add an explicit retention time (e.g. from a CSV file) in batch to molecules imported from a Spectral Library?
  • Is there an existing way to associate spectra from spectral libraries to existing molecules in a document (rather than to insert new molecules from spectra)?
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skyline batch installation
(4 responses) chi nguyen 2024-02-21 16:45
dear Skyline team,
I am trying to install the skyline batch most recent version ( but I was not successful so far. Below are the error details.
many thanks

2024-02-21 16:02:57,329 [INFO] SkylineBatch: Saved configurations were found in: C:\Users\NguyenD14\AppData\Local\Apps\2.0\Data\X823W123.JCK\94EG05GC.7Y7\skyl..tion_e4141a2a22107248_0015.0002_29e5eb00463e294d\Data\\user.config
2024-02-21 16:02:57,943 [ERROR] SkylineBatch: An unexpected error occured during initialization.
System.NullReferenceException: Object reference not set to an instance of an object.
   at SkylineBatch.MainForm.ListViewSizeChanged()
   at SkylineBatch.MainForm.MainForm_Resize(Object sender, EventArgs e)
   at System.Windows.Forms.Control.OnResize(EventArgs e)
   at System.Windows.Forms.Form.OnResize(EventArgs e)
   at System.Windows.Forms.Control.OnSizeChanged(EventArgs e)
   at System.Windows.Forms.Control.UpdateBounds(Int32 x, Int32 y, Int32 width, Int32 height, Int32 clientWidth, Int32 clientHeight)
   at System.Windows.Forms.Control.UpdateBounds(Int32 x, Int32 y, Int32 width, Int32 height)
   at System.Windows.Forms.Control.SetBoundsCore(Int32 x, Int32 y, Int32 width, Int32 height, BoundsSpecified specified)
   at System.Windows.Forms.Form.SetBoundsCore(Int32 x, Int32 y, Int32 width, Int32 height, BoundsSpecified specified)
   at System.Windows.Forms.Control.ScaleControl(SizeF factor, BoundsSpecified specified)
   at System.Windows.Forms.ScrollableControl.ScaleControl(SizeF factor, BoundsSpecified specified)
   at System.Windows.Forms.Form.ScaleControl(SizeF factor, BoundsSpecified specified)
   at System.Windows.Forms.Control.ScaleControl(SizeF includedFactor, SizeF excludedFactor, Control requestingControl)
   at System.Windows.Forms.ContainerControl.Scale(SizeF includedFactor, SizeF excludedFactor, Control requestingControl)
   at System.Windows.Forms.ContainerControl.PerformAutoScale(Boolean includedBounds, Boolean excludedBounds)
   at System.Windows.Forms.ContainerControl.PerformNeededAutoScaleOnLayout()
   at System.Windows.Forms.ContainerControl.OnLayoutResuming(Boolean performLayout)
   at System.Windows.Forms.Control.ResumeLayout(Boolean performLayout)
   at SkylineBatch.MainForm.InitializeComponent()
   at SkylineBatch.MainForm..ctor(String openFile)
   at SkylineBatch.Program.Main(String[] args)
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Skyline Error on MS data file import
(1 response) db432 2024-02-21 04:43

Dear Skyline Support,

I am using Skyline to import MS data collected on waters Xevo TQXS and Skyline fails to open the file with the error below.

"At 12:28 PM:
Could not load file or assembly 'pwiz_data_cli.dll' or one of its dependencies. The specified module could not be found".

I have uninstalled Skyline from the PC, deleted all command files, restarted the PC and reinstalled the most up to date version (Skyline 64-Bit) (cf25ad847) but still the error appears.

I also note that my Skyline files on the PC do not have the Skyline logo but just plain files (see attached PDF file).

Can you please advice on what to do?

 Skyline Errror.pdf 
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Regression fit
(2 responses) danielacgranato 2024-02-16 11:53

We are analyzing our calibration curves in Skyline for peptide absolute quantification. We have observed that Skyline now has different regression fits besides linear. Would like to know, if we should consider any criteria when applying one type instead of the other. Do you have any suggestions?

Thank you very much in advance. Best, Daniela

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Analysis of Sciex MRM3 data from 7500 QTRAP
(3 responses) h l elfrink 2024-02-08 08:01

Dear Skyline team,

For our publication we are considering uploading the targeted MRM3 data (Sciex 7500 QTRAP) that we generated to the Panorama repository. If I understand correctly, then compatibility of the data with Skyline is a prerequisite, but this is not true for our data. When trying to open a data file Skyline cannot find any chromatogram. This issue was raised previously by a colleague of mine a couple of years ago, the proposed solution, using the the MS3 fragment instead of the MS2 fragment did not work for us.

Could you provide another round of feed back on this issue, please? Should we look for an alternative repository to share our data for now?


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Error in generating .mzid file
(2 responses) elvistc 2024-02-15 22:09

Has anyone experienced this error before? I saw another post regarding this and someone mentioned to update the visual C++ version. The current version installed on my computer is correct and I still see this error.

Thank you in advance.

 mzid error.jpg 
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Importing fractionation sample to Skyline
(1 response) GH 2024-02-15 13:23

I have DIA sample which is fractionated and I have total of 12 .raw fractions data files.
My question is how can import raw files to Skyline? is there anyway to import as combine all fractions? or we need to combine before importing?

Thank you, Ghazaleh

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Filter peptide contains specific peptide in library
(2 responses) GH 2024-02-14 11:43

I generated my library from other software and want to import to skyline (import library assay). I want to filter a peptide containing only L (leucine). I tried in peptide setting -->Filter-->Exclude peptides containing. I created the one to be excluded (attached file here). I did refine and advanced, however it does not filter peptides and still see all peptides.
my question is that is it possible to filter peptide for library? or Do you have any suggestion to solve this problem?

Thank you, Gazale

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Comparing MS1 data with and without FAIMS
(2 responses) paul.derbyshire 2024-02-15 04:07

Hi Skyline team,

I've been running some samples on two different Thermo mass specs - Orbitrap Fusion and Orbitrap Eclipse with FAIMS. When I build the data in Skyline I see distinct saw-tooth chromatograms from Eclipse/FAIMS samples but not from Fusion samples (see attached screenshots). Applying smoothing masks the saw-tooth appearance. Is this a effect of the FAIMS ion separation or is there something in transition settings that needs to be applied before data import?

Thanks in advance.

Paul Derbyshire

 Skyline_MS1_no_smoothing.png  Skyline_MS1_with_smoothing.png 
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a-ions from fully SIL labelled amino acids
(4 responses) jfoe 2024-02-14 04:35

Dear Skyline-Team,

I have a workflow where I create spectral libraries from Skyline. I recently noticed that I am missing some a-ions from my SIL-labelled peptides, if the fragmentation is next to the SIL label.

The reason is, that while the common b- and y- ions fragment neatly at the peptide bonds, the a-ion loses a C-atom that belongs to one of the adjacent amino acids.
Skyline always subtracts a 12C. When the amino acid is fully SIL, this should be a 13C. I suspect this is tough to implement in a general sense but it is very common that SIL are fully labelled so maybe skyline could implement an exception where a 13C is removed if there is no 12C at all in the amino acid.

I am including a screenshot that hopefully shows you why this causes me to lose signal. In that specific case there is actually some peak at the wrong a6+, too, but I am missing the main, correct a6+ peak. In effect, this is quite bad because the spectral library will end up with a bad dotp when detecting the natural non-labelled peptide because it expects only a very weak a6+ ion.
Also, I am including the skyline file.
Skyline version is x64 on Windows.

Let me know if anything is unclear.

Best wishes,

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How to get Skyline to produce different molecule calibration curves in a single instance?
(2 responses) manesh chand 2024-02-14 15:46

I have 115 molecules that I want to calculate concentrations for using Skyline. We have ran a calibration curve of all 115 molecules pooled together at varying concentrations and performed a serial dilution that we ran on our SCIEX 6500 QTrap, files are labelled inslicht 753884 cal 0-9. I followed the tutorial steps for the small molecule quantification and I am able to input the analyte concentration values for one of the 115 molecules on the document grid view and get a calibration curve for. However, this does not allow me to add the other 114 molecules that have different concentrations inside those cal 0-9 files that I want to calculate concentrations for using a singular Skyline instance. Is there a way I can format the document grid view in Skyline to where I can input the other 114 molecule known concentrations into the analyte concentration column without having Skyline auto filling those values down. I have attached pictures of the molecule setting and document grid view, skyline files, and raw data. Thank you for the help!

 OxyEndo Skyline  OxyEndo Skyline  OxyEndo Skyline Template.skyd  OxyEndo Skyline Template.skyl 
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Spectrum annotation inconsitencies
(2 responses) jfoe 2024-02-14 05:10

Dear Skyline-Team,

I often try to identify peptides via skyline. In this effort, the "Show peak annotations" feature of the spectrum view can be really handy. But unfortunately, I really struggle to understand how it works, i.e. what leads it to match some peaks and not match others.

I am uploading two screenshots, one (spectrum_annotation_issue_control.png) shows how the transitions are matched from the canonical way of having transitions active in the targets panel. Note e.g. y7 and especially y8 are matched nicely between 900 and 1100 m/z.
Switching to the peak annotation view (spectrum_annotation_issue.png), I lose these matches (red box). I struggle to find any reason why this is so inconsistent.
The view is really helpful in many situations and e.g. in the example reveals the b3 match, which I didn't have enabled and I think it's a great feature!

Skyline version is x64 on Windows.

Let me know if anything is unclear.

Best wishes,

 spectrum_annotation_issue_control.png  spectrum_annotation_issue.png 
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Spectral Library and Data processing
(6 responses) damlaaygun09 2024-02-12 04:46


I'm trying to use Skyline version for my thermo.raw proteomics datas. I have 2 diseased and 1 healthy data, and each one has 3 fragmentation. I want to get group comparision result LFQ and also need shared and different proteins between subjects. I watched tutorials from YouTube (MQSS 2023 - Dmitry Alexeev). I solved many problem but some points are still unknown for spectral peptide library. I have downloaded from Peptide Atlas and NIST but software is not accept these file types. I'm not sure about am I wrong when I download the library? The other problem is when I can get result for group comparision I see the adjust p-value as 1.00 so I don't know what is the actually correct steps for my aim. Can you help me?Thank you!


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Building a spectral library using msp
(11 responses) laura corveleyn 2021-02-02 03:24


I am trying to build a spectral library in Skyline using an msp file based on predicted spectra. Since we work with histones, we derivatize our peptides using propionylation, so all unmodified lysines and N-terms are blocked with a propionyl group. Therefore, Propionyl (K) and Propionyl (N-term) are fixed modifications, unless another variable modification is present. However, Skyline does not seem to recognize the modification "Propionyl" from the msp, although I also defined it as a modification in the peptide settings. All other modifications (e.g. Acetyl, Dimethyl,..) that I defined, are used correctly in the library. After some research, we figured out that Skyline recalculates the precursor m/z for each peptide (based on its sequence) and doesn't use the one that is stated in the msp. So for a peptide that carries a propionyl group, the m/z is recalculated as if the peptide would be unmodified because Skyline does not recognize the propionyl.

In the attachment you can find a short example of a predicted msp and the Skyline document with the corresponding library I built with this msp. In the msp you can see that the Propionyl group in the header is stated in the exact same way other modifications are, so I don't understand where the problem is.

Thank you in advance for helping me out!



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Chromatogram information unvailable
(4 responses) gazelezi 2024-02-13 12:42


I am a new Skyline user and trying to get some chromatographic information bout several target small molecules. Following the instructions from the site, I was able to insert a transition list and import raw file results from one of my test runs. Initially, I was able to see the chromatogram pieces of information for all the target molecules except for one. After several trials, I wasn't able to fix the issues.
So, I decided to reload the transition list and the raw files. After that, I could not see any chromatogram information for all target molecules. I would appreciate any help.

Attached is the file I am working on.

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building a MS2 transition list
(6 responses) p de blaauw 2024-02-09 01:27

Hello skyline,
I have been using a transition list for small molecules in Skyline for high-resolution data (MS1 scan) for years now. We now have another machine, namely an orbitrap where we generate MS2 spectra in besides the MS1 full scans. What is the best way to create a transition list to analyze the (known) MS2 fragments in Skyline?"

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uploading pdresult files without raw files
(1 response) ssedigh2 2024-02-12 12:18

Hi ,

I was wondering if there is a way to open a PD file in skyline if I don't have access to the raw files?
Thank you for the ongoing support.

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DIA to SRM assay gradient
(3 responses) akhilabrai 2024-01-22 21:37

Hi Skyline team,

I was looking at the tutorial on developing methods for SRM/MRM assay from DIA chromatogram libraries. I had a list of precursors of my interest. It was suggested to run iRT in the qqq instrument. I planned to run Pierce iRT in the Sciex QTRAP in triplicates.
My questions are

  1. What concentration of iRT to be taken?
  2. How to choose the gradient and the run time while running in the QTRAP?

Please help me out.

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When importing Agilent .D files is there a way to read the Sample Group information?
(2 responses) cian monnin 2024-02-05 13:17

Hello Skyline Team!

When making a worklist on Agilent's Data Acquisition software there is a Sample Group column. Is there a way to import or populate a column with this information in Skyline? It's stored in the the sample_info.xml as

    <DisplayName>Sample Group</DisplayName>

Specifically the <Value>20x_MEF_NPRL2</Value> is of interest for us.


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autoQC explicit retention time window
(3 responses) halwaseem 2024-02-06 10:26

I am running serum samples using an MRM method on an Agilent triple quad. Only product ion data is collected (at specified RT windows). There is no full MS data. I would like to use autoQC to monitor the signals of the deuterated internal standards spiked into a quality control sample. I have tried including and excluding the explicit retention times but Skyline isn't correctly integrating the ISTD peaks due to inter-batch retention time shifts(~0.2-0.8 min). Even with a window that captures the shift, Skyline is going in an peak picking noise at the explicit retention time instead of the nicer peaks.

I have attached 3 examples of 3 different compounds in the autoQC transition list that are being properly/improperly integrated.

Since this is for SST, I would prefer for this to be automated and not have to go in and manually adjust the peaks so that they show up correctly in Panorama. Is there something in the settings that can be adjusted to address this issue? Is there a way to set a minimum peak intensity for autoQC integration? There are many examples where there is an obvious peak in the RT window, but Skyline integrates noise instead.

If a iRT calculator was added to molecule settings, would Skyline be able to apply that as raw files came in via autoQC?  autoQC issue.docx 
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PDA Data in Thermo Raw File Prevents Skyline Import
(6 responses) ddrruss 2024-01-31 08:44

I am using the most recent version of skyline, on a windows 10 OS.

I have a PDA detector inline and am collecting UV as well as MS1 and MS2 (DDA using a thermo ascend).
I am using a byonic mzid file to build my library.
When I try to upload my raw file I get an error that says something along the lines of PDA chromatogram not recognized. It looks like Skyline is also trying to import the PDA data from my raw file in addition to the mass spec data. I can send the raw data file for inspection.

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Unable to import Spectra from Bruker DDA analysis.tdf or analysis.tdf_bin using BlibBuild
(42 responses) lubwen 2024-01-08 10:31

I ran into this error of 'No spectra were found for the new library' when importing DDA data from Bruker's raw analysis.tdf (or analysis.tdf_bin) data.

I am using a tab-delimited .ssl result file (954.ssl, see attached), with the following example lines:

file scan charge sequence
HT_20221220_Vividion_sampleB12_1000ng_Slot1-19_1_954.d/analysis.tdf_bin 177793 3 [+42.0]AAAAAAVGPGAGGAGSAVPGGAGPC[+324.2]ATVSVFPGAR
HT_20221220_Vividion_sampleB12_1000ng_Slot1-19_1_954.d/analysis.tdf_bin 176745 3 [+42.0]AAAAAAVGPGAGGAGSAVPGGAGPC[+324.2]ATVSVFPGAR
HT_20221220_Vividion_sampleB12_1000ng_Slot1-19_1_954.d/analysis.tdf_bin 176808 3 [+42.0]AAAAAAVGPGAGGAGSAVPGGAGPC[+324.2]ATVSVFPGAR
I am attaching the error screenshot to this thread. The original tdf and tdf_bin files are too large (~760MB) for this forum.

Could someone help me track down the issue?

Thanks a lot!
Bingwen Lu
 Error.png  954.ssl 
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Different calibration curve concentrations and fittings for different molecules
(1 response) cian monnin 2024-02-05 13:07

Hello Skyline team!

We are doing relative concentration (mix of approx 260 authentic standards in a 9 point curve). Some standards are at different levels. Is there away to have different values for different molecules other than using the Concentration Multiplier?

Is there a way to select different Regression fit for different molecules?


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DIA with overlapping windows, MSFragger output files, import peptide search creates Warning: Did not find spectrum
(3 responses) jennifer schwarz 2024-01-31 04:35

Dear all,

I am using overlapping windows in DIA and demultiplex the spectra with MSConvert.
The created mzML files I use for the search with FragPipe(v21.1)/MSFragger (4.0).
The created interact-*.pep.xml I use for the "import peptide search" (Skyline .
This created the error shown in the screenshot. "could not find scan number" and "did not find spectrum".
Different score values don't make a difference.
This issue was not there in cases when I did not have overlapping windows and did not need to use MSConvert first.

All relevant files are in one folder.
I have the following files:
interact-naming_rank1.pep.xml files
naming_rank1.pepXML files
the mzML files from MSConvert used for the search
and the Thermo raw files.

The difference to the runs without prior usage of MSCovert is that those files are having the naming:

 Screenshot 2024-01-31 131943.png 
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Importing Thermo Biopharma Finder Search Results to Skyline
(3 responses) Yao Chen 2024-02-02 00:25

Dear Skyline Team

Would it be possible to create a targeted search list in Skyline from Thermo's BioPharma Finder search results?


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Peak inversion over y-axis with new skyline version
(1 response) dhaupt 2024-02-02 08:11

Hi Skyline Team,

I recently downloaded the latest update ( and now all of my peaks appear split with the earlier half of the peak being above the y-axis and the later half falling below the y-axis. The peak areas themselves don't appear to be affected, however, I can't seem to figure out how to fix this. I sent multiple files to a colleague using an older version of skyline and they observed typical peak shapes. Wondering if there is a way to disable this or if there is a way to revert to an older version of skyline. Will attach a screenshot of an example chromatogram along with my skyline project so you can check my settings. Thanks for your help.

Dan Haupt

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Building a spectral library from Proteome Discoverer Output
(7 responses) r g beniston 2013-07-24 09:00
To whom it may concern (so to speak),

I'm trying to use the Mascot search results from within Proteome Discoverer 1.3/1.4 on OrbiTrap Elite generated .raw files (Nodes used are: Spectrum Files, Spectrum Selector, Mascot, Annotation and peptide validator) to build a spectral library for future use. I've exported the PSM results to a .pep.XML file and tried to build a library in Skyline v1.4 but I get the error message that the file is not from one of the recognized sources and that no spectra were found for the new library. Help, new to Skyline, and use of spectral libraries generally. Is there a get around?
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Refine/Results/Max transition peak rank setting
(6 responses) Zac 2024-01-31 10:59

Refine/Results/Max transition peak rank...i have being setting this value to 3 to try to filter results to only show the top 3 ranked transitions. I have noticed that for most peptides i get rank 1, rank 2 and rank 3, but for some peptides there may be a different rank included. Is this a bug or is another transition rank included if there is no signal for one of the top 3 ranks? In some cases all three top transitions have signal but they are still not chosen.

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(4 responses) vanyabangera 2024-01-28 06:01

Hello Team,

I have generated tryptic peptides using skyline.

Could you please confirm whether the method I followed is correct or not?

a) I have edited peptide settings with and without (none) background proteome
b) Edit>import>FASTA>and paste by peptide sequence from the FASTA file

I would require clarifications on background proteome and edit>import>FASTA options. At first, I had the whole FASTA file of an organism as background proteome and pasted the whole FASTA file of an organism in edit>import>FASTA options. It gave me some tryptic peptides.
Secondly, I gave none as background proteome and imported a peptide sequence of interest on edit options. It gave me some tryptic peptides

But different results. why is that so? What exactly do background proteome and edit>import>FASTA options do in this case?

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Data processing on Skyline
(2 responses) nbekhti 2024-01-22 07:31

Hi Skyline team,

I have a bench of questions on the data processing using skyline, below:
(1) Is it possible to do baseline correction on Skyline?
(2) Is it possible to have noise canceling on Skyline (Smoothing)? I can see on: View>transform>Savitzki-Golay smoothing, is this the right tool to use for this purpose?
(3) Before doing the integration, does skyline apply resolving? meaning identify shoulder peaks and split into individual features?
(4) Does skyline have alignment tool, meaning aligns detected peaks in different samples?

Thanks a lot for your help,

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isotopes/labeled forms not reported in mProphet command line export
(3 responses) Will Thompson 2024-01-30 11:44

We have been looking at utilizing the mProphet command line export to help fix incorrect candidate peak assignments, and we notice that none of the isotope labeled, iRT, or surrogate standards are in the report. Is this on purpose or a bug? This removes the ability to fix misassignments of some of the isotope labeled standards in our workflows, particularly for small molecules where some isotope labeled standards share a mass. Skyline document attached; specifically the mProphet entries are missing for molecules in the 'iMT' and "IS" molecule lists.



view request
Chromatogram information unavailable JUST for the Heavy Label
(1 response) cvarcini 2024-01-30 11:41


I'm encountering an issue with my internal standard, a heavy label of one of my molecules, in Skyline. While I can see all the chromatograms, the one for the heavy label isn't processing properly. I've attempted this with both Skyline and Skyline Daily, experiencing the same issue on both platforms. I've uploaded two different transition lists, one with the heavy label having the same name as the molecule, and another with a different name. Unfortunately, neither has worked. I've verified the raw data, and I'm confident the peak is present.

How can I resolve this problem and successfully use Skyline to quantify samples using my internal standard?  VitD.skyd  VitD.skyl  TransitionList.csv  TransitionList2.csv  Std12.raw  HeavyLabelPeak.png 
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How to analyze Shimadzu .lcd files?
(7 responses) AWY 2024-01-19 21:37

I outsourced LCMS running and received .lcd files for targeted metabolites.

Files contain (samples with replicates, blanks , standard curv / dilution series ).

I don’t have access to shimadzu software to process targeted LCQQQ .lcd files. This was not possible with MSDIAL, I heaerd the best open source software for processing targeted LCQQQ data.

How can I analyze .lcd files from targeted metabolics?

I would be grateful if you could share any tutorial please.

view request