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
Adding measured peptides to iRT standard curve
(11 responses) romeally 2020-01-16

I have 224 peptides into which I spiked a pierce iRT standard mix and ran it as 3 methods to cover my retention time calibration. When I go to add the non-iRT peptides to the iRT calculator I get an error saying I have insufficent correlation and no peptides will be added to the iRT database. This is PRM data and I am not using a library at this point, so I do not have any fragment correlation but the dotp for the precursors all looks pretty good. The tutorials are all based on triple quad with transitions calling the peptides. Is this something I have to do? I ran these all back to back on the same column with the iRT peptides spiked in so I don't know why there wouldn't be correlation. Is my data bad or am I doing something wrong??? I am attaching a screen this a different procedure with PRM than MRM data? I do see my intercept for my peptide mix is lower than the intercept for my iRT standards...could this be my issue?

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Unable to export method to Masslynx 4.2 SCN895
(6 responses) Tran Tran 2020-01-16

Skyline ( 64-bit)
Masslynx V4.2 SCN895/ Waters Xevo TQD
Windows 7 professional 64 bit

Skyline is installed at the same computer as the instrument . I was trying to export CE optimization using the File/ Export /Method/Multiple methods but received an error ( screen shot attached). However, I was able to export into the .csv format using File/Export/Transition List

Waters Tech Support was contacted but they don't have a solution at the time. Masslynx security update isn't installed either.
Wonder if anyone else observed this issue and was able to resolve it.
Thank you,

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SKYLINE BUG for display and peak area reporting when Non-Quantitative transitions are included
(11 responses) Will Thompson 2020-01-14

Hi Brian, Brendan et al

Please see attached to this message a reasonably detailed account of a pretty bad bug in peak area reporting which seems to occur when non-quantitative transitions are included in Skyline documents. It affects reported total peak areas, such that external measurement of results (such as stats or against calibration curves) will not match up what is returned by Skyline.

As always, let us know if you have any questions.


Will  SKYLINE BUG reporting when Non Quant Transitions Included.docx 
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Scheduling Lumos Targeted Method Development
romeally 2020-01-15

So I am trying to build a scheduled method for 224 peptides which are all +2 charge. When I go into build the multiple methods to get the scheduling I selected 30 precursors maximum per run thinking this should be about 8 injections, but under Thermo Fusion it says I need 172 methods to get there. If I switch it up to QE or Agilent QTOF it says 8. I did a test export and I found 172 .csv files some with only 1 peptide in them. Is this a bug? Should I use the QE settings? What is the most isolation precursors I should use in method building for a Lumos instrument? In any event 172 methods can not be I have some weird setting I am missing? This is with fixed carbamidomethyl and all +2 charge so it should be exactly 224 precursors.

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Skyline does not recognize all peptides identified with MaxQuant
(14 responses) Sven F 2014-08-21
Hi, I like to do some quantification of phosphorylated peptides with the Skyline software. For identification I use the MaxQuant software v1.4.1.2 To quantify my data, I import the msms.txt from the MaxQuant search into Skyline. Because I need just the data from one single protein, I erased data of all other proteins from the .txt file. Skyline builds up a peptide library based on the MaxQuant search, but also there are some peptides, which can be found in the library but are not identified by Skyline in the MS spectrum. So there are some phosphorylated peptides identified by MaxQuant but are not found by Skyline. So in the end, MaxQuant gives me a sequence coverage for this protein about 86%, wheras Skyline is approximately at 20%.
I already checked the parameters like modifications ect, but I could not find a solution. Also I tried to import peptide boundaries using a modified verdion of the evidence.txt as described formerly here on this forum, which occurs in an error, saying the peptide could not be found.

I attached the Skyline an msms file so hopefully you can take a look on them.

Thanks a lot.

Best regards,
 msms.txt  Pex5_CK2_200u_100u_20140729.skyd  Pex5_CK2_200u_100u_20140729.blib 
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mProphet issues
(3 responses) Tobi 2020-01-13

Dear Skyline Team,

in the attached skyline daily I want to highlight some things I would be happy to get your feedback.

When exporting mProphet features unfiltered it seems to list all potential peaks for a single peptide at various retention times and with different z-scores and p-values, however, the q values for all peak candidates are the same despite different p-values, is that a bug?

Also, the checkbox to filter for highest scoring peak can be highly useful but also lead to errors. Problem here is that when correcting peak picking (by hand or with Avant-Garde) this export still reports the highest scoring candidate but not the actually picked peak. Having this filter ( only integrated peak instead of highest scoring or both) would be very nice as it would deliver desired results for both automatic and manual peak picking.


 mProphet unfiltered.csv 
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Exploris PRM data import into skyline
(5 responses) Fynn 2020-01-08

Dear Skyline-Team,

I am using a PRM method with multiplexing (MSX) for targeted measurements on a "Q Exactive HF-X" mass spectrometer. Here everything works well and I can observe precursors as well as transitions. After transferring the method onto the "Orbitrap Exploris 480" platform and loading of the raw files into skyline (and Skyline daily) I still see the precursors, but no transitions. When I open the raw files with "SeeMS" I can see both, the MS1 and MS2 scans. Since the skyline method worked for the HF-X runs I am a bit lost why it is not working for the Exploris runs. Do you have any suggestions how to solve the data import problem?

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Wrong position of amino acids?
(1 response) fcsigloch 2020-01-13

Dear Skyline team,

I find the way of numbering amino acids of a protein in Skyline somewhat confusing: The first amino acid is given as position 0. This behaviour is especially misleading, when trying to locate point mutations or PTMs, where the Skyline position is reported to be one less than the normally reported one.

An example: if, say, a deamidation is detected on the first N of F2QME5 (see attached picture), it would be reported to be at position 3. But the N is actually amino acid #4.

Is this a bug or a feature of Skyline?


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Add q value annotation check box missing in the Reintegrate form
(2 responses) minliecn 2020-01-10

Hi Skyline team,

I am trying to add q value annotation so that I can export the q value in Skyline report. But I do not find the Add a value annotation check box in my Reintegrate form. I am running Skyline(64-bit) on Windows 7. Do you know how can I fix the reintegrate form issue? I attached the expected reintegrate form and the actual integrate form in case these are useful.


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Using Skyline image/logo in graphical abstract for publication
(1 response) Alok 2020-01-08
Hello Skyline team,

Is it possible to use Skyline image in graphical abstract of a paper? We think it is good to include the logo as we will be making all Skyline files publicly available after the acceptance.

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Customs Adducts // GC MS Agilent import
(2 responses) Christina lucas 2020-01-06

I try to import GC MS Data (SingleQuad) to skyline, as I really like the software. The approach presented by Pawel Sadowski for Shimadzu and Thermo does not work for Agilent Data.

So far my I was to create different adducts for the basepeak molecule, but this is not possible if you just want to create a mass additon that is not in the adduct list.
I tried [M(-5.0)+H] or likewise, it does not work with the actual version.

If I try to add more than one precurser, skyline starts to label them

I attach a file of the agilent data, maybe that makes it aesier to solve the problem... 
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The document format version 19.12 is newer than the version 19.1 supported by Skyline (64-bit)
(2 responses) Sadr 2020-01-08

Please can you help me with file format version error below;



Failure opening I:\core\proteomics\data\group_folders\Mass Spectrometry DATA\Q_Exactive_HF\2019\12_December\QE_HF_PR1271_REB_12122019\SKYLINE\
The document format version 19.12 is newer than the version 19.1 supported by Skyline (64-bit)
OK More Info
System.Reflection.TargetInvocationException: There is an error in XML document (2, 2). ---> System.InvalidOperationException: There is an error in XML document (2, 2). ---> pwiz.Skyline.Util.VersionNewerException: The document format version 19.12 is newer than the version 19.1 supported by Skyline (64-bit)
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadXml(XmlReader reader) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 532
   at pwiz.Skyline.Model.SrmDocument.ReadXml(XmlReader reader) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2099
   at System.Xml.Serialization.XmlSerializationReader.ReadSerializable(IXmlSerializable serializable, Boolean wrappedAny)
   at Microsoft.Xml.Serialization.GeneratedAssembly.XmlSerializationReaderSrmDocument.Read1_srm_settings()
   --- End of inner exception stack trace ---
   at System.Xml.Serialization.XmlSerializer.Deserialize(XmlReader xmlReader, String encodingStyle, XmlDeserializationEvents events)
   at System.Xml.Serialization.XmlSerializer.Deserialize(TextReader textReader)
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass948_0.<OpenFile>b__0(IProgressMonitor progressMonitor) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 311
   at pwiz.Skyline.Util.ProgressWaitBroker.PerformWork(ILongWaitBroker broker) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Util\UtilUI.cs:line 123
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 232
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1868
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 180
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 132
   at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 305
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Importing peak boundaries with Skyline Runner
(2 responses) estancliffe 2020-01-06

Hi Skyline Team,

I have begun using the command line version of Skyline to automate the processing of a large metabolomics dataset. With the user interface, I am able to import the peak boundaries to make sure the correct peaks are being integrated; however, with Skyline runner I am unable to do this. Right now, I am able to import my transition list and all of my datafiles and generate a skyline document with Skyline runner, but then I have to manually go in and open the skyline file and import the peak boundaries and then export the final report of the peak areas. If I was able to import peak boundaries through the command line, I would not need to open the interface at all. I don't know if this is a simple fix on your end, but it would help me out a lot!

Thank you!


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Peak picking and transitions
(1 response) aqassab 2020-01-06

I am currently working on a project designed to discover peptide bio-markers using SRM. I observe some peptides peptides with five well represented transitions, other peptides with less transitions. Based on the changes observed between samples, a few peptides show one transition better than other transitions selected in the method. I have a few questions related to this:

  1. What is the minimum number of transitions recommended to use in the data analysis?
  2. For peptides with maximum of three transitions selected, one or two transitions are not located under the main peak area (the area under the transition with the highest intensity), is it recommended to delete those "poor" transitions and use only two or maybe just one transition for some peptides?
  3. When transitions in actual samples (please see the figure attached) do not line up with the transitions found in the heavy "spiked" peptide, should I extend the boundaries to cover the area of all transition selected in the endogenous peptide in the sample, or should I place the boundaries based on the transitions observed for the heavy "spiked" transitions?



 peak picking issue.emf 
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Adding to my transition list (small molecules) does not show the new molecules
(1 response) mazzouny 2020-01-06

I hope these questions are not redundant. Whenever I add to my transition list, everything I add, is not getting analyzed. I need to re-insert a new list and import the RAW files from scratch in order to quantify a new compound in my small molecules list. Are there any re-analyze option ?
Also, when I export the transition list, it shows that there is no CE chosen (because I'm doing MS1 quant) and it export it as a generic format. When I import the same file again, I get an error. Any idea?
I'm using version 19.10.193, small molecules installation.

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Targeted Calibration Curve Value Import
(1 response) gsbyram 2020-01-06

Hey Everyone,

I have a targeted steroid assay which quantifies roughly 35 compounds. The compounds contained in the calibration curve have different concentrations based around their reference range. Is there a way to individually assign concentration values for each compound in a calibration standard instead of a blanket value for all compounds?

West Coast Metabolomics Center
UC Davis
Oliver Fiehn Laboratory

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CE optimization library different precursor and product z
(2 responses) Erik 2020-01-03

Hi All,

I more often measure peptides that have a predominant breakage point (mostly proline) and thus the most abundant ions are formed from the same ion but in two different charge-states. Can I store the CE value of multiple product ion charges from the same peptide ion fragment (y6+ and y6++ for instance) in the library?
Similarly, but less relevant can I do the same for a peptide sequence with different precursor charge?

I was not able to find this anywhere in the tutorial or support forum. Hopefully this is possible without changing the software too much. I suggest one or two additional columns with precursor z and product z. Or is it possible to provide the charge in the "product ion" column?


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Error while running MSstats and AvantGardeDIA external Tools
(4 responses) Chinmaya k 2020-01-02


I have installed both MSstats and AvantGardeDIA with all the R versions and libraries successfully. However, I am facing some issues while running both of these external tools right now. Such as;

MSstats - As you can see in the attached file. The MSstats function exports the results to a Temp directory with all the basic column information in it. But, the Fragment Ion column contains both fragment ions and precursors identified in the DIA search with respective "Area". According to MSstats, precursor information is not required for DIA analysis and how to exclude precursors from being exported by the MSstats as MSstats input file?

AvantGardeDIA - Whereas here in AvantGardeDIA, this tool is not able to change the working directory. I do not know what exactly is the reason here. I have also attached the screenshot of the current set working directory for AvantGardeDIA\Run_AvG_Module\GlobalRefinement below. Why this tool is not able to change the working directory and how can we resolve this issue?

Thank you,

 AvantGardeDIA_Error  AvantGardeDIA_Working_Directory.PNG  MSstats_Error.txt 
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Different peak height, area in Skyline and in Xcalibur
(3 responses) hilaire 2020-01-02

I am analyzing Q-Exactive HF PRM Data with Skyline.
In Skyline after importing of raw data, for compound G, the Skyline XIC is totally different from what I can see in Xcalibur.
For 78.9585, the height in Skyline is 880, while in Xcalibur it is 5.02e4.
For 492.9919, the height in Skyline is 75346, while in Xcalibur it is 1.98e4.
What is the cause of the error and how can I improve it??

I have attached the skyline file, transition setting and the XIC, MSMS snapshot from Xcalibur for your reference.

 Test.skyd  QExHF03_438 493 XIC.PNG  QExHF03_438 286 MSMS.PNG  QExHF03_438 79 XIC.PNG  QExHF03_438 Skyline XIC.PNG  Transition setting.PNG 
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xuezhangzhi 2020-01-02
Dear Skyline team
 I am trying to analyze hundreds of MRM dataset to use skyline, but I didn’t acquire the transitions of Decoy peptides in the MRM experiment in prior as suggested by the mprophet method. Can I still use the mProphet model for peak picking, or could you provide me some suggestions?Now, I just following the tutorial for manual evaluation.
I am wondering if can I automate pick manual peak and avoid of the bias (such as ( how good RT, ppm accuracy etc.)) by any statistical model? Do you have any suggestion to automate manual peak picking with statistical model for MRM dataset using Skyline?

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Poor System Utilization by Skyline
(15 responses) 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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method match tolerance
(2 responses) kbagramyan 2019-12-17

Hi Brendan,

I am glad that I found the question that I wanted to ask you too. We recently performed an MRM analysis using 6495 LC-MS Triple Quadrupole (Agilent). We optimized the MRM transitions for the quadruply charged precursor ions of the light and the heavy peptides. Here, are our transition settings used in the method: 545.3 (precursor, +4) and the transitions are 509.0 (b, 4+); 640.0 (b, 3+); 678.3 (b, 3+). The Skyline recognized the two of transitions such as 508.5155 and 639.67. The third transition of 678.3 in our method was 677.6849 in Skyline transitions giving 0.6151 Da difference. Unfortunately, this was the most intense peak but wasn't accepted by Skyline because I couldn't extend the method match tolerance above 0.6.
The data look very good and I was wondering if there is a way to extend the method match tolerance, so I can include that transition for accurate quantification?
I hope I was clear and was able to describe the problem.
Thank you in advance,


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mass tolerance
(2 responses) zhengemma 2017-03-29
Can the method match tolerance be set above 0.6 under the Transition settings? Is there a way to do?

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Analyzing timsTOF data with FragPipe/MSFragger search results
(9 responses) nesvi 2019-12-13

Dear Skyline team,

I am trying to extract MS1 quantification from timsDIA PASEF data. I have 4 raw files (.d folders) that were processed using FragPipe/MSFragger/Philosopher. Which, by the way, works great for these data.

I am not able to build a spectral library using the DDA-MS1 quant workflow, with Import - Peptide results etc for building the list form the search results. Neither using MSFragger pepXML files nor using interact-.pep.xml files after PeptideProphet work. When using interact-.pep.xml files, I get an error that it cannot find spectral files. When using pepXML, it finds the files to get the spectra (from file_calibrated.mgf files produced by MSFragger) but gives an error at a later stage.

However, I am able to trick Skyline into extracting MS1 quant from DDA files by going through the DIA workflow. When going that route, I am able to select pepXML files, Skyline finds the corresponding spectral files, and it can build the library. I can also select .d DDA files and perform quantification. However, with this trick, Skyline tries to quantify both precursor and y/b ions (because it is a DIA workflow), which takes a lot of time. I just need precursor quantification from MS1, but removing b,y ions from the list of ions to quantify gives me an error.
Also not that using interact-*.pep.xml files (instead of pepXML from MSFragger) still does not work when using the DIA workflow.

So, ideally, I would like to being able to

  1. Load PeptideProphet pep.xml files, and not just the MSFragger pepXML files.
  2. Being able to use DDA-MS1 quant route rather than DIA route when working with DDA data.

Admittedly, I am an inexperienced Skyline user. So I may be doing something wrong. However, Brett Phinney seemed to confirm the errors I am getting with DDA files.

I could get someone in my team to work directly with the Skyline team to resolve these issues, or to learn how to do this analysis properly.

Thank you,
Alexey Nesvizhskii
University of Michigan

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One peptide, two heavy labels?
(3 responses) ztflaten 2019-12-13

Hey Skyline Forum,

I have a peptide with two labels. One is a N15 labelled full protein that is added to monitor digestion, the other has a single amino acid label. They share the same light peptide, but I've had to "duplicate" it to have both modifications present in Skyline. Is there a better way to do this? The issue ends up being if I change the integration window for one, it ends up having a different light peak area than the other.

I've attached a screenshot of what this looks like in my work space.



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MSstats error
(4 responses) rbbsky123456 2019-12-11

Hi, everyone. I met a problem for MSstats analysis with both DIA and PRM data. Here is the error messages:
** Reading the data for MSstats.....
** Peptides, that are used in more than one proteins, are removed.
** Truncated peaks are replaced with NA.
Error in SkylinetoMSstatsFormat(raw, removeProtein_with1Feature = TRUE, :
** Please check precursors information. If your experiment is DIA, please remove the precursors. If your experiments is DDA, please check the precursor information.

Can't finish analysis.

Skyline version is daily(64-bit)19.1.338 and Msstats is install as external tools.



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MSstats download error
(2 responses) 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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mProphet scoring of removed peaks
(2 responses) Tobi 2019-12-11

Dear all,

I wanted to report some strange behavior of Q-values and z-scores as shown in the attached screenshot and the uploaded file ,,mProphet scoring on removed peaks". (I expect it to show up pls let me know if not)

When working with DIA and peptide identification in skyline, when removing peaks manually (right-click, remove peak) and via Avant-Garde DIA, those peptides yield Q-values and Z-scores as if nothing happened. I even checked with a different scoring model and see values changing aka refreshing, but peptides where peaks were removed (see RT as well as dotp = 0) still show values from prev. existing peak boundaries. I would expect something like #N/A.

Could you please take a look at this? I guess a workaround would be to filter peptides where dotp > 0.00 or where RT fails.

Can you please also think of extending the document grid for exporting all intermediate values of the scoring? Or is there already a way to score Skyline curated data with Percolator? (When decoys might not be normally distributed etc.).

Looing forward getting in touch with you.


 mProphet Scoring on removed peaks.JPG 
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Strange idotp behaviour in small molecule mode
(5 responses) Chris Ashwood 2019-12-11

Dear Skyline support,

I've noticed a couple of times that the values of idotp can change drastically if certain precursor transitions are turned off and on again. In the attached Skyline document, the idotp values are all around 0.3-0.45 (pretty low for how they visually look) yet if I disable and then re-enable the monoisotopic precursor transition, my idotp values increase at least two-fold (0.84 - 0.94).

The final idotp values look to be correct based on the workaround, but I could imagine this could cause some trouble for people that don't know about it and automatically filter their transitions based on idotp.

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Apply Boundary limits to all replicates
renecoig 2019-12-11

When it is necessary to change the boundary limits of a selected peak across all replicates, this is very time consuming, and it seems like this could be automated pretty easily. It would be great to have a place where you could type in an upper or lower boundary (or both) and have it apply to all. Or even better, apply to selected replicates. This would cut down on hours of QC work done by hand currently within this software, where the automated peak selection was not working well.

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Cannot import FASTA database with sequences in lowercase
(1 response) fcsigloch 2019-12-10

When trying to import a FASTA protein database with some sequences in lowercase, Skyline fails to process the database, but gives no error message. It took me some time to find out what the problem was. Converting all sequences to UPPERCASE solved the problem.

It would be great, if you could provide support for lowercase libraries or at least give some meaningful error message.

Greetings from Bavaria,

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EIC coelution problem
(10 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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Updated external tools and interactive tool documentation
(3 responses) Chris Ashwood 2019-12-07

Dear Skyline team,

I am preparing a GUI that helps improve accessibility for users to perform design experiments and analyze data in the small molecule mode of Skyline. I would like to have it as an interactive tool, so I've read the documentation that has been written but noticed that the documentation is quite dated (2013 and 2015 for external tools and interactive tools, respectively). Are there any updated versions of these documents? I would like to know if there are new commands that can be used with the SkylineTool.dll and if these tutorials are relevant for small molecule/mixed modes.


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Problem importing MS1 data
(2 responses) ekaterina mostovenko 2019-12-09

Dear Skyline,

I am trying to import MS data acquired on Waters Synapt G2 Si and I keep on getting the following error:

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

I set all settings in Full-Scan to none and I still get the same error.

I also tried to import DDA data, it was also unsuccessful, I did change my Transition Settings to DDA/TOF and got:

"No scans in *.raw match the current filter settings."

Does it mean that Skyline can't match spectra to the predicted transitions? Or am I doing something wrong?

Thank you!

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Prosit in skyline
(6 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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Small Molecules Analysis
(1 response) abangak 2019-12-09

I am analysing some small molecules data with skyline. However, they are in .mzdata.xml format which cannot be recognized by skyline. How do I convert these files to a supported format in skyline?

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"TNG" error when exporting optimization methods for Thermo Altis QqQ
(2 responses) wangqingok 2019-12-08

Hi Skyline team,

We are trying to export the peptide optimization methods using Skyline and the methods will be used on a Thermo Altis. For some particular peptides, with repeated amino acids there will always a "TNG" error showing up and the whole exportation procedure will quit. Attached please find a screenshot demonstrating the details of the error. Can you help with some solutions?

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Problems with method export
hober 2019-12-09

I'm using Skyline for screening of recombinant proteins for method development on our SRM instrument (TSQ Altis).
In this workflow, the method export has worked really well most of the time. I have a Skyline document with all my targets in the target list and then export my methods using One method per protein, since my proteins are in independent samples. Occasionally, when I have longer peptides, the collision energy which Skyline tries to write to the method files exceeds the collision energy possible on the instrument (<65). This is especially a problem when doing the CE optimization. This causes the entire methods export to abort and I cannot even retrieve files for the proteins which don't cause the error.

Would it be possible to introduce an upper limit in regards to collision energy?


view request
(49 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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DDA analysis from Bruker Amazon SL data
(8 responses) rliyana 2019-12-07

I am try to load data from Bruker Amazon SL using mzML (raw) and .dat (MASCOT). Having issues. Appreciate someone can help me with this. I am attaching files of interest with this. Thanks !!

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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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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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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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protein quantification report
zhangfa 2019-11-28
Hi Skyline Team,
Can Skyline provide a built-in label‐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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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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