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.

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Showing: limited to 100 requests
Storing DP values (Declustering Potential) within Skyline
(3 responses) kathrin lauter4951 2016-02-23
Hi Brendan!

I am using Skyline and I am really appreciating this software!

As I am doing Optimisation of the CE and DP values for the peptides i am analysing, I was happy to see that there is the possibilty to store optimized CE values in a database which makes life much easier.

Now I would like to know if it is/will be possible to also store optimized DP values. In the "optimization library" I just see the opportunity to store CE values or Compensation Voltage...

Thanks, Kathrin
view request
ERROR: No spectra were found for the new library.
(10 responses) proteomica2 2023-03-13
Dear all

I am having problems with Skyline daily. I have an DDA experiment acquired in Timstof which I want to study peptides label with NEM and d5NEM. After loading file data and searching, Skline is not able to build the library and return the next message:

ERROR: No spectra were found for the new library.

Command-line: C:\Users\Carlos\AppData\Local\Apps\2.0\41VM9PKA.2W2\L22PKGOR.GPN\skyl..tion_e4141a2a22107248_0016.0002_bdd2fb77bf0c781e\BlibBuild -s -A -H -o -c 0.95 -i Cys_NEM_D5NEM -S "C:\Users\Carlos\AppData\Local\Temp\tmp9C39.tmp" "E:\Proteomic-Projects\Ricardo_CysNEM\combined\txt\Cys_NEM-D5NEM.redundant.blib"
Working directory: E:\Proteomic-Projects\Ricardo_CysNEM
OK More Info
System.IO.IOException: ERROR: No spectra were found for the new library.

Command-line: C:\Users\Carlos\AppData\Local\Apps\2.0\41VM9PKA.2W2\L22PKGOR.GPN\skyl..tion_e4141a2a22107248_0016.0002_bdd2fb77bf0c781e\BlibBuild -s -A -H -o -c 0.95 -i Cys_NEM_D5NEM -S "C:\Users\Carlos\AppData\Local\Temp\tmp9C39.tmp" "E:\Proteomic-Projects\Ricardo_CysNEM\combined\txt\Cys_NEM-D5NEM.redundant.blib"
Working directory: E:\Proteomic-Projects\Ricardo_CysNEM
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\pwiz\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 161
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\pwiz\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 428
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 161
After that, Skyline starts to load data again and the software goes into loop.

Could it be a possible solution?

Thanks in advance.

Carlos Fuentes
view request
Recovery of Skyline "Import Results" process after system crash
(1 response) tryphoncosinus 2023-03-21


I am processing DIA search on a large batch using a high end server. All went fine but at a certain point during result import of pepXML/mzML files, the system memory went full and a crash occured.

Is there a way in Skyline to recover a session so that it continues the import process after the last imported result file ?

Thank you.

view request
"Import DIA Peptide Search" pipeline in Skyline start page
(3 responses) tryphoncosinus 2023-03-15


I have a collection of pepXML and mzML files previously obtained using File/Import/Peptide Search menu, including Oxidation and Carbamidomethyl modifications.

My goal is to import these files in Skyline for full visualization. The pipeline cited in title seems to be a tool created for that purpose.

I am following the indications Skyline is asking but there is only one modification displayed in the "Add Modifications" window (Oxidation). I also need Carbamidomethyl. When I try to add it in the list, I get "The modification Carbamidomethyl already exists".

Could you confirm that this popup window is asking NEW modifications I may need without discarding Oxidation and Carbamidomethyl I used to generate pepXML and mzML files ? This point is confusing me because all I can do on this step is to check or uncheck Oxidation modification, nothing else.

Thank you for your help.

view request
Trouble viewing MS2 data from Bruker maXis for systyem suitability applications
(4 responses) paul mathews 2023-03-08

Hi Skyline team,

I am in the process of building SS protocols across our LC systems using Skyline and Panorama AutoQC function. In this request I am investigating DDA MS2 data acquired on a bruker maXis 2 for a single compound, resperine, ran on with a c18, 4 min uplc method with bruker autoMSMS detection.

I have followed the tutorials for small molecule quantitation and generated two skyline documents, one from scratch and one using the "Auto MSMS_MS and MSMS filtering" template. In the scratch generated document, I am unable to see MS2 information. In the modified template, I can see my MS2 transition of interest, but the chromatogram appears wonky with a straight line. It seems selecting "DIA" or "none" I see in another post this may just be a bug. Is there something I can do within my transition settings or transition list to get the transition ion (which appears in the MS2 scan of m/z 609) to appear and be useful for SS? We may ultimately just quantify on the MS1 TIC, but for further use (including using GCMS data) I'd like to find a way to use a fragment ions in quantitation, without a MRM method.

Attached are screenshots of the skyline documents. For the auto MSMS template file there are two shots, one with DIA and one with the targeted(obsolete) method (which strangely disappears once you modify the template)

 skyline automsms troubleshooting.PNG  skyline resperine troubleshooting.PNG  skyline automsms troubleshooting_DIA.PNG 
view request
New BioReplicate column?
(1 response) franziska vollmy 2023-03-14

Hi all,
I'm looking to generate an external calibration curve but the column called $BioReplicate I used in the past to specify the replicates of a given point no longer exists. This means that the replicate data points are simply aggregated and the individual points do not show up on my curve. Which column should I use instead?

I'm using Skyline version

I'm attaching a screenshot of what the document grid looked like in the past.


view request
lack of weight 1/X calibration curve
(1 response) giuseppe corona 2023-03-13
Hi, working on molecule side i found a problem using the calibration weight option: when it is selected this latter option no caibration curve can be obtained and appeared the following error message " matrix have to positive define". When the weight option is selected as " none " the calibration curve appared and performed as aspected.
 how may be solved this issue?
thank in advance
 Skyline Lack of weithting 1-x or 1-x2.pptx 
view request
Unintended behavior when performing CE optimization with more than 10 product ions for a precursor
(2 responses) Chris Ashwood 2023-03-10

OS/Skyline version: Windows 10, Skyline-daily

Mode: Small molecule data

I hope you're all doing well. I am performing QQQ CE optimization for a single compound per run, with 14 product ions and 10 collision energies. As it currently stands, I have to modify the transition list after importing into Thermo software (Xcalibur 4.6 on an Altis Plus) as the export results in more than 10 rows with the same compound name and precursor m/z which causes an error. My understanding of the cause of the problem is below.

As Thermo method editor is limited to only a maximum of 10 rows with the same compound name and precursor m/z, Skyline appends a .2 to the end of transitions when there are more than ten. E.g. When I have 14 product ions and am performing steps in collision energy, there are ten product ions listed as "moleculeName(-charge).4", and four listed as "moleculeName(-charge).4.2". This behavior is expected and works fine.

The middle CE for a given molecule and product ion is just "moleculeName(-charge)" and, as I have 14 product ions for that name, there are ten product ions listed as "moleculeName(-charge)", and four listed as "moleculeName(-charge).2". This naming convention causes a problem as the second step in collision energy optimization uses the same suffix. E.g ten product ions listed as "moleculeName(-charge).2" and 4 product ions listed as "moleculeName(-charge).2.2". As a result, I then have a total of 16 compounds named "moleculeName(-charge).2", which is 6 more than allowed by the vendor software. In addition, it's confusing as I have to work out which of the "moleculeName(-charge).2" are from step 0 or step 2 in the CE optimization.

I currently append a B to the end of 4 of the compound names, but I'm not sure if Skyline can then read those files correctly. One possible solution could be changing the naming convention for CE optimization to add a 0 for the middle step in CE optimization, giving 10x "moleculeName(-charge).2" and 4x "moleculeName(-charge).0.2" in the example I'm encountering above instead of 16x "moleculeName(-charge).2". This should prevent any collisions in the naming as the transitions will then uniquely named.

Please find attached an example of the transition list that gets exported from Skyline, note the middle step for the last four product ions have the .2 suffix.


view request
building spectrum library issue
(9 responses) chen qian 2023-03-08

Hi Skyline team,

I am trying to build the spectrum library based on the searching results of the heavy labelled peptides. When I uploaded the mzid file, I got the error message shown in the attachment. I am not able to understand the issue based on the error message. Can you help take a look and advice what is the issue?


 Skyline error.PNG 
view request
Fail to integrate small molecule peaks
(7 responses) ho-tak lau 2023-02-22

Hi Skyline Team,

I am trying to analyze some small molecule data acquired on TimTOF pro2 in MRM mode (Skyline-daily (1e3a4e917)). However, Skyline is not able to integrate the peaks. DataAnalysis from Bruker showed that the peaks are there. What change can I make to have Skyline to integrate the peaks?

Thank you in advance.


 transitionList.xlsx  DataAnalysis.pptx 
view request
Fragpipe output parsing error
(1 response) ho-tak lau 2023-03-08

Hi Skyline Team,

I found a bug parsing Fragpipe 19.1 result using Skyline When I load the interact-filename.pep.xml with filename_uncalibrated.mgf in the same folder. Skyline was unable to find the .mgf. I have to rename the file to filename_uncalibrated_uncalibrated.mgf for Skyline to find it.


view request
Peak integration wrong for a single replicate, but for thousands of precursors
(2 responses) joshuasmith 2023-03-07

Hi all,

I am having an issue where Skyline is integrating peaks incorrectly for one out of 6 replicates, but for thousands of precursors in the file. This is data from the same support ticket as a previous issue with importing the DIA-NN spectral library ( - thanks Nick for helping on that.

I've attached screenshots of the issue. I have tried refining the peak picking several times and I think I have a really good peak scoring model, but it still is mis-integrating peaks. Sometimes it does pick the correct peak (slide 4), but this is uncommon. I obviously would prefer not to have to manually go through roughly 10,000 precursors and correct integration on what appears to be 80-90% of them. One way I tried to do this more quickly was by looking at run-to-tun regression and finding outliers, but this didn't work. As you can see in slides 5-8, as highlighted with the red box on the regression plot, there isn't much deviation for the misintegrated peaks, and so no good way to target the worst cases first. It's so many anyway that it wouldn't end up being a "targeted" fix.

One thing I did notice is that the replicate that has a lot of integration issues does seem to be unusual in that it has more ID matches for than other replicates, and they tend to skew towards the leading tail of the peak. See slides 8-10. Could that be the issue? Not sure why that would have happened, other than that was the first sample in the file list run through DIA-NN. The spectral library is based on all 6 runs, but the DIA-NN log does say:
"DIA-NN will optimise the mass accuracy automatically using the first run in the experiment. This is useful primarily for quick initial analyses, when it is not yet known which mass accuracy setting works best for a particular acquisition."
I know that this optimization can be turned off by specifying a mass accuracy setting for the run, but I don't know if that would fix the issue I'm seeing.

I have compressed my entire skyline document and can share that if needed, although it's 4 Gb zipped.

view request
cannot see MS/MS data
(2 responses) naymin saw 2023-03-08

Hi Skyline team,

I am trying to develop a standard library with DDA data but I cannot see the MS/MS data in Skyline. I can check only MS1.
Could you please check and advise if somewhere is wrong?

Thanks and regards,
Nay Min 
view request
Importing error "Index was outside the bounds of the array"
(13 responses) rschoenh 2017-08-29

I have a set of wiff files that I'm trying to import into Skyline, and some are imported fine, while others give me an error message (pasted partially below). I used the same Analyst scheduled acquisition method for all files, and I can open up the wiff files in Analyst without a problem. I'm using Skyline version

Thanks so much in advance for your help!


At 7:13 AM:
Failed importing results file '(...path and file name...)'.
Index was outside the bounds of the array.
view request
Issue with importing DIA-NN speclib
(6 responses) joshuasmith 2023-03-06

Hi Skyline team,

I know this issue has been in the forums before (, but I tried the suggestions in that post and I still seem to be having issues.

Basically, I created a spectral library of a basic yeast search with DIA-NN and am trying to import into Skyline. I have moved the speclib and accompanying tsv file to their own folder, so that they are in the same directory and there isn't any confusion on which tsv to import. However, I am getting an error stating that Skyline cannot find the accompanying tsv file. See attached ppt for screenshots. I can upload the speclib and tsv if you'd like.

I'm running Skyline Any advice?


view request
two accounts
(1 response) cabarnescabarnes 2023-03-06

Hi Team,

I have ended up with two separate accounts in your system. One is through my gmail address and the other one, which is more focused on PanoramaWeb is through my work email address. I'd like to consolidate them into one account with my gmail address. Is that possible?



view request
Using previously calculated calibration curve equation
(3 responses) mshubham 2023-03-02

We have calibration curve equations determined for all our molecules (Lipids) in an excel sheet, but we do not have all the raw files. Is there any way in Skyline that I can import the calibration curve equation and calculate the concentrations?

view request
Question of "Peak" Defination
(2 responses) hzhu 2023-03-02

Dear Skyline Team,
This software helps me a lot in analyzing PRM and DIA data. Thank you.
And I am curious about how the software identify peaks in spectra. However I can't find the defination of that anywhere...
For example, this explanation ( makes me clear that how Skyline calculates peak areas, but why the time 14.4- 14.48 is defined as the start and end of the peak to calculate? I wonder if there is a document containing the algorithm about that, like treat the "10% intensity of climax" as the bottom.
Thanks again for your reply!

Best regards,
He Zhu

view request
PRM-PASEF method creation, 'All targets must have an ion mobility value'
(1 response) robert parker 2023-03-01

When creating a method for bruker timsTOF for PRM PASEF I get this message see attached.

The spectral library viewer shows the ionmobility values and I have it selected in transition settings, 'use spectral library ion mobility value when present'..

Is there some where else I need to specify this in skyline so it can creat the method?

bw and many thanks!


view request
Query in DIA peak extraction and Groupwise comparison
(1 response) Sangram 2023-02-28

Hi Skyline Team,

I am analyzing DIA data from for cell line proteome generated on Sciex TripleTOF 6500.

Nick and I have previously tried to understand the Isolation Scheme issue and now I do it by manually inputting it (and have it saved) as we use variable window. But I have noted that when I use only DIA data to generate a spectral library and quantify, Skyline produces only 1800 proteins while from the same files DIA-NN is producing 3500 proteins. But if we use DDA data to build spectral library, then we are able to generate 6500 proteins and quantify. We have also used it to create a groupwise comparison. Now my queries:

  1. We know that DIA produces more data then why are we able to capture much less protein than DDA. What can we check and improve ?
  2. In the groupwise comparison, the proteins of interest (we have RNAseq for the samples and looking for those genes) are all non-significantly expressed across groups. How can that be possible even the proteins of highly expressed genes ? How can we check that and also what method is used to calculate the fold change (need it for writing the methods).

P.S. I have the skyline zip [9Gb] in Onedrive and can share as per your convinience.

Much gratitude for all the help and advice.

Best regards

view request
Mirror plot for modified vs unmodified peptides
lgamon 2023-03-01

Dear Skyline,

Not sure where best to post this. Love the mirror-plot feature for prosit spectra but would really like to be able to compare mirror-plots of modified and unmodified peptides natively in skyline. Is this something you are working on or a feature you could add?

Luke Gamon

view request
Bibliospec not recognize modification from PLGS file
(6 responses) jcaceresvergara 2022-12-02
I'm trying to create a library from a peptide search made using PLGS. I get the error at the end. It does not appear to understand the modification I set, I try to change the final_fragment.csv file or add the modification to my skyline file but nothing seems to work. can you help me with this?

The modification should be a loss of' -2HS' variable on cysteine. I'm attaching my final_fragment.csv file.

ERROR: The modification 'Dehydroalanine+C(4)' on line 1429 is not recognized. Supported modifications include: "12C d0", "13C", "13C N15", "13C d9", "1H d0", "2H d8", "Acetyl", "Amidation", "Biotin", "C-Mannosyl", "Carbamidomethyl", "Carbamyl", "Carboxymethyl", "DUPLEX_TANDEM_MASS_TAG126", "DUPLEX_TANDEM_MASS_TAG127", "Deamidation", "Dehydration", "Dimethyl", "Farnesyl", "Flavin-adenine", "Formyl", "Gamma-carboxyglutamic", "Geranyl-geranyl", "Glycation", "Hydroxyl", "ICAT-C", "ICAT-C13C(9)", "ICAT-D", "ICAT-D 2H(8)", "ICAT-G", "ICAT-G 2H(8)", "ICAT-H", "ICAT-H13(6)", "Isobaric 114", "Isobaric 115", "Isobaric 116", "Isobaric 117", "Isobaric 8plex 113", "Isobaric 8plex 114", "Isobaric 8plex 115", "Isobaric 8plex 116", "Isobaric 8plex 117", "Isobaric 8plex 118", "Isobaric 8plex 119", "Isobaric 8plex 121", "Lipoyl", "Methyl", "Myristoyl", "N-Glycosylation", "NATIVE_TANDEM_MASS_TAG126", "NATIVE_TANDEM_MASS_TAG127", "NIPCAM", "O-GlcNAc", "O-Glycosylation", "O18", "O18 label at both C-terminal oxygens", "Oxidation", "Palmitoyl", "Phosphopantetheine", "Phosphoryl", "Propionamide", "Pyridoxal", "Pyrrolidone", "S-pyridylethyl", "SILAC 13C(1) 2H3", "SILAC 13C(3)", "SILAC 13C(3) 15N(1)", "SILAC 13C(4) 15N(1)", "SILAC 13C(5)", "SILAC 13C(6)", "SILAC 13C(6) 15N(2)", "SILAC 13C(6) 15N(4)", "SILAC 13C(8) 15N(2)", "SILAC 13C(9)", "SILAC 13C(9) 15N(1)", "SILAC 15N(2) 2H(9)", "SILAC 15N(4)", "SIXPLEX_TANDEM_MASS_TAG126", "SIXPLEX_TANDEM_MASS_TAG127", "SIXPLEX_TANDEM_MASS_TAG128", "SIXPLEX_TANDEM_MASS_TAG129", "SIXPLEX_TANDEM_MASS_TAG130", "SIXPLEX_TANDEM_MASS_TAG131", "SMA", "Sulfo", "Trimethyl".
ERROR: reading file 20221122 C418U_iRT MSE JC_LC-05_IA_final_fragment.csv

Command-line: C:\Users\jcaceresvergara\AppData\Local\Apps\2.0\T0VY925W.HAY\GJ5BNRYZ.4Y7\skyl..tion_e4141a2a22107248_0016.0002_c984fd7cba69179e\BlibBuild -s -A -H -o -i lib -S "C:\Users\jcaceresvergara\AppData\Local\Temp\tmp6856.tmp" "D:\lib.redundant.blib"
Working directory: C:\Users\jcaceresvergara\Downloads\PLGS Search Results\PLGS Search Results\Trypsin\20221122 C418U_iRT MSE JC_LC-05_20221202133552
OK More Info
System.IO.IOException: ERROR: The modification 'Dehydroalanine+C(4)' on line 1429 is not recognized. Supported modifications include: "12C d0", "13C", "13C N15", "13C d9", "1H d0", "2H d8", "Acetyl", "Amidation", "Biotin", "C-Mannosyl", "Carbamidomethyl", "Carbamyl", "Carboxymethyl", "DUPLEX_TANDEM_MASS_TAG126", "DUPLEX_TANDEM_MASS_TAG127", "Deamidation", "Dehydration", "Dimethyl", "Farnesyl", "Flavin-adenine", "Formyl", "Gamma-carboxyglutamic", "Geranyl-geranyl", "Glycation", "Hydroxyl", "ICAT-C", "ICAT-C13C(9)", "ICAT-D", "ICAT-D 2H(8)", "ICAT-G", "ICAT-G 2H(8)", "ICAT-H", "ICAT-H13(6)", "Isobaric 114", "Isobaric 115", "Isobaric 116", "Isobaric 117", "Isobaric 8plex 113", "Isobaric 8plex 114", "Isobaric 8plex 115", "Isobaric 8plex 116", "Isobaric 8plex 117", "Isobaric 8plex 118", "Isobaric 8plex 119", "Isobaric 8plex 121", "Lipoyl", "Methyl", "Myristoyl", "N-Glycosylation", "NATIVE_TANDEM_MASS_TAG126", "NATIVE_TANDEM_MASS_TAG127", "NIPCAM", "O-GlcNAc", "O-Glycosylation", "O18", "O18 label at both C-terminal oxygens", "Oxidation", "Palmitoyl", "Phosphopantetheine", "Phosphoryl", "Propionamide", "Pyridoxal", "Pyrrolidone", "S-pyridylethyl", "SILAC 13C(1) 2H3", "SILAC 13C(3)", "SILAC 13C(3) 15N(1)", "SILAC 13C(4) 15N(1)", "SILAC 13C(5)", "SILAC 13C(6)", "SILAC 13C(6) 15N(2)", "SILAC 13C(6) 15N(4)", "SILAC 13C(8) 15N(2)", "SILAC 13C(9)", "SILAC 13C(9) 15N(1)", "SILAC 15N(2) 2H(9)", "SILAC 15N(4)", "SIXPLEX_TANDEM_MASS_TAG126", "SIXPLEX_TANDEM_MASS_TAG127", "SIXPLEX_TANDEM_MASS_TAG128", "SIXPLEX_TANDEM_MASS_TAG129", "SIXPLEX_TANDEM_MASS_TAG130", "SIXPLEX_TANDEM_MASS_TAG131", "SMA", "Sulfo", "Trimethyl".
ERROR: reading file 20221122 C418U_iRT MSE JC_LC-05_IA_final_fragment.csv

Command-line: C:\Users\jcaceresvergara\AppData\Local\Apps\2.0\T0VY925W.HAY\GJ5BNRYZ.4Y7\skyl..tion_e4141a2a22107248_0016.0002_c984fd7cba69179e\BlibBuild -s -A -H -o -i lib -S "C:\Users\jcaceresvergara\AppData\Local\Temp\tmp6856.tmp" "D:\lib.redundant.blib"
Working directory: C:\Users\jcaceresvergara\Downloads\PLGS Search Results\PLGS Search Results\Trypsin\20221122 C418U_iRT MSE JC_LC-05_20221202133552
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\pwiz\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 161
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\pwiz\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 412
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 161
 20221122 C418U_iRT MSE JC_LC-05_IA_final_fragment.csv 
view request
Ion Mobility of Fragments are "0"
(12 responses) Premy 2023-02-26


I am importing and analyzing DIA-IM data using an Ion Mobility library and iRTs built from DDA-PASEF data. When clicking the apex of a peak, a purple IM extracted window is shown for the precursor but not for all fragments (with a label of IM = 0 above them). I'm a bit concerned that the fragments shown are not at the specific IM window but rather the entire IM range.

Is there a way to make sure the same IM extraction window is assigned to the fragments of a target?


view request
Build library from DDA and use the library in DIANN
(1 response) lorrain 2023-02-27

We are trying to build a library for DIANN search. When we export the library, it's only in .blib format and DIANN is not recognizing this format (it only takes .speclib). Is there are way to export the library that can be used in DIANN?


view request
upgrade to skyline daily prevents opening data from Sciex 6500+
(1 response) becker7825199 2023-02-24

Good morning. I updated my skyline daily this morning. Opened my template file, saved as the new data set and then attempted to import data from my Sciex 6500+.
I'm getting an error when importing data that says "the file may be corrupted, missing or the correct libraries may not be installed. [WiffFile::create()] could not load file or assembly ' Culture=neutral. Public key Token 2a79e0d8fd2e4eca or one of its dependencies. system cannot find the file specified.

I went to a different PC declined the upgrade and opened my template, saved with the new file name and imported the same data no problems. I suspect something is broken with the new version of daily.

view request
Skyline daily closes upon import timsTOF DIAPASEF runs
(4 responses) an.staes 2023-02-23


When importing timsTOF DIAPASEF runs in skyline daily, skyline closes down. Any idea what is going on?
List of peptides is not huge, only the promega isotopologues.....

Thank you for looking into this!

Kind regards,

view request
Which product ion is used as Qualifier and Quantifier
(1 response) johannes jaegers 2023-02-24

I use Skyline for metabolic flux analysis. I am tracing the conversion of isotopic labeled substrates into different metabolites. For this purpose I define the count of labeled ions in the precursor ion and let Skyline calculate the Ratio between the unlabeled and the different labeled metabolites in my samples.

Lately I want to trace the fate of nitrogen and on what position they will appear in amino acids (sidechain or headgroup). In this special case the precursor ions have the same mass but differ in only one product ion.

How can I define this ion to be the one being used as the quantifier for the calculation of the ratio?

view request
skyline shows PROSIT library twice in the mirrored image ignoring real data
(2 responses) pavel shliaha 2023-02-22

I am using a prosit library and I would like to see how my spectrum compares to the one in the library (predicted by skyline) using mirror image display. However smth is clearly wrong:

  1. in the mirror image the dotp 1, but in the target tab dotp is 0.89
  2. the name "2022_08_24_BSA_ATG4 vs prosit", but the 2022_08_24_BSA_ATG4 is the name of the library generated using prosit
  3. The mirror image includes the ions that I have removed from the analysis
  4. all the errors are 0 ppm implying that skyline just plot the library vs library

(see the screenshot attached).

Why is skyline not showing the actual spectrum from the current analysis as part of the mirror?

view request
Switching synchronize integration on/off for specific target molecules of a multianalyte method
LeeM 2023-02-24
Hi, I'm a new to Skyline this year and using it for metabolomics. I'm finding it very good, but have my first question:

Is it possible to turn synchronize integration on/off for specific target molecules in select/all replicate samples, rather than turning it on/off for all targets in specific replicates? Am I right that currently it is controlled completely by inclusion/exclusion of replicate samples?

When I have multiple target analytes, I would like to be able to deactivate sync integration for one analyte when reviewing/adjusting integration if sync is not appropriate, but without automatically deactivating sync integration for the other analytes in the same rep sample, whose integration might be
perfectly fine sync'd.

Many thanks for your help.
view request
Defining Isolation Schemes for Bruker timsTOF DIA data
(4 responses) Premy 2023-01-13

Thanks again for all the support with Skyline!

I am developing a DIA-IM Lipidomics pipeline on Skyline and was wondering whether an isolation scheme would need to be defined for Bruker timsTOF data or if the isolation scheme can be read from my raw files using "Results Only"?



view request
Sharing .skyd error
(6 responses) yann guitton 2023-02-23

A colleague sent me a .zip file made with (File/share) from a skyline version. when i try to open it on my computer (same version of skyline)
I have the following error. Can you give us a solution?

Failure opening C:\Users\ygu\Downloads\
The file contains an error on line 4127 at column 8.
The isotope modification type heavy does not exist in the document settings.
OK More Info
System.Reflection.TargetInvocationException: Il existe une erreur dans le document XML (4127, 8). ---> System.InvalidOperationException: Il existe une erreur dans le document XML (4127, 8). ---> System.IO.InvalidDataException: The isotope modification type heavy does not exist in the document settings.
   à pwiz.Skyline.Model.Serialization.DocumentReader.ReadLabelType(XmlReader reader, IsotopeLabelType labelTypeDefault) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:ligne 1413
   à pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionGroupXml(XmlReader reader, Peptide peptide, ExplicitMods mods) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:ligne 1328
   à pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionGroupListXml(XmlReader reader, Peptide peptide, ExplicitMods mods) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:ligne 1322
   à pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideXml(XmlReader reader, PeptideGroup group, Boolean isCustomMolecule) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:ligne 969
   à pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideListXml(XmlReader reader, PeptideGroup group) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:ligne 904
   à pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideGroupXml(XmlReader reader) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:ligne 874
   à pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideGroupListXml(XmlReader reader) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:ligne 653
   à pwiz.Skyline.Model.Serialization.DocumentReader.ReadXml(XmlReader reader) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:ligne 617
   à pwiz.Skyline.Model.SrmDocument.ReadXml(XmlReader reader) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\SrmDocument.cs:ligne 2150
   à System.Xml.Serialization.XmlSerializationReader.ReadSerializable(IXmlSerializable serializable, Boolean wrappedAny)
   à Microsoft.Xml.Serialization.GeneratedAssembly.XmlSerializationReaderSrmDocument.Read1_srm_settings()
   --- Fin de la trace de la pile d'exception interne ---
   à System.Xml.Serialization.XmlSerializer.Deserialize(XmlReader xmlReader, String encodingStyle, XmlDeserializationEvents events)
   à pwiz.Skyline.SkylineWindow.<>c__DisplayClass735_0.<OpenFile>b__0(IProgressMonitor progressMonitor) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\SkylineFiles.cs:ligne 328
   à pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:ligne 254
   --- Fin de la trace de la pile d'exception interne ---
   à pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\Util\Util.cs:ligne 1965
   à pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:ligne 202
   à pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:ligne 140
   à pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) dans C:\proj\skyline_22_2\pwiz_tools\Skyline\SkylineFiles.cs:ligne 345
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DIA-MS analysis: Replicates not showing up
(9 responses) nbaig 2023-02-20


I am performing DIA data analysis. I notice that only one replicate is showing up while the others are not visible. How do I resolve this issue?

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The system cannot find the file specified
(5 responses) sascha 2023-02-20

Dear Skyline people,

I am sorry in advance to haunt you with ghosts from the pasts, but I have a problem with importing Thermo Fisher raw files in one our systems by using your last version of Skyline 32-bit. We have two systems which run Windows 7 32 bit.

System 1

  • Xcalibur Foundation 3.0 SP2
  • Xcalibur 3.0
  • Q Exactive Plus Orbitrap MS 2.9

System 2

  • Xcalibur Foundation 2.0 SP1
  • Xcalibur 2.2 SP1
  • TSQ Quantum 2.3.0

I want to import an SRM run into Skyline to check system performance. While it works flawlessly with system 1, I get the following error on system 2:

At 1:15 PM:
Failed importing results file 'C:\Xcalibur\data\Faelle\Test_TSQ\230210\Test_LXQ_Q1_SRM_pos_230210.raw'.
[SpectrumList_Thermo::spectrum()] Error retrieving spectrum "controllerType=0 controllerNumber=1 scan=2": [ThermoRawFile] The system cannot find the file specified.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Xcalibur\data\Faelle\Test_TSQ\230210\Test_LXQ_Q1_SRM_pos_230210.raw'.
[SpectrumList_Thermo::spectrum()] Error retrieving spectrum "controllerType=0 controllerNumber=1 scan=2": [ThermoRawFile] The system cannot find the file specified. ---> System.Exception: [SpectrumList_Thermo::spectrum()] Error retrieving spectrum "controllerType=0 controllerNumber=1 scan=2": [ThermoRawFile] The system cannot find the file specified.
   at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, Boolean getBinaryData)
   at pwiz.ProteowizardWrapper.MsDataFileImpl.HasSrmSpectraInList(SpectrumList spectrumList) in C:\proj\skyline_20_2\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1064
   at pwiz.ProteowizardWrapper.MsDataFileImpl.get_SpectrumList() in C:\proj\skyline_20_2\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 538
   at pwiz.ProteowizardWrapper.MsDataFileImpl.get_HasCombinedIonMobilitySpectra() in C:\proj\skyline_20_2\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 599
   at pwiz.Skyline.Model.Results.FileBuildInfo..ctor(MsDataFileUri msDataFileUri, MsDataFileImpl file) in C:\proj\skyline_20_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1430
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 211
   --- End of inner exception stack trace ---

It doesn't matter where I try to import the results file from, it is never found. Do you have any idea why this might be the case? Is the Xcalibur version on system 2 too old?

I attached the file in question and the used skyline file for you.

Thank you very much for your help!

view request
SRM method creation for TSQ altis plus
(1 response) kmsd 2023-02-22

I have made several lists which contain protein/peptide/ms1/ms2/rentention time using skyline from my discovery untargeted proteomics data. Now I want to transfer them to TSQ altis plus for monitoring them. Can you show me tutorials for this task?


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Analysis of hela-PRTC data from tsq altis plus
kmsd 2023-02-22

I need help with processing hela-PRTC SRM data from tsq altis plus using skyline. Is there any tutorial available?


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Skewness and kurtosis calculation
(2 responses) chloe cloteau 2023-02-21

Dear Skyline team,

I am performing SRM data analysis for small molecules on the beta version of skyline daily. I would like to display the skewness, kurtosis and shape correlation values in my table.

While I have integrated the columns in the report, the cells appear empty whatever the compound despite the presence of a chromatographic peak. I also searched the check box "Calculate skewness" in the advanced section in the settings, without success.

How can I solve this issue?

Thank you in advance for your feedbak.
Best regards.

Chloé C.

view request
Retention times do not fit with scan times in ID picker
(25 responses) staab-weijnitz 2023-01-27

Hi all,

I want to use Skyline for MS1 filtering and quantification of collagen PTMs from crude samples, conceptually similar to what we did here:

Right now, I am trying to analyze crude cell ECM from cells where I knocked down a gene (n=2, four samples in total). This is basically a test run, to see whether we get enough coverage of collagens and other proteins of interest.

We use MyriMatch (2.2.19172) for peptide search of thermo .raw files and obtain results in .pepXML format and check them in ID picker (3.1). After the "fine PTM search" where we restrict the search on proteins present in the sample and include decoys in the fasta file, I loaded a merged session of my four files in IDpicker and exported the spectral library (.sptxt file) from the merged idpDB file.

I imported this .sptxt file into Skyline ("Use existing library") and then loaded the four thermo .raw files after conversion to .mzMXL via MSConvert (3.0). This seemed to work.

However, now, when I crosscheck the IDs and the retention times I get in Skyline, they are most of the time completely off compared to the scan times for the same peptides in ID picker.

Is there anything obvious I have been doing wrong? I would appreciate your help on this. Happy to upload files or give more information, of course, too, if needed.

Many thanks,

view request
EICs replicate order distorted with the use of Ctrl + Shift + Up/Down (arrow key)
(2 responses) Juan C. Rojas E. 2023-02-14

Hi Skyline team,

I am looking back at a Skyline document that has 100+ runs in it. The Ctrl + Shift + Up/Down (arrow key) is very useful to go through the different runs based on the document order. However, I have noticed that it completely changes the arrangement of runs in the Replicates window. Any ideas why?

I noticed this a year ago, but tested it with the newest Skyline-daily available ( and see that the problem persists.

As always, thanks for your time and help!
Juan C.

view request
Peak annotation in Skyline
(4 responses) mnissa 2023-02-14

Dear Support Team,
I had a doubt regarding the peak annotation is Skyline.
Under "Transform" option, there are four different options: None, Interpolated, Second derivate, Savitzky-Golay Smoothing.
In each view, the number of data points shows a different value (as in the attached picture)
While selecting the peak, which view should be considered for analysing the peak width or to know the number of data points in the peak? Can we consider the Smoothing view for this as that shows the maximum number of data points.

I look forward to this.

Best Regards

 Skyline-peak-transform view.png 
view request
Request improved Precision on Retention Time Prediction Values
(1 response) Will Thompson 2023-02-14

Dear Skyline Team. Currently the max precision calculated for retention time prediction (reported in "Predicted Result Retention Time" field), is hundredths of a minute. For very fast runs with very narrow peaks, it seems like it would be useful to allow a more precise prediction. The current precision of 0.01 minutes is only 0.6 seconds, which for some separations is nearly the 4sigma peak width. Please see attached an example of a file where I think having a more precise result of the prediction algorithm would be useful for putting the prediction closer to the apex of the peak. Or please let me know if I've just not configured something correctly. Thanks for your help!


view request
Distorted peaks
(4 responses) VM26 2023-02-13
I recently acquired the PRM profile of one protein that I have run on Q Exactive. However, upon importing the RAW file onto Skyline, I am unable to get any good intensity of inclusion list peptides. I only find one transition per peptide with a very low intensity (e3-e5) with a green dot in the target section while the rest show red dots. What could be the possible cause?

The data has been acquired on the following specifications:
Instrument: Q Exactive (Positive mode)
PRM: 150 min with Chrom peak width of 30sec
Isolation window: 2.0m/z
Resolution: 17500
IT 50 sec
view request
Points Across Peak vs Raw Number of Points
(1 response) anton poliakov 2023-02-14

Hellow World! I am looking at a SureQuant result and I found a parameter called Raw Number of Points, which is the number of ms2 spectrums acquired for a particular target. However, there is a separate parameter called Points Across Peak. When I look into the numbers there are far fewer Points Across a Peak than Raw Number of Points. This brings up a question: exactly what does the parameter Points Across Peak mean?

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Error: no decoy PSMs were provided.
jalbin28068 2023-02-13

I'm attempting to do a DDA search with MSFragger. My overall workflow is as in the thread I started last week (FATAL: An exception occurred: 'c' is not a valid amino acid), and works fine with those old data. The same workflow also works fine with most of the new data I have collected. With some files, however, I will get 0 PSMs returned from the decoy search, which results in an error message when Percolator gets to that file (Error: no decoy PSMs were provided). I have attached the log from an attempt at running the search with one of the files in question (several files will trigger the same error message). This might, in part, reflect the small size of my database and relatively low sample complexity (289 peptides), but is there some way that I can adjust settings to encourage MSFragger / Percolator to find at least 1 PSM among the decoys so the search can complete with all files?

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FATAL: An exception occurred: 'c' is not a valid amino acid
(11 responses) jalbin28068 2023-02-08

I'm trying to import DDA files and complete a search within Skyline. I can get the search to complete sometimes with MSAmanda, but when I use MSFragger, I usually get the error, "FATAL: An exception occurred: 'c' is not a valid amino acid." I'm not sure where this is coming from. There are no "c" in my fasta; I'm searching a peptide library that lacks cysteines, and I have checked / resaved-as / etc. the fasta without effect. I think it might be related to modifications because I could get MSFragger to complete when I set the maximum variable modifications to 0 (though there were few identified peptides when I tried that compared with when I have searched in MSAmanda or MaxQuant). My library has fixed C-terminal amidation, but otherwise should not have any modifications. I've tried making a custom C-terminal amidation modification instead of using the built-in modification without effect. I've attached the full MSFragger log from my latest attempt if that would be helpful. Does anyone know where this error is coming from an how to fix it?

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Batch processing and command line
(2 responses) tryphoncosinus 2023-02-08

Dear team,

Quite new with Skyline. I would like to run DIA analysis with quite large set of MS data. The idea:

  • Prepare analysis configuration/libraries using Skyline and Skyline batch programs under a standard Windows machine.
  • Execute this configuration on a powerful server using only the command line through Skyline.exe / SkylineBatch.exe, i.e., without any graphic UI that is not available on this machine (I checked I can execute SkylineCmd.exe --help on it).
  • Go back with result files on the standard machine for Skyline visualization.

Is there any way to achieve this idea or alike and process the workflow with no more user intervention? Or suggestions ?

Thank you for your help.

view request
Skyline can't load the interact-*.pep.xml and *_uncalibrated.mgf files from FragPipe 19.1 coupled with MSFragger 3.7
(17 responses) fcyu 2023-01-18

It works for the interact-*.pep.xml and *_uncalibrated.mgf files from FragPipe 18.0 coupled with MSFragger 3.5. But not the files from the latest MSFragger. Could you please help to test, and tell me if Skyline or MSFragger need to be adjusted? All files have been uploaded to the attachments.



view request
Skyline compatibility with GC-QTOF
(12 responses) higgi022 2023-01-05

I am curious if Skyline could be used to generate data collected on a GC-QTOF. My instrument (Agilent 7200 GC-QTOF) is not capable of generating data using MRM\PRM acquisition modes - all data represents full product ion scans using EI ionization.

Would I be able to use Skyline to extract select ions from the full scan and compare their ion intensities relative to a spectral library entry to detect compounds and obtain quantitation?

view request
No quantification data on isotopically labelled internal standard
(2 responses) wangn2 2023-02-06

Hello Skyline support team,

I have an issue when extracting MS2 fragments integration for quantification. The quantification results (showed in the "quantification" column from the "peptide ratio result" report) of my target peptides are correctly showed, but for the isotopically labelled (+8 for Lysine and +10 for Arginine) internal standards, the quantification showed "#N/A", even though the chromatogram was correctly extracted. I wonder if this is due to some settings about isotopic labelling that I've ignored.
I attached a snapshot from the "peptide ratio result" report preview. Any suggestions would be greatly appreciated!

view request
Error SpectrumList_mzXML
(8 responses) CoB 2023-02-03
Dear Skyline Team,

I am using Skyline and I have a problem with looking into the MS spectra of my imported files.

I have mzXml files that contain a subset of a given retention time window and m/z range (subsetted in MSconvert).
These files work just fine in Skyline.
After retention time alignment using an external tool, the files can be readily imported without error in Skyline again and I can evaluate these files. However, when I click on a peak to see the underlying MS information the error specified below occurs.

Thanks a lot for your help in this matter.


[SpectrumList_mzXML::spectrum()] Index out of bounds.
OK More Info
System.Exception: [SpectrumList_mzXML::spectrum()] Index out of bounds.
   at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, DetailLevel detailLevel)
   at pwiz.ProteowizardWrapper.MsDataFileImpl.GetCachedSpectrum(Int32 scanIndex, DetailLevel detailLevel) in C:\proj\skyline_22_2\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1232
   at pwiz.ProteowizardWrapper.MsDataFileImpl.GetSpectrum(Int32 spectrumIndex) in C:\proj\skyline_22_2\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 931
   at pwiz.Skyline.Model.Results.ScanProvider.GetMsDataFileSpectraWithCommonRetentionTime(Int32 internalScanIndex, Boolean ignoreZeroIntensityPoints, Nullable`1 centroidedMs1, Nullable`1 centroidedMs2) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\ScanProvider.cs:line 210
   at pwiz.Skyline.Model.Results.MsDataFileScanHelper.BackgroundScanProvider.Work() in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\MsDataFileScanHelper.cs:line 441
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In Targeted Method Refinement Tutorial I keep Getting a Corrupt RAW file error
(3 responses) sorenf 2023-02-01

Hi Skyline Team,

I have been getting myself familiar with skyline by doing the tutorials and when I was working on the Targeted Method Refinement Tutorial I got stuck. Whenever I need to import results I end up getting a Corrupt RAW file error. I have tried it with both with Method Refinement supplemental and if I skip that I get the same error when I try to import in the "Scheduling for Efficient Acquisition" step.

I am running skyline 64-bit 22.2 on Windows 11 using Parallels on a 2020 MacBook Air.

I have tried to access the file from a few different ways;
*direct by clicking on the tutorial inside the skyline app,
*downloading it from the website,
*using the hyperlink in the PDF that opens when you start the app (

It has also persisted through restarting skyline and using a completely fresh .sky file.

Error message:

At 3:13 PM:
Failed importing results file '\Mac\Home\Downloads\MethodRefineSupplement\MethodRefineSupplement\worm_0001.RAW'.
[RawFileImpl::ctor()] Corrupt RAW file \Mac\Home\Downloads\MethodRefineSupplement\MethodRefineSupplement\worm_0001.RAW
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file '\Mac\Home\Downloads\MethodRefineSupplement\MethodRefineSupplement\worm_0001.RAW'.
[RawFileImpl::ctor()] Corrupt RAW file \Mac\Home\Downloads\MethodRefineSupplement\MethodRefineSupplement\worm_0001.RAW ---> System.Exception: [RawFileImpl::ctor()] Corrupt RAW file \Mac\Home\Downloads\MethodRefineSupplement\MethodRefineSupplement\worm_0001.RAW
at filename, MSData result, Int32 runIndex, ReaderConfig config)
at 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\skyline_22_2\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 197
at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 291
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 199
--- End of inner exception stack trace ---

Many Thanks,
view request
Skyline integrates low signal peak but exports a zero value
(5 responses) halwaseem 2023-02-02


I am using Skyline Daily v to integrate small molecules using a transition list. I noticed for a few metabolites, that I manually integrated, the exported Skyline report shows zero values even though there is a peak that has been integrated. In all cases these are low signal peaks. However, within the same dataset, Skyline allows integration of noise from blanks and samples that have even lower than the biological replicates that was "zeroed out".

In the retention time comparison graph, there is a retention time window showing the sample that has a zero value. The peak area graph doesn't have a line/bar for that sample because Skyline is recording it as a zero. Please refer to attached document.

Thank you for your assistance. Let me know if there are any other details you'd like from me.


 Skyline Issue_RockefellerU_02022023.docx 
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Spectrum IDs
(1 response) paul.derbyshire 2023-02-01

I would like to include the number of spectrum IDs per peptide when exporting a document report. For example, if I have 8 samples and each chromatogram has 1 ID then I would be looking for a value of 8.

Hope you can help.



view request
DIA results from SCIEX 7600 zenoTOF instrument
(4 responses) lorrain 2023-02-02

We are testing some standard peptides using SWATH DIA on SCIEX 7600 zenoTOF instrument. Our transition setting is attached. After importing the datafiles, for some peptides we were able to see MS2 fragments; for some peptides, there is no fragment data. I am wondering if this is due to our DIA method settings or skyline transitions settings. Would you please help?

 Transition settings.PNG  peptides with and without fragments.PNG 
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Instructions for normalizing with TIC
(1 response) dave lee-2 2023-02-02

Hi all,

I have a PRM experiment where I am trying to normalize each sample according to their respective TIC.
I have looked in the Skyline forum where the suggestion is to take modify the document settings and enable calculation of the Total Ion Current Area.
Subsequently, setting the "Explicit Global Standard Area" column to the TIC area

However, I have come across a couple of issues -> The "Explicit Global Standard Area" is under the "calculated" tab rather than "editable" in the document settings so I can't seem to copy and paste.

Ultimately, what I would like is when I view the peak areas of my peptides (with respect to the other replicates); I would like to normalize against the TIC -> Currently, the only options available are "Total", "Maximum" and "None"
And then to take these values through to MSstats

Is there any resource that provides step-by-step instructions in what I am attempting to do?

The version of Skyline I'm using is:

view request
Fitting cross-linked peptides to an iRT scale
(2 responses) Juan C. Rojas E. 2023-01-16

Hi team,

I am trying to fit some cross-linked peptides to an iRT scale (i.e. calculator) generated from endogenous peptides. Unfortunately the column had to be changed, resulting in different retention times. However, I am trying to compensate for this using the background proteome to define some iRTs.

Skyline allows me to add the peptides within the "Edit iRT Calculator" by clicking "Add Results..." but when I try to click OK I am getting a error message (attached).

You will know best, but to me it seems is that here Skyline is not recognizing cross-link modifications?

Any suggestions for a workaround or is it something that has to be patched?

As always, thanks for your time and help.
Juan C.

view request
Showing Chromatogram information unavailable after importing results
(3 responses) mnissa 2023-01-30

Dear Support Team,
When I import the result files, while looking at the results, it seems as if the whole target list is not at all monitored in all the samples and it shows "Chromatogram information unavailable". However, I have used the same method to run all the samples.
When i checked the raw file, it is ok and have been monitored for all the precursor masses. I tried to import the results in a new Skyline document, some of the files which didn't show up before, they showed up and I could see the data for all the precursors in the target list. But the issue was still there if I remove those files or reimport.
As i have shown in this attachment, the peak is being shown for only sample 1 and not for sample 2-4. If I try starting the whole thing again in a new document It can happen the next time that I see the results for sample 3 or 2 or 4 and not for sample 1. So, it is just happening randomly. As this is not happening to a single or same set of raw files each time, I am not able to guess the issue.
Kindly help me fix this.

Best regards

 Result import issue.PNG 
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Exporting isolation list for Thermo Eclipse
(2 responses) mnissa 2023-01-30

Dear Support team,
I am using Skyline-daily (64-bit)
I am planning for a PRM experiment on Thermo Scientific Orbitrap Eclipse mass spectrometer. How can I export isolation list for this instrument.?

Best regards

 Isolation list Instruments.jpeg 
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Click to peak chromatographic peak broken?
(2 responses) Juan C. Rojas E. 2023-01-30

Hi all,

I am currently analyzing some DDA- and DIA-PASEF data with the most recent Skyline-daily version and I am not being able to click on proposed peak boundaries as I used to.

Is someone else facing this same issue? Or is it my document and/or data the issue?


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Not seen glycopeptides working with data from MSFRagger (DDA MS1 filtering)
(12 responses) cpavan 2023-01-18

Dear Skyline staff,

I’m trying to analyze a dataset by DDA MS1 filter to compare XIC of glycopeptides. The peptide search comes from MSFragger – glycol search –. I can create a library with different peptides from the proteins ID but I can see the glyco peptides identified by MSfragger, which is actually my interest in the analysis.
I have tried to add the glycosylation by peptide setting but Still, I only see unmodified peptides ( only Carbamidomethylation and met ox -.
Any suggestion to solve that?

thank you so much in advance,


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Split retention time window
(1 response) dennisjakob 2023-01-27

I was wondering if it's possible to implement a slit retention time window for the explicit retention time.
In some cases we have shoulders or a bit of tailing. Of course this is not ideal but just the case sometimes.
adjusting the retention time window with different values would help in these cases.
Best, Dennis

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Finding unique peptides
(10 responses) prajita 2023-01-23

I have 15-20 peptides that I would like to confirm specificity against Human proteome. I took following steps:
Settings->Peptide setting->Digestion->background proteome
and have added human Fasta sequence.
Then I took following steps:
Edit-> Unique peptides
Under this option, I could see some peptides that have blue checks meaning they are not unique. However, if I keep scrolling I see a limited number of proteins in human. I am not sure if this option is covering all the human proteins. Can you please provide an insight to this?

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the problem of merging skyline documents with one raw data in different spectral libraries
(3 responses) zhao 2023-01-19

Suppose I have a raw data, named N1, which generates 300 sky files in 300 spectral libraries. How should I merge these 300 sky files? I try import-document and set it to merge with existing results by duplicate name, but the new results after import have no peak. I want to ask how to merge the sky files of different spectral libraries.
best wishes.

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Skyline not opening
(1 response) henry 2023-01-19


We have user whose skyline app continues to crash. When She attempts to open it, she gets the attached error message telling her to check the log in Event View, also attached. I've already uninstalled and reinstalled the program and uninstalled windows updates.

 Skyline error.rar 
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Raw CCS values and Overriding with Explicit IM Values
(2 responses) Premy 2023-01-05

Thanks so much for all the developments in Skyline. This tool has been an amazing support for my DDA and diaPASEF data!

I'm currently working with DDA-PASEF data (Bruker timsTOF) to build an ion mobility library for a set of lipid standards. I had a few questions regarding the 1/K0 and CCS values presented in the report.

  1. Is there a way to get the raw CCS values (in case there are slight shifts in CCS between replicates)?
  2. In the case that the IM extraction window needs to be corrected by overriding with "Explicit Ion Mobility Value", is there a way to also recalculate the CCS value in the report? It currently still reports the original CCS value and the newly corrected 1/K0 value.

I hope this makes sense and thank you in advance!


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Windows 10 22H2 and Windows 11 22H2 Compatibility
(1 response) taperez 2023-01-11


Im trying to find out if the newest skyline upgrade is compatible with windows 10 22H2 and/or windows 11 22H2?



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Import FASTA file
(7 responses) prajita 2022-12-19


Can you please let me know to import FASTA file is this the only way:
Or is there any other way?

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Non-Canonical Amino Acid
(12 responses) lulmer 2022-10-24

I have a unique use case working with the non-canonical amino acid, benzoylphenelaylanine, and I'm having trouble importing my results into skyline. I'm trying to import comet/peptideprophet search results that have the benzoylphenelaylanine non-canonical amino acid defined (attached interact.pep.xml). When I import these results there is a suggested modification that makes the peptide with the non-canonical amino acid viewable in skyline (attached jpegs 1 and 2 described the modification). However, when I try to reopen the skyline document with that modification I get the error "Variable modifications must specify amino acid or terminus" (attached error.jpeg). I've attached the skyline document that gives this error as well. Any suggestions on how to make these comet/peptideprophet search results with the non-canonical amino acid compatible with skyline would be greatly appreciated.

Lindsey Ulmer
 suggestedmod_01.JPG  suggestedmod_settings_02.JPG  210727_hspb5_b9_nouv_t_mon_interact.pep.xml  error.JPG 
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Skyline Batch error if decimal symbol is a comma
(6 responses) roman sakson 2023-01-06

Hello Skyline team,

I would like to report an error issue that might not have been mentioned here yet.

I am working with Skyline Batch, where I shared a bcfg file and tried to import it back into Skyline Batch on a different computer. Both systems were using Skyline Batch While trying to import the bcfg file I got an error complaining that the version of my file is newer than the version of the program, which was, of course, wrong (see screenshot).

Since the version number look weird with a comma in the error message, I checked the windows 10 region settings and realized that the decimal symbol on my German system was set as comma and the digit grouping symbol was set as dot. Reversing this in thw windows settings resolved the issue. I am not sure, whether something can or should be done here but I thought I better report this.

Thank you for this great software,

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Selecting Prosit in Library Match window results in unexpected error
(2 responses) tmaile 2023-01-04

Hello Skyline team,

I accidentally clicked on Prosit in the Library Match window which lead to the window blanking out and becoming completely unresponsive, showing the message "Prosit is not properly configured".
Right clicking on the blank window resulted in an unexpected error report (submitted):

Object reference not set to an instance of an object.

Source code location:
System.NullReferenceException: Object reference not set to an instance of an object.
at pwiz.Skyline.Controls.Graphs.GraphSpectrum.get_SpectrumInfo() in C:\proj\pwiz\pwiz_tools\Skyline\Controls\Graphs\GraphSpectrum.cs:line 1466
at pwiz.Skyline.SkylineWindow.pwiz.Skyline.Controls.Graphs.GraphSpectrum.IStateProvider.BuildSpectrumMenu(Boolean isProteomic, ZedGraphControl zedGraphControl, ContextMenuStrip menuStrip) in C:\proj\pwiz\pwiz_tools\Skyline\SkylineGraphs.cs:line 1159
at ZedGraph.ZedGraphControl.contextMenuStrip1_Opening(Object sender, CancelEventArgs e)
at System.Windows.Forms.ToolStripDropDown.OnOpening(CancelEventArgs e)
at System.Windows.Forms.ToolStripDropDown.SetVisibleCore(Boolean visible)
at System.Windows.Forms.ToolStripDropDown.Show(Control control, Point position)
at System.Windows.Forms.ContextMenuStrip.ShowInternal(Control source, Point location, Boolean isKeyboardActivated)
at System.Windows.Forms.Control.WmContextMenu(Message& m, Control sourceControl)
at System.Windows.Forms.Control.WndProc(Message& m)
at System.Windows.Forms.UserControl.WndProc(Message& m)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
Exception caught at:
at System.Windows.Forms.Application.ThreadContext.OnThreadException(Exception t)
at System.Windows.Forms.Control.WndProcException(Exception e)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
at System.Windows.Forms.UnsafeNativeMethods.CallWindowProc(IntPtr wndProc, IntPtr hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
at System.Windows.Forms.UnsafeNativeMethods.CallWindowProc(IntPtr wndProc, IntPtr hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
at System.Windows.Forms.NativeWindow.DefWndProc(Message& m)
at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
at System.Windows.Forms.Control.WndProc(Message& m)
at System.Windows.Forms.UserControl.WndProc(Message& m)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
at pwiz.Skyline.Program.Main(String[] args) in C:\proj\pwiz\pwiz_tools\Skyline\Program.cs:line 312

Reloading the Skyline file did not fix the issue, I can see my spectra fine via View>Spectral_Libraries. I imported my library into the Targets list via the Spectral Library Explorer, but this also did not have any effect on the Library match window staying blank.
It seems like the window is stuck for now. Is there a workaround for this?


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Issue in Spectral Library building from Timstof pro data (dda .d files)
(1 response) suresh choudhary 2023-01-05

Dear Skyline team,

I am trying to build spectral library from timsDIA PASEF data. I have 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 and DIA 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. Can some one help me to get the solution of it.

 Screenshot 2023-01-05 183838.png  Screenshot 2023-01-05 184104.png 
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Skyline for crosslinked peptides MSe Waters data analysis
(1 response) floriana capuano28104 2023-01-05


I have found a previous post suggesting that Apex3D pepxml files can be uploaded in Skyline. I am planning to use skyline to identify two crosslinked peptides and I would like to use the output of UNIFI data search to build an ion library and gain confidence in peak picking. I understand from previous posts that it is possible to use PLGS csv output, however at this stage I would not know how to add additional modifications in PLGS. I can see that UNIFI allows to export results in mzml and mgf format. Is there any chance I could directly use these to built the ion library? UNIFI is doing a targeted analysis to match spectra to peptides from one or two proteins added to the processing method. Would this have an impact on dotp or confidence in peak picking? Many thanks, Floriana

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Opening DIA for small molecules ((bbCID (MS/MS) and MS)) from Bruker impact II
(1 response) mohamed kaddah 2023-01-03

I am trying to open bbCID (MS/MS) and MS from Bruker, it is a type of DIA. I applied this method for degradation of pharmaceutical compound (small molecule). What is the procedure, how I can insert the range not a list of masses?

Can I inspect the data manually? is the step seeing the precursor and products overlayed available? can I use the software to extract specific ions during by inspection?

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Modifications for MSGF+ search within Skyline; chemical formulas don't seem to be accepted
kvancott2 2023-01-01

I am working with v22.2.0.351 and trying to run a MSGF+ search.

However, when running the searches, I got multiple errors related to my digest and modification settings.

Digestion: I had made a customized trypsin with proline digestion, and that was not recognized. I had to revert back to the default Trypsin digest setting (no cut when Pro follows KR). Once I did that, that error went away. My customized digest setting was "-1", which was out of the expected range of 0-9.

Modifications: I got multiple errors for modifications that were defined by their chemical formula in my Peptide Settings. It did not like "H3N" that was defined for pyroGluQ. It did not like "H2O" defined for pyroGluE. It also did not like "O - HN" that was defined for deamidation. I tried clearing all modifications from my Peptide Settings, and re-entering all the default modifications that had these chemical formula definitions, but that did not help. I ended up deleting all the chemical formulas from the modifications, just leaving the numerical values in there, and now the searches run as expected.

Below is the error information I got for the deamidation and pyroGluQ chemical formula instances:

MS-GF+ Release (v2021.09.06) (06 September 2021)
Java 17.0.1 (Oracle Corporation)
Windows 10 (amd64, version 10.0)
java.lang.NumberFormatException: For input string: "O - HN"
at java.base/jdk.internal.math.FloatingDecimal.readJavaFormatString(
at java.base/jdk.internal.math.FloatingDecimal.parseDouble(
at java.base/java.lang.Double.parseDouble(
at edu.ucsd.msjava.msutil.AminoAcidSet.parseConfigEntry(
at edu.ucsd.msjava.msutil.AminoAcidSet.getAminoAcidSetFromModFile(
at edu.ucsd.msjava.msdbsearch.SearchParams.parse(
at edu.ucsd.msjava.ui.MSGFPlus.runMSGFPlus(
at edu.ucsd.msjava.ui.MSGFPlus.main(
Search canceled.

MS-GF+ Release (v2021.09.06) (06 September 2021)
Java 17.0.1 (Oracle Corporation)
Windows 10 (amd64, version 10.0)
java.lang.NumberFormatException: For input string: "-H3N"
at java.base/jdk.internal.math.FloatingDecimal.readJavaFormatString(
at java.base/jdk.internal.math.FloatingDecimal.parseDouble(
at java.base/java.lang.Double.parseDouble(
at edu.ucsd.msjava.msutil.AminoAcidSet.parseConfigEntry(
at edu.ucsd.msjava.msutil.AminoAcidSet.getAminoAcidSetFromModFile(
at edu.ucsd.msjava.msdbsearch.SearchParams.parse(
at edu.ucsd.msjava.ui.MSGFPlus.runMSGFPlus(
at edu.ucsd.msjava.ui.MSGFPlus.main(
Search canceled.

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Using R script in Skyline Batch
(14 responses) schen19 2021-08-27

Dear Skyline Team,

I was just trying to set up a configuration for Skyline Batch. I added the path to the R script in "R script file path" then clicked "OK". However, I did not see the path appears afterwards. I have attached some screenshots to illustrate the problem.

Am I missing some steps? Is there anything I can do to fix this problem?

Thank you in advance.

 Rscirpt path.jpg  Configuration.png 
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Calculation of asymmetry factor and tailing factor of chromatographic peaks
(2 responses) Juraj_Lenco 2019-08-06
Hi, it would be great, and I guess appreciated by many users, if Skyline could determine asymmetry factor and tailing factor of chromatographic peaks. Skyline can determine peak widths at 50% intensity or at baseline so far. Unfortunately, the width at 50% intensity is not sufficient for assessing chromatographic peaks quality. The peak width at baseline is almost never used in LC-based analytical chemistry since it is challenging to determine real peaks boundaries . The peak width at baseline should be thus replaced by asymmetry factor and tailing factor that are very popular methods of measuring peak shape in chromatography.
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Feature request: color dotP labels based-on value
(1 response) gabe 2020-04-06

We analyze datasets with many samples (typically 96) and rely critically on the dotP values displayed above each Peak-Area bar. Depending on the monitor and resolution, sometimes the dotP text is so small that it's very difficult to read. It would be helpful if these values could optionally be colored based on the value -- something like >0.9 green, between 0.7 and 0.9 orange, below 0.7 red. Ideally these would be user-configurable.


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Document Grid: Molecules missing Predicted Retention Time and/or Average Measured Retention Time when using a Spectral library
(11 responses) jtsorren 2022-12-13


As the titled states I am using skyline-daily in molecule mode. When I add all of my molecules from a spectral library from the Spectral library Explorer I find that if I navigate to View -> Document Grid -> Reports -> Molecules the columns with either Predicted Retention Time or Average Measured Retention Time contain #N/A.

I am wondering if one of the columns should have picked up the molecule retention time given that the spectral library contains scans at explicit retention times. Maybe there is a setting I am not correctly using...

Also I am wondering if this is the cause of some incorrectly selected peaks for precursor that fall outside of the observed retention time seen in my spectral library for some molecules.

Thank you!

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Internal standard, relative response factor and quantification
(1 response) naymin saw 2022-12-20

Hi Skyline community,

I am using Skyline for small molecules quantification.
For the quantification of some metabolites, I don't have standard but I have two internal standards in the samples and relative response factors of my interest metabolites.
Is it possible to implement these data in Skyline workflow for the quantification of large-scale metabolites?
I have not found the documents in Skyline and please advise me.

Thanks and regards,
Nay Min

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An error occurred trying to download Skyline
(3 responses) amahan 2022-12-19

An error occurred trying to download ''.
See the setup log file located at 'C:\Users\amahan\AppData\Local\Temp\VSDF115.tmp\install.log' for more information.

when I enter "" directly in browser I get xml code

<?xml version="1.0" encoding="utf-8"?>
<asmv1:assembly xsi:schemaLocation="urn:schemas-microsoft-com:asm.v1 assembly.adaptive.xsd" manifestVersion="1.0" xmlns:asmv1="urn:schemas-microsoft-com:asm.v1" xmlns="urn:schemas-microsoft-com:asm.v2" xmlns:asmv2="urn:schemas-microsoft-com:asm.v2" xmlns:xrml="urn:mpeg:mpeg21:2003:01-REL-R-NS" xmlns:xsi="" xmlns:asmv3="urn:schemas-microsoft-com:asm.v3" xmlns:dsig="" xmlns:co.v1="urn:schemas-microsoft-com:clickonce.v1" xmlns:co.v2="urn:schemas-microsoft-com:clickonce.v2">
<assemblyIdentity name="Skyline.application" version="" publicKeyToken="e4141a2a22107248" language="neutral" processorArchitecture="msil" xmlns="urn:schemas-microsoft-com:asm.v1" />
<description asmv2:publisher="Skyline" asmv2:product="Skyline" asmv2:supportUrl="" xmlns="urn:schemas-microsoft-com:asm.v1" />
<deployment install="true">
<deploymentProvider codebase="" />
<dependentAssembly dependencyType="install" codebase="Application Files\Skyline_22_2_0_312\Skyline.exe.manifest" size="142364">
<assemblyIdentity name="Skyline.exe" version="" publicKeyToken="e4141a2a22107248" language="neutral" processorArchitecture="msil" type="win32" />
<dsig:Transform Algorithm="urn:schemas-microsoft-com:HashTransforms.Identity" />
<dsig:DigestMethod Algorithm="" />
<compatibleFrameworks xmlns="urn:schemas-microsoft-com:clickonce.v2">
<framework targetVersion="4.7.2" profile="Full" supportedRuntime="4.0.30319" />

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An error occurred trying to download ''.
(2 responses) kguehrs 2019-01-22

I tried to install Skyline on my computer running Windows7 64bit. The installation did not start giving the error message you can find in the attached screenshot together with the log file the message is referring to. Can you give me any advice to bypass the error and install the new Skyline version. Is there any possibility that the error is related to settings of the locales or regions because I run the English keybord and locales on the computer my IT installed a German version of Windows 7 on. If not I will keep the actual version 4.1 which is currently working without any problem.

I looked into the support issues list but I did not find any request with the same error description.

Thanks for your assistance.

 190122_installation_error.png  install.log 
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XIC overlay
(2 responses) sstoychev23513 2022-12-16

Hey guys,

Is it possible to overlay the XIC of two samples/runs in Skyline of have to export the data?


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AutoQC-no updating of raw files
(1 response) danielacgranato 2022-12-16

We have just installed AutoQC to monitor our System suitability runs, but although we were able to generate a file with 3 runs in Panorama, it does not update the new runs added to the folder. It does not move forward from "waiting for files". Could you help us troubleshoot this issue? I have attached the skyline file document (.zip) were using and the error log information. Please let us know if you need other files. Thank you very much. Best, Daniela

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Generating GC-MS "transition list" from .msp
(9 responses) brynnsundberg 2022-12-14

Hello Skyline team!

I have looked at the user instructions to view GC-MS data in Skyline, but I'm stuck without an initial transition list. The dream is to import my NIST .msp file and to be able to generate "transitions" from the 1710 small molecule spectra it contains to find them in my GC-MS results (using the strategy of selecting DIA in the MS2 transition settings and using a fake precursor mass). Unfortunately, the library explorer doesn't recognize the spectra in my .msp file, maybe because it's missing precursor mass and charge information. Is there a way to make this work? Do I have to learn how to code so I can pull the information from the .msp file and create the transition list I need? Any help you can offer will be greatly appreciated!


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files with dual polarity
(7 responses) naymin saw 2022-12-09

Hi Skyline community,
I am new to Skyline and I found some issues with dual polarities. My .raw files are positive and negative polarities in one file. When I prepare the standards, QC and blank samples in positive mode, it worked well.
However, when running in negative mode, there is no chromatogram found.
Could you please advise how to work with these dual polarities .raw files? Or please check the transition lists I have prepared and let me know if anything is not correct.

Many thanks,
Nay Min

 Transition list Pos.PNG  Transition list neg.PNG 
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Keyboard Shortcuts
(14 responses) thomlau 2011-09-16
Hi all,

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


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Spectronaut assay library import failed
(9 responses) Rita 2022-12-08


I tried to import a large Spectronaut Mouse library - MouseRefSWATH_iRT.csv by Krasny, L. et al 2020 ( Skyline was able to read .csv and check the file for errors but the process ended up with an error message (please see the attachment). I have Skyline running on 64-bit Win10 Pro 128 GB RAM. Would you have any suggestions?


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Negative transitions (and precursor) of a peptide
(5 responses) dilip singh 2022-12-12

Hi Skyline Team,

I am working on project in which inactive protein undergoes PTM (carboxylation) and leads to active protein (carboxylation at gamma position of glutamate (Glu) residues). I am not getting good response in positive ionization mode and I want to try in negative ionization mode.

Is it possible to have the negative transitions (and precursor) of a peptide in skyline? if I generate data in negative mode, Is skyline able to read that data?

Kindly suggest.


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DDA search hangs on "Running percolater..." with a specific FASTA file
(8 responses) Remco van Soest 2022-09-20

Skyline (64 bit) (c99206313)

When using the "Import DDA Peptide Search" workflow Skyline hangs on "Running percolator..." after the search has successfully finished. I have to manually end the "percolator.exe" task in order to make the software respond again.

The issue seems related to the FASTA file (attached; downloaded from NCBI; sequence is correct). If I manually remove line 11 and 12 everything works just fine. It is not the length, as everything works fine as well if I replace line 11 and 12 with A's.

Why does the percolator task hang when using a specific FASTA file? I don't see anything wrong with the file, and obviously want to be able to use the full sequence for my search.


 sequence (2).fasta 
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Skyline on apple IOS
New to Skyline 2022-12-08


As i cannot use the Prosit server on the Skyline software on Trust PC, i thought i could access it through my Laptop, however i am unable to do this on my laptop, is this due it being an Apple IOS and not on Windows? is there a way to overcome this?

Kind regards.

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MGF files with product ion charges are ignored during spectral library build
(14 responses) jtsorren 2022-12-01


I am using skyline-daily in molecule mode and using the built in feature to build a spectral library from a .ssl file and mgf files.

In mgf files you may now explicitly denote the product ion charge for measured peaks in individual scans. I am wondering why when I build the spectral library and add the molecules with transitions to the target space that the product ions are all assumed to have 1+ charge.

Thank you 
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Error message: Failure opening - "the file contains an error on line 13438 at column 9"
(6 responses) katherine wongtrakul-kish 2022-12-06

Hoping you can help me get back into my skyline file please... I added a new molecule to my transition list recently ( (HexNAc)2 (Deoxyhexose)1 (NeuAc)1 + (Man)3(GlcNAc)2 with m/z 876.8) by copying and modifying an existing one.

Usually this works alright if I'm copying and creating multiple entries for isomers, but on this occasion I modified the mass as well to make an entirely new glycan molecule -- which was perhaps where my mistake was?!

I managed to integrate peaks for the new glycan, but then at some point hit a clipboard error. When I closed and tried to re-open the file, I couldn't (error message in the attached screen shot).

I've also attached the SKYL file. Thanks for any advice you can give
 Total Ascites_EpCAM+_CD45_NG.skyl  skyline error 5Dec.PNG 
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SureQuant with two FAIMS CV voltages on Orbitrap Fusion Lumos - ZigZag shaped peaks on Skyline
(16 responses) aheinone 2022-08-11

Dear Skyline Team,

We are starting to do SureQuant on Orbitrap Fusion Lumos with FAIMS by using two CV voltages (-50 and -70). We are dealing in total with about 2000 peptides, but < 500 peptides at a time in one Skyline document. We have imported the raw files to Skyline for further processing. We have built Ion Mobility Libraries by using the results. Some peaks are drawn correctly, but in many cases product ion peaks are jagged (zigzag shaped in MS2 level). But still precursor peaks (MS1 level) look ok. It seems that this happens with CV -50 peaks. Probably Skyline takes data points from the both CV values for some peaks? We have tried many different settings for MS/MS filtering, but we haven't found any solution. We tested to change the CV values manually in the Ion Mobility library, but it doesn't solve the problem. And the CV values seem to be correct in the library. If we split the original raw files to two separate files according to the CV voltage, and then import them to Skyline, the peaks are fine. But if we build the Ion Mobility Library by using the two separately imported files, it didn't solve the problem. Is there any other way to handle multiple CV values than split the files according to the CV voltage. We would appreciate for any help with this, thanks.


view request
Waters Synapt G2-Si - multiple issues to access CCS values
(9 responses) nicolas macorps 2022-08-31
Dear Skyline support,

I have been trying to use Skyline to analyse data obtained with a Water Synapt G2-Si with ion mobility (IM) experiments. Experiments are performed with the HDMS mode (ion mobility, elution time and full scan MS only). The ion mobility technique is TWIMS that use a calibration curve (mob_cal.csv file in the sample root) that the proprietary software uses to correlate measured drift times to CCS values.
I want to use to have a solid workflow with this software to quantify PFAS (small targeted molecules) in various samples. To confirm that I am indeed looking at a PFAS, I need to access CCS values (which include the need to create an IM library) so I can then have it when exporting a "report'.

I encountered some issues when using Skyline features which is why I am opening this support feed. You can find uploaded in the "File Sharing Folder for Skyline Support" my folder under the name "". This file contains *.raw data from the Water Synapt, *.sky file (with the trageted list molecules in a *.csv in case it is needed) and snapshots of some error message I got along my tests.

1) Skyline crashing when importing *.raw files :
When trying to import Synapt *.raw files (220621008.raw and 220621009.raw in the *.zip folder), skyline crashes at the importation step (software closes without error message). This issue was encountered and reported in another support feed. The proposed solution in this feed was to convert *.raw into *.mzML files, but it seems that this approach doesn't allow for CCS values when creating IM library.

I went looking into the *.raw folders and found that when changing _FUNC002.DAT into _FUNC002.temp (in other words I "remove" this function from the *.raw file) I was now able to import without crashing the *.raw file and was able to obtain chromatograms with full scan spectrum and drift time (ms) distribution.
I initially thought that this "Function 2" contained "lockmass" data which can cause some issues when converting into *.mzML so I thought that removing this function could fix the problem. However I cannot be sure that this function is indeed the "lockmass" when looking in the "_extern.inf" file nor in the "_HEADER.TXT". The only information I have on these files are for the function 1 : "MOBILITY MS FUNCTION". So function 2 may also contain IM data from the HDMS experiments but I cannot be sure.

In any case, I kept going with modified *.raw files with the change of _FUNC002.temp (220621008_modify.raw and 220621009_modify.raw) to try to obtain CCS values

2) Negatives CCS values when creating IM library with "use results"

Now having something to work with, I tried creating the IM library for my molecules. I was happy to see that I could obtain some CCS values by doing so. Unfortunatly they are all with a negative sign and as such completely incorrect. I had two thought on that :
- Conversion of drift time into CCS values using the mob_cal.csv file (as done by the proprietary software) is not performed in Skyline in this manner or Skyline needs another type of file to do so.
- We need the function 2 to be active to have CCS values

For the latter, I tried to reactivate the function 2 in the *.raw file and reimport them. Doing so led to no crash so I followed with the creation of an IM library. I ended up with an error message "Failed using results to populate ion mobility library: [SpectrumList_Waters:spectrum()] Bad index: 18446744073709551615" (you can find the snapshot in the *.zip file and the extended error message in a *.TXT file in the *.zip).
Further trying with reimportation led to a crash of the software as mentionned in my first point.

I hope you have enough elements with this description to help me ?
- Do you know why Waters HDMS experiment *.raw files would make the software crash ?
- Do you know why I have negative CCS values when creating the IM library with the "use results" feature?

Let me know if you need anything else from me.

Best regards,
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Gas-phase-fractionation and iRT
(16 responses) Joerg 2022-11-18

I have a problem with iRT alignment with my gas-phase fractionation-SWATH-runs. As I am doing GPF for the SWATH-analyses some of the iRT peptides (Pierce C18) are fragmented only in fraction while others are fragmented in fraction 2 and so on, but none of the peptides are fragmented in the different gas-phase-fractions. I have full-MS-spectra for the entire precursor region in every run, though. Is there a possibility to align the runs according to the precursor signals or do I have to fragment each peptide in each run?
Many thanks!

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New to SkyLine- In silico
(11 responses) New to Skyline 2022-11-29

Hi All,

I am new to skyline. I am doing a research project in measuring a particular hormone. Is there a 101 document on how to use the Software, I have been through the tutorial (targeted method editing) but unable to replicate it for my project. We have the water Xevo. I wanted to do an in-silico for my peptide but dont know how to conduct it. Thank you for any support.

Kind regards,

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Results found 2 samples instead of 34 samples from the msms file
(5 responses) yael hirschberg 2022-12-02

Hello Skyline Team,

I ran 34 samples on Maxquant and tried to import the msms file on Skyline. However, Skyline only continued with 2 samples and 17 replicates of each. I attach a screenshort of the import and the msms file. What did I do wrong?

Thank you,

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timsTOF Scheduled PRM Data Import
(11 responses) robwsprung 2020-02-19

Dear Skyline Team,

Thank you for your continued development of an excellent software package.

I am having trouble importing scheduled PRM data from a timsTOF Pro instrument. When I look at the results in Compass DataAnalysis, it appears that the method worked as expected - MS1 signals for the expected isotope labeled peptides are observed and MS/MS spectra are acquired at the retention times of interest. However, when importing into Skyline Daily ( no precursor and no product ions are observed.

I've successfully imported other PRM runs from the instrument. Only this scheduled method is causing problems. Any insight you could provide would be appreciated. Thanks.


 timsTOF MHC  timsTOF MHC settings.pptx 
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Prosit Server Unavailable with Skyline Latest Version
(1 response) cm748 2022-12-01

I have just updated Skyline to the most recent version and am getting the "Server Unavailable" error with Prosit (attached image), as are my colleagues so I don't think it's a problem my end with connection etc. Could you advise?

Best wishes,

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Enhancement Requests: Filter a Range of Fold Changes and Multi-Proteome Homologous Peptide Filtering
philip remes 2022-11-30


I had an idea for an enhancement request. There is a workaround involving highlighting potentially 100's of lines and deleting them, it just isn't as nice. I created a targeted assay in a two-proteome mixture, and before performing a dilution curve that would take a few days, wanted to confirm with a quick experiment that the peptides chosen were from one proteome and not the other. Two replicates were injected and imported at each of two concentrations, and a Group comparison performed between the concentrations. The sorted Bar plot does a good job to show which peptides do not exhibit the expected fold change. Using Refine / Advanced / Group Comparison and entering a Fold change value appears to be useful in the case where one is interested in peptides that change by more than some ratio, up or down. But in this case, I am interested in accepting peptides with a range of acceptable fold change values, for example between 0.5 and 1.5, and rejecting the others. Does that make sense? Something you could add to your queue of new features?

A related feature request for these types of two-proteome experiments is the ability to filter out peptides that are homologous between organisms. This comes up also when using a carrier proteome: one wants to make sure not to pick peptides that could possibly exist in the carrier proteome. One could imagine a filter tool that allows to enter a path to another fasta file, and to filter out any peptides in common between the current document fasta and that one.


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BS3: how to insert in two peptides, which are probably cross-linked, two different site of BS3 modification?
(1 response) francesca grassiscalvini 2022-11-30


I would like to verify an option of cross linking between two peptides:

ATAQDNPKSATEQSGTGI (with BS3 linked on K) and SYCR (with BS3 linked on S which is the amino-term)

Is it possible in the panel modify, clicking on the sequence of one of the two peptide, insert two different site of modification for the two peptides?

or is it possible to insert my modification with K as a position and also C term, in the same modification? I know that is possible but then the program search for K only in the position C term and in the peptide ATAQDNPKSATEQSGTGI it's not on the c term

many thanks

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