support

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.  

In order to post to the forum, you'll need to sign-in or if you don't yet have an account sign up. Forgot your password? You can reset it using the "(forgot password)" link on the sign-in page.

You can also follow the Skyline support board through email updates after you sign up.

When you post a question, please include the following information:

  • A detailed description of your problem or question, including instructions for re-creating any problem that you are encountering. Screenshots are often helpful.
  • Your operating system, and the version of the software that you are using.
  • Any other information that may help us to answer your question, including whether you are working with proteomics or small molecule data.

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

If you are including a Skyline document, please use Skyline's File | Share menu item (choose "Complete" if asked), which prepares a single zip file with your document and all the needed supporting files in it. Then upload that .sky.zip file to the Uploads page. If the actual raw data files are needed to illustrate a problem, those will need to be zipped up and uploaded separately.
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Showing: limited to 100 requests
Refining CE optimizations with smaller step size
(1 response) rschoenh 2024-06-17 04:44

Dear Skyline Team,

This is Regine from the Paulovich lab with another question about CE optimization. As part of our CE optimization process, we do a preliminary CE optimization run using a 3V step size. We then want to do a refinement run with a 1V step size. In the Transition Settings, we checked the “Use optimization values when present” box and we have “Optimize by: Transitions” chosen. However, we noticed that the csv file that we export for the refinement run does not have the center CE value corresponding to the best CE value that was found in the 3V data for each transition. Instead, the resulting csv has the same center value defined for each transition, despite the fact that the center value is quite different for some of the transitions. How can we force it to use the previous, 3V data to choose a best CE value as the center CE value for the 1V step size optimization? (We've been manually adjusting the csv files so far before running our refinement mass spec run.)

I'm attaching a couple of files that show this problem.

Thanks very much in advance for your help!

~Regine

 3_HWAP_8_LDHB_L_EDRN2_R2.sky_for_Skyline_troubleshooting_RMS.zip  3_HWAP_8_LDHB_L_EDRN2_R2_test.csv 
view request
Skyline software information
(2 responses) eo03nb 2024-06-14 04:35

Hello,

I need to provide information to my institutions IT services in order to download Skyline.

Please may you provide me with the following information:

  1. Software terms and conditions URL
  2. Software privacy policy URL
  3. Software accessibility information URL
  4. Cyber Security information - for example Cyber Essentials or ISO27001/ISO27002 series and if these do not exist then a copy of the application development history,

Thank you,
Nicole

view request
.pdResult File Import Issue
(4 responses) wes rogers 2024-06-14 14:12

Hi Skyline Team,

When building a library from a .pdResult file, I have noticed a discrepancy between the number of “high confidence” peptides found in PD and the number of peptides imported in. When using a Percolator q-value of 0.01 or 0.05, the number of peptides imported into library is often a fraction of the “high confidence” peptides found by CHIMERYS. I am using Skyline daily 23.1.1.520 and Proteome Discoverer 3.1 with CHIMERYS searches primarily. I have attached a few examples. I would be happy to provide additional info or upload the .pdResult files. Do you mind taking a look at this?

Thank you,

Wes

 .pdResult File Import Issue.pdf 
view request
Partly failed import of MRM3 data
(7 responses) h l elfrink 2024-05-02 04:13

Dear Nick, Brendan and/or colleagues from Skyline support,

To satisfy a prerequisite of a journal, reviewer and to attempt to comply with FAIR data principles, we are trying to upload MRM3 data to Panorama. I have previously asked for help to upload the data making a method, we have found a work-around and have succesfully uploaded data to Skyline in a method.

Unfortunately, a substantial part of the data-files failed to upload, while others do. Uploading the 'faulty' data provides this error message:

At 10:05:
Failed importing results file 'C:\Users\Hyung\Surfdrive\Projects\2020\P20-0001_COVID-19\Data\20240425_Exp134_RepositoryUpload\220503_Exp111_batch02\renamed_files\220503_019_MRM3_30089A1_10uL.wiff2'.
[WiffFile2Impl::getInstrumentSerialNumber()] Object reference not set to an instance of an object.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\Hyung\Surfdrive\Projects\2020\P20-0001_COVID-19\Data\20240425_Exp134_RepositoryUpload\220503_Exp111_batch02\renamed_files\220503_019_MRM3_30089A1_10uL.wiff2'.
[WiffFile2Impl::getInstrumentSerialNumber()] Object reference not set to an instance of an object. ---> System.Exception: [WiffFile2Impl::getInstrumentSerialNumber()] Object reference not set to an instance of an object.
at pwiz.CLI.msdata.ReaderList.read(String 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\pwiz\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 200
at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 295
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 191
--- End of inner exception stack trace ---

It seems to me that the built-in msconvert module is throwing an error, pertaining the serial number of the MS. I checked the original Sciex data file that fails in Sciex OS (version 2.1.6.59781), and it does not seem to be corrupted, at least it opens normally. The MS data and metadata (serial number in the 'sample info') are also retrievable using Sciex OS (explorer module).

I also tried to convert the data in MS convert (version: 3.0.24094-d2966db), but I am not able to execute it properly. It also returns an error ([WiffFile2Impl::setSample()] Value cannot be null.). I think my colleague has previously reported this. I also tried to look in previous reported Skyline issues if I could find an answer to my question, but I was unable to find a solution for this problem.

Back to the Skyline upload: Is there a solution to upload all of our files?

Best regards,

Hyung Elfrink
Leiden University, Metabolomics and Analytics Centre.

P.S. I have attached my method file and I can share the data files per FTP.
P.P.S This is a link to the data that succeeded to upload, to Skyline and subsequently Panorama: https://panoramaweb.org/Leiden University - MAC/COVID-19 Mass Spectrometric MRM3 repository/targetedms-showPrecursorList.view?id=204275

 Skyline.rar 
view request
Export dot p of library spectrum vs midas data
(5 responses) heyang 2024-05-30 10:40

Hi,

Could anyone tell us how to export dot p value after compare library std spectrum vs midas data. We can find this value which present in spectra match (see attached). But can't find a way to export these values.

Thanks,
Heyi

 dotP value of library spectra vs midas.png 
view request
About some transitions not being detected
(4 responses) riri riri h 25 2024-06-09 21:49

When the measurement file (.lcd) obtained from the Lab solution was imported into skyline, peaks were detected only for some transitions (with green marks), but not for others.
However, on the lab solution, all transitions showed peaks.
Any solutions would be appreciated.

The version of the software : 64-bit Skyline 23.1
Measurement and analysis method : proteomics data

Below is a screenshot of the skyline screen.

 About some transitions not being detected.png 
view request
PD results does not work for spectral library
(11 responses) jueun c kim 2024-06-11 11:54
Hi Skyline Team,

I used skyline-daily to import PD.result files or mzML files from Proteome discoverer to build library on Skyline -Daily, but I keep getting an error message. Please help. Here is my workflow:

Open and save Skyline Daily --> Import FASTA of my protein --> Settings --> Library --> Built --> Data Source "Files" --> Input Files "files.pdResults" OR also Tried "files.mzML" which I learned is not a valid file . For Files.pdResults, the error says MassSpectrumItems table.

Please Help. Below is the error message I get when trying to import the spectra from PD to Skyline.
---------------------------
Skyline-daily
---------------------------
ERROR: This file does not contain q-values. You can set a cut-off score of 0 in order to build a library from it, but this may cause your library to include a lot of false-positives.
ERROR: reading file NfL_DDA_Combined.pdResult
ERROR: no such table: MassSpectrumItems

Command-line: C:\Users\jueun.c.kim\AppData\Local\Apps\2.0\XO3CK2OC.ECG\X7QOLBOA.ELX\skyl..tion_9286511f3362df93_0017.0001_479d187256242aac\BlibBuild -s -A -H -v warn -o -c 0.95 -i NfL_PD_Spectral_20240611 -S "Z:\Jueun Kim\NfL\PD and Skyline\Spectral Library\NfL_PD_Spectral_20240611.redundant202406111144.stdin.txt" "Z:\Jueun Kim\NfL\PD and Skyline\Spectral Library\NfL_PD_Spectral_20240611.redundant.blib"
Working directory: Z:\Jueun Kim\NfL\PD and Skyline\Spectral Library
---------------------------
OK More Info
---------------------------
Skyline-daily (64-bit) 23.1.1.520 (80747de92)

System.IO.IOException: ERROR: This file does not contain q-values. You can set a cut-off score of 0 in order to build a library from it, but this may cause your library to include a lot of false-positives.
ERROR: reading file NfL_DDA_Combined.pdResult
ERROR: no such table: MassSpectrumItems

Command-line: C:\Users\jueun.c.kim\AppData\Local\Apps\2.0\XO3CK2OC.ECG\X7QOLBOA.ELX\skyl..tion_9286511f3362df93_0017.0001_479d187256242aac\BlibBuild -s -A -H -v warn -o -c 0.95 -i NfL_PD_Spectral_20240611 -S "Z:\Jueun Kim\NfL\PD and Skyline\Spectral Library\NfL_PD_Spectral_20240611.redundant202406111144.stdin.txt" "Z:\Jueun Kim\NfL\PD and Skyline\Spectral Library\NfL_PD_Spectral_20240611.redundant.blib"
Working directory: Z:\Jueun Kim\NfL\PD and Skyline\Spectral Library ---> System.IO.IOException: ERROR: This file does not contain q-values. You can set a cut-off score of 0 in order to build a library from it, but this may cause your library to include a lot of false-positives.
ERROR: reading file NfL_DDA_Combined.pdResult
ERROR: no such table: MassSpectrumItems

Command-line: C:\Users\jueun.c.kim\AppData\Local\Apps\2.0\XO3CK2OC.ECG\X7QOLBOA.ELX\skyl..tion_9286511f3362df93_0017.0001_479d187256242aac\BlibBuild -s -A -H -v warn -o -c 0.95 -i NfL_PD_Spectral_20240611 -S "Z:\Jueun Kim\NfL\PD and Skyline\Spectral Library\NfL_PD_Spectral_20240611.redundant202406111144.stdin.txt" "Z:\Jueun Kim\NfL\PD and Skyline\Spectral Library\NfL_PD_Spectral_20240611.redundant.blib"
Working directory: Z:\Jueun Kim\NfL\PD and Skyline\Spectral Library

Output:
Reading results from NfL_DDA_Combined.pdResult.
ERROR: This file does not contain q-values. You can set a cut-off score of 0 in order to build a library from it, but this may cause your library to include a lot of false-positives.
ERROR: reading file NfL_DDA_Combined.pdResult
Reading results from NfL_DDA_Oxi_0530_Labelled.pdResult.
ERROR: no such table: MassSpectrumItems
100%

 ---> System.IO.IOException: ERROR: This file does not contain q-values. You can set a cut-off score of 0 in order to build a library from it, but this may cause your library to include a lot of false-positives.
ERROR: reading file NfL_DDA_Combined.pdResult
ERROR: no such table: MassSpectrumItems

Command-line: C:\Users\jueun.c.kim\AppData\Local\Apps\2.0\XO3CK2OC.ECG\X7QOLBOA.ELX\skyl..tion_9286511f3362df93_0017.0001_479d187256242aac\BlibBuild -s -A -H -v warn -o -c 0.95 -i NfL_PD_Spectral_20240611 -S "Z:\Jueun Kim\NfL\PD and Skyline\Spectral Library\NfL_PD_Spectral_20240611.redundant202406111144.stdin.txt" "Z:\Jueun Kim\NfL\PD and Skyline\Spectral Library\NfL_PD_Spectral_20240611.redundant.blib"
Working directory: Z:\Jueun Kim\NfL\PD and Skyline\Spectral Library
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass, Boolean forceTempfilesCleanup, Func`3 outputAndExitCodeAreGoodFunc, Boolean updateProgressPercentage) in C:\proj\pwiz\pwiz_tools\Shared\CommonUtil\SystemUtil\ProcessRunner.cs:line 203
   --- End of inner exception stack trace ---
   --- End of inner exception stack trace ---
   at pwiz.Common.SystemUtil.ProcessRunner.ThrowExceptionWithOutput(Exception exception, String output) in C:\proj\pwiz\pwiz_tools\Shared\CommonUtil\SystemUtil\ProcessRunner.cs:line 263
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass, Boolean forceTempfilesCleanup, Func`3 outputAndExitCodeAreGoodFunc, Boolean updateProgressPercentage) in C:\proj\pwiz\pwiz_tools\Shared\CommonUtil\SystemUtil\ProcessRunner.cs:line 245
   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 445
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 163
---------------------------
view request
Bibliospec version 3 does not support my DIA-NN library file
(2 responses) bora onat 2024-06-12 11:00

Hello,

I'm trying to build a new library in Skyline using a DIA-NN predicted library output. I get the error, saying Bibliospec supports up to version 3, but my library file is version 8. How do I correct this issue? Thank you.

view request
Precurser Selection Small Molecules
(7 responses) matthew.r.russell 2024-06-12 02:01

Dear Skyline

I have been experimenting with small molecule data import into skyline daily 23.1.1.520 from a waters HDMSe experiment. I have m/z, charge state, RT and ion mobility information, but not molecule identifications. I would like to have multiple precursor ions imported. Although I appreciate skyline can't calculate isotope dot product with no formula I thought it would be able to import [M+1], [M+2] etc signals. Tweeking the transition settings for filter and Full-scan back and forward is not triggering these precursor inclusions on the list. Is it possible to set these precursor transitions up at all?

view request
Missing peak area measurement bars in Skyline Replicate plots when optimizing CEs on a 6500 Qtrap
(3 responses) rschoenh 2024-06-13 04:54

Good morning,

this is Regine in the Paulovich lab. We have been optimizing CEs for numerous peptides using a 6500 Qtrap and have seen that not all peak area measurements for the different voltage steps are being shown for some transitions on the Skyline Replicate plots, when viewed using the Single Transitions setting. We're attaching a file that shows this issue for the GTELWER peptide, for the light y5+ transition. Only 4 measurements are shown, whereas for the other transitions, all the voltage step measurements are shown. We've checked the raw data using the Qtrap's Analyst software, and the data are there, they are just not showing up in Skyline.
Would you please look into this issue so that all measurements show up in the Replicate plots? Thank you very much in advance!

Best,
Regine

 1_HWAP_8_LAMP1_NOTCH2NLA_HH_R3_for_Skyline_troubleshooting_RMS.sky.zip 
view request
Modifications on N and P with 13C and 15N in Peptide VINKQTPNRQIW
(1 response) teerapat rojsajjakul35372 2024-06-13 07:34

I have all amino acid labelled in peptide VINKQTPNRQIW with 13C and 15N. When I used Peptide Settings, modifications, N and P have the same mass, and can’t distinguish one to another in skyline and skyline doesn’t pick up the signals.

view request
How to add acetylation modification to the K-terminal in Skyline document?
(1 response) caixue 2024-06-13 01:11

I have identified several K-acetylated peptide sequences in the Fragpipe analysis of my data. I would like to see the peak groups and spectra of these peptide sequences in Skyline.

The peptide segment acetylated on the K-terminal is shown below:
ALAAGGYDVEK[42.0106]NNSR
GK[42.0106]YYAVNYPLR
IEGEGSVLQAK[42.0106]LK
ISIC[57.0215]SSDK[42.0106]R
NGLSLAALKK[42.0106]
NGLSLAALK[42.0106]K
n[42.0106]SETAPAETATPAPVEK[42.0106]SPAK

I insert the peptides as below:
ALAAGGYDVEKNNSR
GKYYAVNYPLR
IEGEGSVLQAKLK
ISICSSDKR
NGLSLAALKK
SETAPAETATPAPVEKSPAK

And I also set modifications in peptide setting as attached figure 1. However, the peptide in the document failed to display the acetylation marks, as illustrated in attached figure 2.
I don't know where the operation went wrong.
I look forward to your reply.
Best wishes.

 skyline_figure1.png  skyline_figure2.png 
view request
Request: Consistency between note importing and exporting
(2 responses) Chris Ashwood 2024-06-06 23:14

Dear Skyline Team,

I hope you've had a great ASMS. I've been working on my reports, and noticed that when I import a transition list, only one form of note is available, "Note". However, when I try to export the same note from the document, there are four different note types (transition, precursor, molecule, molecule list). Skyline seems to default that "Note" in the transition list import is a "Transition Note", and this cannot be changed in the transition list import stage. This becomes problematic when exporting MS1 data with isotopes because only the monoisotopic line in the export features the imported note.

Could the transition list import feature please be updated to include all existing note forms? I would like to say that a note is a precursor note, ensuring the note is specified across different isotopes coming from the same precursor.

Cheers,
Chris

view request
Modifications show same masses when rounded
(2 responses) hassan hijazi 2024-06-12 08:29

Dear Skyline team,

When having two modifications in the same Skyline document, and we choose to see modifications as mass difference, the mass difference will be the same for modifications of masses when rounded to first decimal digit will identical.

Example 1:

Formyl (K) [+27.994915] and Dimethyl (K) [+28.0313] will both appear as [+28] when choosing: View > Modifications > Mass Difference.

Example 2:
The same applies to trimethyl (K) [ +42.04695] and acetyl (K) [+42.010565] .

If I want to export 'Peptides' report from the Document Grid in the 'Peptide Modified Sequence' column, both can be seen as the same peptide if the 3-letter format was not chosen.

Could you please keep the rounding to the 2nd decimal digit?

Thank you,
Hassan

view request
Error importing TimsTOF data in mzXML format, exported from PEAKS
(1 response) julian matytchak 2024-06-11 02:29

Dear support team,

We are trying to build a spectral library from Bruker TimsTOF data, analyzed and exported as mzXML in PEAKS studio 11.

When adding data to a new spectral library in Skyline, the same error happens every time the data is being read from the mzXML file.
I attached a compressed folder where you can find the pep file, the exported mzXML file as well as a text document with the entire error code log.

Note that the data is from run 142 and the error code is from loading data from run 141, though the data is practically identical and the same issue occurs with both.

We are running Skyline (V23.1.1.520) as well as PEAKS Studio 11 on windows10.

Thank you in advance,

Julian Matytchak
Lab technician at the Temmerman lab
KU Leuven
Belgium

 DDA_run_142_with_Error.zip 
view request
Export hundreds of MS1 chromatrograms
(1 response) nesgio 2024-06-11 05:50

Hi guys,

I am trying to export all the chromatograms of the species detected with a contaminants template published by Rardin (https://link.springer.com/article/10.1007/s13361-018-1940-z) in a RAW sample acquired in full scan.
However, when I "Right Click --> Copy Data" on the chromatogram and I have only 100 intensities while the documenport the group name (i.e., protein.name) TIC for each contaminants - would it be possible? I ts contains more than 700 of "MS1 filtering". I would like to click on "Export --> Chromatogram --> Precursor" but I do not see "Retention Time" and "Intensity" column.

Many thanks and best regards,

Nesgio

view request
"chromatogram information unavailable"
(3 responses) luzalonsodasques 2024-06-10 08:31

Good afternoon,

I am writing you because I cannot view the chromatograms after importing the files.

The message that i see is: Chromatogram information unavailable.

I tried to change in settings -> Transition list -> instrument -> Method match tolerance m/z ... but it didn' t work.

I also tried in edit -> modify molecule -> Explicit values -> RT window ... but it didn't work

I am using Skyline (64-bit) 23.1.0.268 (e59d0dc56)

Waiting for your answer,

Thank you in advance,

Luz Alonso Dasques.

 Test_01.sky 
view request
Error code 203-CPUA
(1 response) chelec everest 2024-06-10 04:08

Hi,
We have Skyline oven mod.: ECOE61T2D0.
A few times coming error code 203-CPUA: ACS IC broken on ACU.

Could you pls give advice, what is problem?
The unit is working only 0ne year.

 20240609_085935.jpg 
view request
Unable to build PEPTIDE spectral library using .pep.xml and .mzxml files downloded together from PEAKS 11
(5 responses) ciaranmc 28 2024-06-05 11:51
Dear Skyline supporters,

I am attempting to build a spectral library using pep.xml and .mzXML files downsloded from PEAKS 11 (derived from DDA run on TimTOF, filtered for MS2 peptides at 1% FDR)

However, despite rerunning my peaks analysis to try again, I am repeatedly met with the skyline error included below (which suggest discrepancy between the pep.xml and mzXML files).

Both files were exported from PEAKS at the same time and from the same PEAKs database search (so they should be compatible).

Please let me know if you have any suggestions, thanks!

Cheers,
Ciaran

---------------------------
Skyline
---------------------------
ERROR: index 3128 out of range in mzXML file '.\C240501_C.elegans_Lib_100ng_17min_dda_141.d.mzxml'

Command-line: C:\Users\ciara\AppData\Local\Apps\2.0\7GXGT77K.ABK\AJO9DCLR.Z52\skyl..tion_9286511f3362df93_0017.0001_cdc87d1507aa7380\BlibBuild -s -A -H -v warn -o -i P -S "C:\Users\ciara\Dropbox\KU_Leuven\1_Prohormone_convertase_project\Skyline\2024.06.05_initial_spectral_library\Peptide_library_ran_seperately\Peptide_library_142\P.redundant202406050814.stdin.txt" "C:\Users\ciara\Dropbox\KU_Leuven\1_Prohormone_convertase_project\Skyline\2024.06.05_initial_spectral_library\Peptide_library_ran_seperately\Peptide_library_142\P.redundant.blib"
Working directory: C:\Users\ciara\Dropbox\KU_Leuven\1_Prohormone_convertase_project\Skyline\2024.06.05_initial_spectral_library\Peptide_library_ran_seperately\Peptide_library_141
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: index 3128 out of range in mzXML file '.\C240501_C.elegans_Lib_100ng_17min_dda_141.d.mzxml'

Command-line: C:\Users\ciara\AppData\Local\Apps\2.0\7GXGT77K.ABK\AJO9DCLR.Z52\skyl..tion_9286511f3362df93_0017.0001_cdc87d1507aa7380\BlibBuild -s -A -H -v warn -o -i P -S "C:\Users\ciara\Dropbox\KU_Leuven\1_Prohormone_convertase_project\Skyline\2024.06.05_initial_spectral_library\Peptide_library_ran_seperately\Peptide_library_142\P.redundant202406050814.stdin.txt" "C:\Users\ciara\Dropbox\KU_Leuven\1_Prohormone_convertase_project\Skyline\2024.06.05_initial_spectral_library\Peptide_library_ran_seperately\Peptide_library_142\P.redundant.blib"
Working directory: C:\Users\ciara\Dropbox\KU_Leuven\1_Prohormone_convertase_project\Skyline\2024.06.05_initial_spectral_library\Peptide_library_ran_seperately\Peptide_library_141
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass, Boolean forceTempfilesCleanup, Func`3 outputAndExitCodeAreGoodFunc, Boolean updateProgressPercentage) in C:\proj\skyline_23_1\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 181
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_23_1\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 433
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 161
---------------------------
view request
error importing SPEC-lib file ( Import DIA Peptide search)
(1 response) floor leurs 2024-06-06 00:39
When I select 'import DIA peptide search', I import a SPEC-LIB file that I created in DIANN. but then I get an error (it worked fine a few weeks ago).

It also makes a text file (which was never formed before): skyline.redundant202406060903.stdin.txt
The error says that the TSV report must be in the same directory (but it is...)

Note: it does work in the 'normal' skyline (but not in skyline daily)
 
Error:
Skyline-daily (64-bit) 23.1.1.520 (80747de92)
System.IO.IOException: ERROR: unable to determine DIA-NN report filename for 'library.TSV.speclib': the TSV report is required to read speclib files and must be in the same directory as the speclib and share some leading characters (e.g. somedata-tsv.speclib and somedata-report.tsv)

Command-line: C:\skyline.redundant202406060925.stdin.txt" "C:\skyline.redundant.blib"
Working directory: C:\---> System.IO.IOException: ERROR: unable to determine DIA-NN report filename for 'library.TSV.speclib': the TSV report is required to read speclib files and must be in the same directory as the speclib and share some leading characters (e.g. somedata-tsv.speclib and somedata-report.tsv)

Command-line: C:\4NPBM2VL.RVQ\JNTWPZ9V.1HO\skyl..tion_9286511f3362df93_0017.0001_479d187256242aac\BlibBuild -s -A -H -v warn -o -c 0.95 -i skyline -S "C:\skyline.redundant202406060925.stdin.txt" "C:\skyline.redundant.blib"
Working directory: C:\

Output:
Reading results from library.TSV.speclib.
ERROR: unable to determine DIA-NN report filename for 'library.TSV.speclib': the TSV report is required to read speclib files and must be in the same directory as the speclib and share some leading characters (e.g. somedata-tsv.speclib and somedata-report.tsv)
100%

 ---> System.IO.IOException: ERROR: unable to determine DIA-NN report filename for 'library.TSV.speclib': the TSV report is required to read speclib files and must be in the same directory as the speclib and share some leading characters (e.g. somedata-tsv.speclib and somedata-report.tsv)

Command-line: C:\4NPBM2VL.RVQ\JNTWPZ9V.1HO\skyl..tion_9286511f3362df93_0017.0001_479d187256242aac\BlibBuild -s -A -H -v warn -o -c 0.95 -i skyline -S "C:\skyline.redundant202406060925.stdin.txt" "C:\skyline.redundant.blib"
Working directory: C:\
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass, Boolean forceTempfilesCleanup, Func`3 outputAndExitCodeAreGoodFunc, Boolean updateProgressPercentage) in C:\proj\pwiz\pwiz_tools\Shared\CommonUtil\SystemUtil\ProcessRunner.cs:line 203
   --- End of inner exception stack trace ---
   --- End of inner exception stack trace ---
   at pwiz.Common.SystemUtil.ProcessRunner.ThrowExceptionWithOutput(Exception exception, String output) in C:\proj\pwiz\pwiz_tools\Shared\CommonUtil\SystemUtil\ProcessRunner.cs:line 263
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass, Boolean forceTempfilesCleanup, Func`3 outputAndExitCodeAreGoodFunc, Boolean updateProgressPercentage) in C:\proj\pwiz\pwiz_tools\Shared\CommonUtil\SystemUtil\ProcessRunner.cs:line 245
   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 445
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 163
view request
detection of too long peptide (34 residues)
(4 responses) wangrui2011pku 2024-06-03 18:34

I’m running a PRM of only one long peptide (~34 residues) with thermo orbitrap 480. Although pairs of ion fragment under charge+6 precusor, it's too difficult to get a high quality of PRM result as following attachment. Because there is almost no any signals about +2/+3/+4 precusors, I only choose the highest signals of +6 precursor which also have only some y ions detected. How could I improve my PRM method about this long peptide?

 Screenshot 2024-06-04 092538.png 
view request
Small molecules - Individual regression weighting for each analytes?
(3 responses) johannes kutzler 2023-10-14 10:54

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

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

Thank you for your help in advance
Hannes

view request
Skyline is missing some MRM chromatograms and spectra in my Sciex MIDAS files
(4 responses) vonroep 2024-06-04 09:30

Hallo Skyline Team,
I recently started using Skyline to investigate my MIDAS runs (MRM triggers MSMS) . I started with simple old experiments and want to change over to complex naturally crosslinked proteins. However already in the simple runs I find discrepancies between Skyline and my Analyst software. I guess some of my parameters are not set appropriately. However after 4h of tutorial reading and testing I am still stuck with the missing MRMs. I attached a document with the screen shots of Analyst and Skyline and I also attached the Skyline experiment.

Thanks in advance for your time and help.
Kind regards

Edda von Roepenack-Lahaye

 Hallo Skyline Team.docx  Test MRM2.sky  Test MRM2.skyd 
view request
Deep MRM Server Error
gdrumm 2024-06-04 05:09

Hello there,

I am a research assistant who is attempting to find a way to streamline our data analysis and peak integration process. I came across skyline recently, after having used MassHunter for a while, and was intrigued by the Deep MRM tool. However, when I attempt to use it, I am met with a server error (specifically, an internal server error: code 500). I was wondering if anyone might have any insight on how to fix this. Thank you in advance!

view request
Feature finding feedback for Daily
(6 responses) Chris Ashwood 2024-05-17 20:27

Dear Skyline team,

Hope you're doing well. I'm excited by the new feature finding feature but have some hopefully helpful feedback regarding its use. Specifically, I work with negative mode small molecule data, so this may just be an unintended use case.

Skyline assumes the feature finding will return protonated molecules, so it returns the chemical formulae with M+H adducts, despite Hardklor feature finding being performed on a negative mode only raw file. This then causes an issue when it tries to import the raw file as only positive mode transitions are present in the transition list. When I manually modify the adducts and chemical formula to reflect negative mode, the correct molecules are observed and extracted. So Hardklor/Bullseye might be working fine, and only changes to the transition list processing may be needed.

If small molecule use is unintended, it might be helpful to exclude it from the small molecule mode view.

Happy to provide all the associated files if desired.

Cheers,
Chris

view request
Failed importing results file .wiff: Sequence contains no elements
(6 responses) george wang cal 2024-05-31 08:06

Dear Skyline support,

I am running a targeted, scheduled lipidomics (900 total transitions between + and - mode) MRM on a SCIEX 6500+ Qtrap, each batch consisting of ~80 total runs. A handful of those runs are standards; I am using a splashmix which is commercially available and contains ~14 deuterated lipids, 1 per class. On 2 separate batches, I have encountered this error only when importing standard runs - my sample runs have all imported with 100% success rate. My previous batch, I ran 3 cal curves consisting of 7 points each, and 1 of those runs failed to import (1/21 = 5% failure rate). On my latest batch, I ran my highest point 4 times throughout, and 1 of those runs resulted in that error (25% failure). When I use Analyst to view their TICs, the expected peaks are present at the same RTs and intensities of those which successfully imported, so I know data was collected, but it's just not importing. So it seems to be intermittent and it's unclear why.

Any idea what's going on and how I could resolve this?

Thanks,
George

view request
Skyline crashes when trying to add peptide modifications
(1 response) areiter30500 2024-05-30 20:03

Hi, I'm unable to add any modifications to existing peptides in the target list. When I right-click the peptide and "Modify...", there is no change to the peptide or m/z values below. When I create a copy with a new modification, it just adds the exact same peptide below. Also when I right-click and select "Pick children", I get the "(Not responding)" error and the software crashes.

This used to work perfectly fine. I've been using Skyline-daily 23.1.1.459, and it was working fine last week. I updated to 23.1.1.503 and it still doesn't work. I also uninstalled and reinstalled, but that didn't work either.

I've been using Skyline for a long time, and have never encountered any issues like this.

Thank you for your help!

view request
Exporting no retention time to transition list
(5 responses) Jeff Whiteaker 2024-01-05 10:57

Hi,
Recently updated to the latest daily version of Skyline (23.1.1.353).
I tried exporting a scheduled transition list from the attached document using SCIEX settings.
The values for retention time were not exported (i.e., zero in all rows).
It works if the document is shared using a previous version (23.1).

Maybe I missed something? Many thanks for your help tracking this down.
Best,
Jeff

 Her2ADC_RAS_QC_240106.sky.zip 
view request
Overintegration problem
(5 responses) luzalonsodasques 2024-05-27 07:50

Good afternoon, my name is Luz Alonso

I am writing to you because I have the following problem with Skyline (64-bit) version 23.1.0.268 (e59d0dc56).

The baseline of my chromatogram, instead of being fixed at zero, starts at another point on the y-axis.

Therefore this influences my peak integration process due to it not only integrates the peak but also from the y-axis point to zero.

I attach a picture of my problem.

Looking forward to your early response

Best,

Luz.

 12.JPG 
view request
Building libraries with Koina causes error
(5 responses) msylvest 2024-05-21 05:32
Dear Skyline team,

I was excited to see the additional prediction options that come with Koina. Unfortunately, any attempt to build a library fails (error response attached).
I tried most combinations of intensity and iRT models, with and without iRTs. The server is "online". I had no problems with all previous builds using Prosit (even though I have some peptides with mods unknown to Prosit).

Cheers
Marc
 Error log Koina library building MSY240521.txt 
view request
Disulfide Bond as modification
(2 responses) thcsam 2024-05-21 06:51

Dear Skyline,

I currently would like to analysis the disulfide bond pattern for one single protein.
I originally though if i added disulfide bond as crosslinker as indicate in (https://skyline.ms/wiki/home/software/Skyline/download.view?entityId=ac4c6cc4-97c6-1038-8aae-e465a393ee52&name=NicholasShulman2020ASMSPoster.pdf) then all possible linkage of disulfide bond within the same protein will be listed. It turned out I have to add the linked peptide one by one. Since it have only 2 Cys in the protein, the first segment contain 3 variable modification which generate a total of 8 possible peptide, where second segment contain no modification. Considering these two cysteine containing peptide, it will have 88 (1st segment disulfide link to 1st segment) plus 18 (1st segment disulfide link to 2nd segment) plus 1 (2nd segment disulfide link to 2nd segment), 73 possible disulfide peptide is generated.(assume no misscleavage). 73 is still reasonable for me to add it one by one.
I have few question.

  1. Did i miss some feature of skyline that do the above automatically instead of adding one by one?
  2. If I do not miss feature of skyline, would this feature be added? As if No. of cysteine and no. of modification of cysteine containing peptide
    increase, then the manual addition of crosslink peptide seem to be impossible.
    Really appreciate and looking forward for your response.

Best regards,
Sam

view request
Koina dotp refinement filter
rj8 2024-05-21 14:51

Is there a way to filter peptide IDs using a Koina dotp filter (comparing the Koina spectrum prediction to the spectral library spectrum as a 'refinement')?

view request
Nothing (nether chromatography peaks nor statistic graphs )showed up after transition list and results were imported.
(3 responses) q zhang 2024-05-21 02:22

Hi, Skyline teams!
My LC-MS data is glycome from mice liver tissue. After I import my transition list (29 glycan precursors in MS1, and 21 fragment under each precursors in MS2) and raw data, there's nothing showing up in the window. There's nothing in chromatography, peak area graph and retention time graph. Please see the screenshot in the word file I attached.
I was guessing this issue might be due to too much glycans in transition list, so I deleted 4 glycan precursors in MS1, 25 glycan precursors left, and start with a new skyline file with the same setting and importing steps. Then, it worked, and everything showed up.
However, I think I still need to figure out the solution, and want to know how can it work by using my originally complete transition list.

 Skyline display problem.docx 
view request
Calibration curve
(1 response) mdsir 2024-05-19 18:26

We would like to draw a calibrarion curve for Adenin, and this is where we were stuck.
We aligned the peaks for the molecule and the peak area shown below seems appropriate for the calibration curve but it's not shown.
It's also same for other molecules that we are targeting.
We'd appreciate if we can get some guide for this problem.
Thank you so much.

 Capture.PNG  Adenine peak intensity.PNG  Calibration curve concentration.PNG 
view request
Bruker API does not support Unicode in filepaths
j-kindermans 2024-05-17 02:07

Greeting

When trying to import data from my last experiment, I get this error for all my samples (not blanks or Hela QC) of this experiment:

Failed importing results file 'C:\Users\PPC-P\Documents\1M_REF_F3_1.5µl_S4-C1_1_12079.d'.
[ReaderFail] [Reader_Bruker::read()] Bruker API does not support Unicode in filepaths ('C:\Users\PPC-P\Documents\1M_REF_F3_1.5µl_S4-C1_1_12079.d')

My data is DDA with ion mobility (timsTOF HT). My skyline file and sample data are saved in the same folder.
Does anyone have experience with this error? Could it be that my data is corrupted?

Best regards,
Jana Kindermans

view request
disulfide crosslinked homodimeric also covalently modified peptide does not appear to work
(5 responses) warham 2024-05-14 08:08

So this will probably go to Nick,

So disulfide homodimeric peptides load beautifully through the modify peptide interface. And crosslinks between different peptides can also take other chemical modifications in the insert peptide interface. On a mAb there is this sequence:

THTCPPCPAPELLGGPSVFLFPPKPK

And in evaluating it's disulfide integrity, this peptide

THTCPPC[+57]PAPELLGGPSVFLFPPKPK-THTCPPC[+57]PAPELLGGPSVFLFPPKPK-[DSS@4,4]

would be useful but I get the error that the peptide is not recognized by the software. I tried adding another copy of the sequence but there is no field for specifying different proteins in the same peptide entry on the insert peptide field. Also the insert peptide field does not seem to support multiple crosslinks between two peptides which does happen with disulfides.

Is there a way to do this that I'm missing? If it's too specific an issue, that's fine. But I thought that since there are a fair number of disulfide oligomers, it might affect others too.

Best,
Lance

view request
Area Under the curve
(1 response) naimamuntu 2024-05-14 04:33

Hi,

I am currently working on a project that involves analyzing data. I am interested in the area under the curve for a product of interest. Could you please guide where to find the area under the curve for a particular precursor and one product in Skyline. I am using Skyline 21.01.0.278 for reference.
In this particular project, I am using the Chromatogram Precursor M/Z Chromatogram Product M/Z respectively, 730 and 660.25
I am sending you one skyline file so that you can see what kind of analysis we are doing.

Thank you for your attention
-Nitha

 MYH7_Skyline_NSR_LV_43024.sky.zip 
view request
Error importing DIA-NN search results with newest Skyline-daily update
(3 responses) Shawn Rice 2024-05-13 06:40
Greetings,
After updating Skyline-daily, I have not been able to read search results for DIA-NN. This includes search results that have previously been imported into Skyline, so the results files should be fine. The version of DIA-NN is the same that has previously allowed for the successfully imported into Skyline. The error message is copied below.

Thanks,
Shawn

---------------------------

---------------------------
ERROR: could not find precursorId 'AVGVPALGFSPMNR2' in speclib; is 'enolase.report.tsv' the correct report TSV file?
ERROR: reading file enolase.report.lib.tsv.speclib

Command-line: C:\Users\srice\AppData\Local\Apps\2.0\LC9H788V.2T0\ENRYOW2B.Y1Z\skyl..tion_9286511f3362df93_0017.0001_7a74d73c81fd676c\BlibBuild -s -A -H -v warn -o -c 0.95 -i skyline -S "E:\Belani_240509\Exp._Control_Enolase\skyline.redundant202405130929.stdin.txt" "E:\Belani_240509\Exp._Control_Enolase\skyline.redundant.blib"
Working directory: E:\Belani_240509\enolase
---------------------------
OK More Info
---------------------------


System.IO.IOException: ERROR: could not find precursorId 'AVGVPALGFSPMNR2' in speclib; is 'enolase.report.tsv' the correct report TSV file?
ERROR: reading file enolase.report.lib.tsv.speclib

Command-line: C:\Users\srice\AppData\Local\Apps\2.0\LC9H788V.2T0\ENRYOW2B.Y1Z\skyl..tion_9286511f3362df93_0017.0001_7a74d73c81fd676c\BlibBuild -s -A -H -v warn -o -c 0.95 -i skyline -S "E:\Belani_240509\Exp._Control_Enolase\skyline.redundant202405130929.stdin.txt" "E:\Belani_240509\Exp._Control_Enolase\skyline.redundant.blib"
Working directory: E:\Belani_240509\enolase ---> System.IO.IOException: ERROR: could not find precursorId 'AVGVPALGFSPMNR2' in speclib; is 'enolase.report.tsv' the correct report TSV file?
ERROR: reading file enolase.report.lib.tsv.speclib

Command-line: C:\Users\srice\AppData\Local\Apps\2.0\LC9H788V.2T0\ENRYOW2B.Y1Z\skyl..tion_9286511f3362df93_0017.0001_7a74d73c81fd676c\BlibBuild -s -A -H -v warn -o -c 0.95 -i skyline -S "E:\Belani_240509\Exp._Control_Enolase\skyline.redundant202405130929.stdin.txt" "E:\Belani_240509\Exp._Control_Enolase\skyline.redundant.blib"
Working directory: E:\Belani_240509\enolase

Output:
Reading results from enolase.report.lib.tsv.speclib.
ERROR: could not find precursorId 'AVGVPALGFSPMNR2' in speclib; is 'enolase.report.tsv' the correct report TSV file?
ERROR: reading file enolase.report.lib.tsv.speclib
100%

 ---> System.IO.IOException: ERROR: could not find precursorId 'AVGVPALGFSPMNR2' in speclib; is 'enolase.report.tsv' the correct report TSV file?
ERROR: reading file enolase.report.lib.tsv.speclib

Command-line: C:\Users\srice\AppData\Local\Apps\2.0\LC9H788V.2T0\ENRYOW2B.Y1Z\skyl..tion_9286511f3362df93_0017.0001_7a74d73c81fd676c\BlibBuild -s -A -H -v warn -o -c 0.95 -i skyline -S "E:\Belani_240509\Exp._Control_Enolase\skyline.redundant202405130929.stdin.txt" "E:\Belani_240509\Exp._Control_Enolase\skyline.redundant.blib"
Working directory: E:\Belani_240509\enolase
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass, Boolean forceTempfilesCleanup, Func`3 outputAndExitCodeAreGoodFunc, Boolean updateProgressPercentage) in C:\proj\pwiz\pwiz_tools\Shared\CommonUtil\SystemUtil\ProcessRunner.cs:line 188
   --- End of inner exception stack trace ---
   --- End of inner exception stack trace ---
   at pwiz.Common.SystemUtil.ProcessRunner.ThrowExceptionWithOutput(Exception exception, String output) in C:\proj\pwiz\pwiz_tools\Shared\CommonUtil\SystemUtil\ProcessRunner.cs:line 248
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass, Boolean forceTempfilesCleanup, Func`3 outputAndExitCodeAreGoodFunc, Boolean updateProgressPercentage) in C:\proj\pwiz\pwiz_tools\Shared\CommonUtil\SystemUtil\ProcessRunner.cs:line 230
   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 433
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 161
---------------------------
view request
"No chromatogram available"
(2 responses) nbekhti 2024-05-10 06:09

Hi Skyline team,

I often have this issue but not sure where it's coming from as I download the same SRM list to Skyline.
It shows "No chromatogram available" for some of the compounds, showing as example "aconitic acid" and noticed that on the Document Grid, the "Average Measured RT" is #N/A for this compounds and was wondering what's the issue and how to fix it. (Images attached)
Thanks a lot

Nihel

 no chromatogram available_skyline.pdf 
view request
Inquiries about MRM Data export
(1 response) mdsir 2024-05-08 18:10

Peak intensity (height) appears in the document grid as a different value from the actual obtained data. I would like to know the reason for this.

 Data_export_height.PNG 
view request
In few data files peak peaking is not working
(3 responses) darshak niper 2024-05-08 12:42

Hi,

In few files peak peaking is not working. We manually look at the TIC and check the data file in mass hunter qualitative analysis, it looks ok. But in skyline processing those file does not show any peaks. In the attached skyline document , the file name "Pos_CC6" where peak peaking is not working.

 Skyline_file.sky.zip 
view request
In few data files peak peaking is not working
darshak niper 2024-05-08 12:40

Hi,

In few files peak peaking is not working. We manually look at the TIC and check the data file in mass hunter qualitative analysis, it looks ok. But in skyline processing those file does not show any peaks. In the attached skyline document , the file name "Pos_CC6" where peak peaking is not working.

view request
Add compound ID in addition to the Molecule Name to Skyline
(1 response) nbekhti 2024-05-05 12:40

Hey Skyline team,

I would like to add to my list of metabolites an internal ID (not an HMDB, kegg.. IDs) and would like to be able to export it with the data at the end of the processing.
I tried to use HMDB ID column but as it is not in the same format it shows an error message. I also tried to use Molecule or Molecule Note, but when exporting the data it does not export that info on the molecule.

Is there a way to enter internal IDs in addition to the molecule name and be able to export them at the end of the processing?
Thank you,

Nihel Bekhti.

view request
Prosit server unavailable
(7 responses) michal zawadzki33223 2024-04-11 04:22

Hello,

I am having an issue with connecting to Prosit server through Skyline (version 23.1.0.455) - see the attached snip for details. I have used the test-netconnection command in Windows PowerShell as recommended by Brendan in one of the previous posts and it looks like my computer is able to connect to the server using port 8500. Any suggestions on how this could be fixed are appreciated.

Kind regards,

Michal

 Prosit server unavailable.PNG 
view request
a small molecule charge state notation question
(1 response) warham 2024-05-01 14:39

I am aware of this page

https://skyline.ms/wiki/home/software/Skyline/page.view?name=adduct_descriptions

and it is helpful.

I have this question, if I know the structure of my fragment ion and it is doubly positively charged with a carbocation at one site and a proton at the other,
what is the notation?

do I use [M+2]
and add a Hydrogen to the fragment molecular formula?

view request
M+X peaks for fragment ions
(3 responses) sasha 2013-05-22 09:16
Dear Brendan,
Is there a way that i can see, or quantify, the isotopic clusters for fragment ions scanned at high resolution?
s
view request
Import speclib from DIANN with customized non-unimod modifications
(4 responses) gianin thomann 2024-04-23 04:20

Dear Skyline Team,

I would like to import a spectral library generated from DIANN into Skyline. The library features some customized non-unimod modifications as well as UniMod:4 (carbamidomethylation on cysteine) as a variable modification. Whenever I try to import via "Import DIA Peptide Search" I receive an error Message (attachement). I assume Skyline does not recognize the modification "Unimod:4", probably due to misspelling, and, hence, will not recognize my customized non-unimod modifications anyways. Is there a way to add such modifications to Skyline prior to import?

I also tried to import the speclib without any variable, customized and non-unimod modifications (and UniMod:4 spelled correctly) which was successful.

Best,

Gianin

 Swissprot_HSapiens-BSA_lib.tsv.speclib  ErrorMsg_Skyline.png 
view request
Exporting images with publication quality
(2 responses) antrixj 2024-04-25 15:29
Hello,
What is the best way or format to export and save images from skyline with publication quality? I know we can copy the metafile directly and use it.
Ideally, I would like to share these images with collaborator (not a skyline operator), such that they can use this vector-based file to edit in AI/Inkscape.
view request
Annotate unknow full scan compound using NIST GC-MS library
(2 responses) wanying cao 2024-04-26 09:06

Hi Skyline team, I used to be a Skyline protein mode user but now switched to GC small molecule omics analysis. Current software I am using can only give me a laundry list of RT with precursor mass without any compound name information. I can import transition list and results file to Skyline but cannot annotate these peaks from RT. So I was trying to figure out how can I actually annotate this peaks using Skyline then output a list of compound peak area list.

So I was thinking to use NIST GC-MS library but clearly I have difficult time find the right one (my compounds are all volatile compounds with <350 m/z, but all library I can find is peptide size). So I was wondering can Skyline annotate those undefined Peaks from GC-MS (Agilent)? If yes, how can we actually do it using Skyline? Thank you very much!

view request
How to bulid the BiblioSpec library
2378572477 2024-04-28 23:47

Hello
I downloadsed Honeybee from the Website (PeptideAtlas - Bulk Downloads)(Database Tables:XML) for building BiblioSpec library last time.This is not the correct download path,is it?
When I want to download spectral libraries from Peptide Atlas(Website:PeptideAtlas - Spectrum Libraries),I don not find Honeybee(honey). I can not download spectral libraries about honeybee,is it?
Now, I wonder the way how to bulid the BiblioSpec library.
Thanks,
Zhaoxuanxuan
Hello
I downloadsed Honeybee from the Website (PeptideAtlas - Bulk Downloads)(Database Tables:XML) for building BiblioSpec library last time.This is not the correct download path,is it?
When I want to download spectral libraries from Peptide Atlas(Website:PeptideAtlas - Spectrum Libraries),I don not find Honeybee(honey). I can not download spectral libraries about honeybee,is it?
Now, I wonder the way how to bulid the BiblioSpec library.
Thanks,
Zhaoxuanxuan
Hello
I downloadsed Honeybee from the Website (PeptideAtlas - Bulk Downloads)(Database Tables:XML) for building BiblioSpec library last time.This is not the correct download path,is it?
When I want to download spectral libraries from Peptide Atlas(Website:PeptideAtlas - Spectrum Libraries),I don not find Honeybee(honey). I can not download spectral libraries about honeybee,is it?
Now, I wonder the way how to bulid the BiblioSpec library.
Thanks,
Zhaoxuanxuan
Hello
I downloadsed Honeybee from the Website (PeptideAtlas - Bulk Downloads)(Database Tables:XML) for building BiblioSpec library last time.This is not the correct download path,is it?
When I want to download spectral libraries from Peptide Atlas(Website:PeptideAtlas - Spectrum Libraries),I don not find Honeybee(honey). I can not download spectral libraries about honeybee,is it?
Now, I wonder the way how to bulid the BiblioSpec library.
Thanks,
Zhaoxuanxuan

view request
Adding Precursor [M-1] trace to all precursors by default
(2 responses) sean jensen 2024-04-25 21:39

Hi!

We use the [M-1] to qualify potential interferences and correct species identification for peptides and was wondering if anyone is able to help me enable the precursor [M-1] chromatogram trace for all precursors in a document by default. I've tried manually enabling the option for each precursor, but this is impractical to do for large analyses.

Thanks in advance!

Cheers,
Sean

view request
Import of search results from Peptideatlas to build the BiblioSpec library
(1 response) 2378572477 2024-04-28 00:52
Hello.

I am trying to build the BiblioSpec library from Peptideatlas. I downloadsed Honeybee(Database Tables: XML) for building BiblioSpec library. After I added the result. file, an error popped up saying not a valid library input file. Can you please let me know how to fix this? Or what is the other way to build the BiblioSpec library(about Honeybee)?

Thanks,
Zhaoxuanxuan
 问题.docx 
view request
"Not Responding" after MS1 filtering
(3 responses) y nishimura 2024-04-25 04:39

I imported 32 interact.pep files from FragPipe closed search output, 32 mzML files, and 1% FDR filtered FASTA generated by FragPipe with 2 missed cleavages into Skyline-daily. After MS1 filtering is completed, Skyline does not respond as soon as I click anything. Checking my PC memory and CPU performance, it does not look like they are the reason. Do you have any suggestions to fix this?

view request
The tutorials in the official website and the tutorials in the software should be consistent, it's too messy now!
(1 response) 839868794 2024-04-06 02:44

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

  1. I went to https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_ms1_filtering_zh, the download link opened a new tab called MS1Filtering-20_1_zh-CHS, the download file was actually MS1Filtering-22_2_zh-CHS.pdf.
  2. In the Skyline, I went to the homepage > tutorials, after clicking on MS1 filtering, I noticed that the automatically created folder is MS1Filtering-22_2, but the actual downloaded is MS1Filtering-21_1_zh-CHS.pdf.

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

view request
How to specify simultanous structural modification for N-term and for a side chain, if the modified sidechain is on the N-terminal amino acid
(1 response) pavel shliaha 2024-04-24 14:31

I have two static (structural modifications):

  1. The N-terminus of the peptide is modified by phenylisocyanite.
  2. The NH2 group of Lys is modified by propionyl group.

it so happens that I have a peptide that has K on the N-terminus:

KQLATKAAR

meaning the first Lys should have both phenylisocyanite and propionyl

how ever when loading the peptide in skyline only adds propionyl, ignoring phenylisocyanite

if I however add Ala to the peptide N terminus

AKQLATKAAR

I do get A modified with phenylisocyanite and Lys with propionyl.

How do I force skyline to add both structural modifications to K in KQLATKAAR?

view request
Quantification with a surrogate analyte
(1 response) dsgk 2024-04-24 03:55

Hi Skyline Team,

I have a biomarker assay where I'm using two differently labelled heavy peptides - one is used as an internal standard, the other as the surrogate analyte used for calibration (because my blank matrix has the light peptide endogenously present). I can't figure out how to get Skyline use the calibration curve of the surrogate analyte for calculating the concentrations of my unknown samples where I have signals coming only from the light peptide and the internal standard. Any ideas how to get Skyline to do this?

Thanks!

view request
Preparation of spectral libraries with the Astral Analyser
(1 response) n dasilva 2024-04-24 01:55

At the moment, we are currently attempting to create a spectral library using Skyline for a PRM experiment; using DDA data of the full proteome and of heavily labelled peptides, acquired using the Astral equipment.

It is possible to import the raw files, although a message saying that there is " no matching instrument configuration for the analyser type" appears.
Also when setting the MS/MS filtering acquisition method, as it is not possible to find the Astral mass analyser as an option, what would be recommended to use in this particular case?

I appreciate all the attention given,
Best regards,
Nádia

view request
Problems with Exporting Peak Intensities
(9 responses) matser 2024-04-19 22:16

Dear Skyline team,

I am a recent user of the platform so please forgive my ignorance. I have been trying to use the platform to identify 13C labelled metabolites from a metabolomics ms/ms run. I created a target list where each compound was assigned a list name and the isotopologues of the compound where different molecules. I have also the peak integration in all my samples. The step I am stuck on is trying to normalize the intensities/peak area to total ion current and then exporting the report. I noticed that the normalized areas of the isotopologues are assigned "#NA" and I only get values for the M+0 (the unlabelled fraction). I would appreciate whatever help I can get with this issue.

Best wishes,

Mikky

view request
Loading transition list not working
(1 response) ylee 2024-04-22 12:18

Hello,

I have been trying to upload the transition list attached onto molecular interface of Skyline.
It only gives me an option to do Ctrl-V for loading the transition list.
Whenever I try to upload it that way, it gives me the following error message.
"Data columns not found in first line"

For some reason, the data columns are not recognized by the software.

Could you please let me know how I can go about resolving this issue?

Thank you.

 YL_transitionlist.xlsx 
view request
regarding MSstatsashiny
(1 response) bandgara22 2024-04-22 10:09

hello sir,
i am trying to statistical analysis using a MSstats shiny but i think there is no availability of tutorial on MSstatsshiny so I am trouble in the uploding the skyline annotaton file please guide me to make a annotaion file for group comparison study.

 MSstats error.png 
view request
How to create a library from the refined transitions in skyline?
(1 response) pavel shliaha 2024-04-20 14:51

I am trying to quantify a protein and I have loaded its sequence in skyline to in silico digest it and filter peptides. I have then created a library using prosit and loaded the raw data and picked the peptides that have good signal, peak shape, good dotp with the prosit library (I ignored the RT component of prosit prediction).

Now that I have a list of high-quality peptides from real data, I would like to use the data to create a new library using the experimentally observed RT and fragmentation spectra. However, I can't find a way to do this.

Hence, my question is: how do I take the experimentally observed data loaded into the skyline and curated there to create a library?

view request
Import of search results from ProteomeDiscoverer to build the spectral library
(2 responses) yanyan qu30905 2024-04-19 09:03

Hello.

I am trying to build the spectra library using the PD searching results. After I added the msf. or pdresult. file, an error popped up saying no Mass Spectrum Items. Can you please let me know how to fix this? Or what is the other way to import the PD searching results?

Thanks,
Yanyan

 Screenshot 2024-04-19 115834.png 
view request
Multiple Skyline report templates combined into single Excel Workbook?
(2 responses) josiah.hutton 2024-04-17 11:28

Hello Skyline Team,

First of all, thanks for such a great tool, I've used Skyline for almost a decade now to design and analyze targeted proteomics dataset.

I have recently been exporting my reports for my datasets as multiple individual .csv documents, as I found it cumbersome to export all the information I wanted (fragment ion peak area, MS1 IM, MS1 peak area, total area, fragment ion dotp score, etc.) in a single report and then going through and separating the information into separate tabs. Currently, I take all the multiple individual .csv reports and combine them into multiple tabs into a single Excel Workbook, as this is sometimes easier to manipulate for downstream analysis and is a somewhat more logical document when provided to collaborators. Would it be possible to modify the Report Export procedure so that I can tell Skyline to output into a single Excel Workbook a document with multiple tabs that contain information from different report templates? For example, a new Merged Report Output option that combines the report template from Report Template 1, Report Template 2, and Report Template 5 as three separate excel tabs in a single document? Or, did I just not adequately search the forum for this topic? I currently have a tool to combine the separate single reports into a single merged document, but I still have to tell Skyline to export 5 different reports, and it would be convenient to have a method within Skyline to export a single merged document.

Thanks in advance!
Josiah

view request
MSstats some R packages failed to download
(5 responses) bandgara22 2024-04-17 00:59

Hello, sir. I am facing to a problem downloading R packages . after multiple trials, some R packages cannot be downloaded in MSstats downloding.

 r packages.jpg 
view request
Report problems
(2 responses) jean-christophe prost 2024-04-17 08:41

Dear Skyline,

Since we are using skyline we always had a bit problems with our reports. Mostly after each update we lost them and had to reimport them.
After Brendan's mail on April 1st, I decided to update again all computer with skyline, after almost 2 years.
Unfortunately a colleague using skyline and a specific template lost her template. gain
She was not able to import it. I tested it on a new Instrument acquiring PC we have since 5 month, where I installed AutoQC for Panorama.
This PC never saw any inhouse template we saved. I tried to import the specific report of my colleague.
As shown in the word file, I could import something, but during import the report name changed ("Report_Template_QQQ_Skyline.skyr" to "Report_JC.skyr") and the imported report did not corresponded anymore of what she paste normally in excel. she could not use this imported report.
I did one other test on my computer. I was able to import one of my templates, but unfortunately stays light-grey, and I am not able to use it.

How could we fix these issues?
Thank you in advance for your support and wish a nice day.

Kind regards
Jean-Christophe

 Report import.docx 
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Transitions not selected even with
jiwon hong 2024-04-18 01:23
view request
Importing raw files from Orbitrap Astral with FAIMS
(2 responses) edf4n 2024-04-17 12:54

Hello Skyline Team,
I have been unsuccessful in importing raw files from our Orbitrap Astral instrument.
The instrument method is DDA acquisition using FAIMS with three CV settings.
I think I need guidance with Full Scan scan settings. What is recommended with Astral data using FAIMS?
Thank you very much!

view request
How to make the integration easy and fast?
(2 responses) nbekhti 2024-04-16 18:50

Hi Skyline Team,

I have a question, I processed data from a sampleset, and need to add few new runs to this skyline file and would like skyline to consider the same integrations done on the previous samples for the new added ones.
Is there a way to automate this process? or should I go through all the compounds one by one and manually reintegrate each peak? thanks a lot!

Nihel

view request
Error Library Building from FragPipe (can't find mzml/mgf)
(3 responses) david schmidt 2024-04-16 04:41

Hi, in Skyline-daily (64-bit) 23.1.1.459 (f9dd888b9), I tried to build a spectral library from FragPipe pep.xml output of a DDA-PASEF search. The error message indicates that Skyline is looking for a data file (mzml/mgf), which is actually present in the same folder. Any idea why Skyline doesn't recognize it? Noteworthy, the error message appears when clicking next on the spectral library tab (i.e. before selecting the raw data).

Thank you, David

 Error_Library_Building.PNG  Files.PNG 
view request
artificial modification
(4 responses) petra janning 2024-04-17 07:14

I tried to quantify a peptide containing an artificial, variable modification adding 'C13 H8 O2 Cl2' to lysine. I have DDA data and performed a MaxQuant-search including the artificial modification. When I tried to build the library using the msms.txt file I got the error message "No matching mod for.... Make sure that you provided the correct modification[local].xml file."

I checked the support and found very similar questions two times in 2020 and 2021. Both times the answer was, that 'BiblioSpec is unable to handle ... and Chlorine modifications in MaxQuant search results', but from the next Skyline realease on the problem would be solved. I am working with Skyline (64-bit) 23.1.0.455 (599e74ad2), but obviously I have the same problem. Could you please help me? You will find the mqpar.xml file attached to this mail.

 mqpar.xml 
view request
modifications are not set correctly
lode denolf 2024-04-17 07:47
Dear,

I am working on a project where I want to see the difference in intensities between peptides labeled with a Heavy acetyl group and not.
When I try to insert the peptide list I will give me an error and I don't know how to adjust my imput so that I can continiue

---------------------------
Skyline
---------------------------
The following modifications could not be interpreted.

C[+103.05421] K[+92.0655] M[+42.01057][+15.99492] M[+46.03275][+15.99492]
---------------------------
OK More Info
---------------------------
System.FormatException: The following modifications could not be interpreted.

C[+103.05421] K[+92.0655] M[+42.01057][+15.99492] M[+46.03275][+15.99492]
   at pwiz.Skyline.Model.ModificationMatcher.CreateMatches(SrmSettings settings, IEnumerable`1 sequences, MappedList`2 defSetStatic, MappedList`2 defSetHeavy, IProgressMonitor progressMonitor, IProgressStatus status) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\ModificationMatcher.cs:line 65
   at pwiz.Skyline.EditUI.PasteDlg.AddPeptides(SrmDocument document, Boolean validating, IdentityPath& selectedPath) in C:\proj\skyline_23_1\pwiz_tools\Skyline\EditUI\PasteDlg.cs:line 277
---------------------------

This is the error I got and it happens to the modifications that contain the Acd4 modification (4Da heavier then Ace) in combination with a sertain fixed modification (Carbamidomethylation, fixed Acd4 on K or Methionine Oxidation).

I also tried it where I gave all the modifications in the peptide modifications tab and specify what modifications are in the sample and which ones should be fixed or variable.

In the latter case, I just had double the ammount of peptides where one would be with an Acd4 label and the other without.

I can send you a dat and raw file for 1 of the samples (in total 32) to have a look if you can find a way around this problem.
I will also send you the list of peptide sequences and also the one where I added the Modifications.

F010708.dat contains the NH2 searches
F010740.dat contains the Acd4 searches


Thanks in advance for the help
Lode Denolf
view request
modifications are not set correctly
(1 response) lode denolf 2024-04-17 06:27
Dear,

I am working on a project where I want to see the difference in intensities between peptides labeled with a Heavy acetyl group and not.
When I try to insert the peptide list I will give me an error and I don't know how to adjust my imput so that I can continiue

---------------------------
Skyline
---------------------------
The following modifications could not be interpreted.

C[+103.05421] K[+92.0655] M[+42.01057][+15.99492] M[+46.03275][+15.99492]
---------------------------
OK More Info
---------------------------
System.FormatException: The following modifications could not be interpreted.

C[+103.05421] K[+92.0655] M[+42.01057][+15.99492] M[+46.03275][+15.99492]
   at pwiz.Skyline.Model.ModificationMatcher.CreateMatches(SrmSettings settings, IEnumerable`1 sequences, MappedList`2 defSetStatic, MappedList`2 defSetHeavy, IProgressMonitor progressMonitor, IProgressStatus status) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\ModificationMatcher.cs:line 65
   at pwiz.Skyline.EditUI.PasteDlg.AddPeptides(SrmDocument document, Boolean validating, IdentityPath& selectedPath) in C:\proj\skyline_23_1\pwiz_tools\Skyline\EditUI\PasteDlg.cs:line 277
---------------------------

This is the error I got and it happens to the modifications that contain the Acd4 modification (4Da heavier then Ace) in combination with a sertain fixed modification (Carbamidomethylation, fixed Acd4 on K or Methionine Oxidation).

I also tried it where I gave all the modifications in the peptide modifications tab and specify what modifications are in the sample and which ones should be fixed or variable.

In the latter case, I just had double the ammount of peptides where one would be with an Acd4 label and the other without.

I can send you a dat and raw file for 1 of the samples (in total 32) to have a look if you can find a way around this problem.
I will also send you the list of peptide sequences and also the one where I added the Modifications.

F010708.dat contains the NH2 searches
F010740.dat contains the Acd4 searches


Thanks in advance for the help
Lode Denolf
view request
two peptide bridges on the same amino acid
(1 response) vonroep 2024-04-17 01:41

Dear support team,

I used Skyline to design peptide transitions for our QTRAP6500+ System (Sciex). I could use the software to develop peptide transitions containing a NOS bridge . However I am not able to design a crosslinking modification involving two crosslinking events to the same amino acid (SONOS bridge). I always get an error message (see attached file). Is there a way around the problem?

Thanks for the support.
Kind regards
Edda von Roepenack-Lahaye

 two crosslinker at one amino acid.pdf 
view request
Skyline 21.1 version
(1 response) naimamuntu 2024-04-15 08:41

Hi,
I'm trying to reproduce the old experiments analyzed using Skyline 21.1. Could you please send me the link to download the old version?

Thank you
-Nitha

view request
TMT neutral losses
(4 responses) aj10 2024-04-11 14:47

Hello,

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

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

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

Thank you for your help!
Alex

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

Hello,

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

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

Thanks
Amandine

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

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

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

Hello Skyline team,

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

Thank you

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

Hi, Skyline Team.

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

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

Scott

 Error.PNG 
view request
AvantGarde tool install
(1 response) dkueltz 2024-04-11 08:21

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

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

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

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

Hi,

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

Thank you so much.
-Nitha

view request
Missing DIA acquisition
(1 response) guilherme pauperiolanfredi 2024-04-11 00:08

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

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

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

Any idea?

Thanks in advance.

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

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

ratio=molecule_peak_height / surrogate_standard_peak_height

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

Regards,
Per

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

Hi Skyline users, a new user here.

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

Thank you so much!

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

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

Comments from results "No precursor ion chromatograms found"

Thanks
VC

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

Hi team,

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

Thank you.
Akhila

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

Hi,
I have a problem with data extraction of our amino acid analysis done by FullMS and AIF on a QExactive. While most of the extracted transitions look fine, one transition is missing for isoleucine both in the internal standard and the analyte (the one that distinguishes isoleucine from leucine). I have no idea why the signal is missing (not even baseline), the signal seems to be present in the spectra, though. A file with a single run is attached. Any help is greatly appreciated!
Best Regards,
Joerg

 Missing_transition.sky.zip 
view request
TQ-XS Data not loadable in Skyline-daily (64-bit) 23.1.1.425 (b5b0d400b)
(3 responses) Marvin 2024-04-04 00:14

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

  • are just spaces for intern names without special characters.

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

appeared...

Do you have any suggestions how to tackle this problem?

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

Best wishes,
Marvin.

 test.sky.zip  MS24-0188_AltDB_074.raw.zip 
view request
BoxCar
(16 responses) ekilic 2024-02-29 13:50

Dear Skyline Support Team,

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

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

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

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

Thank you,

Ece and Soren

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

Hello,

I am writing to inquire about an issue encountered during the execution of AutoQC same as https://skyline.ms/announcements/home/support/thread.view?rowId=53279

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

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

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

Thank you for your assistance in resolving this issue.

Best regards,

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

Hi again,

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

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

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

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

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

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

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

 2024_0118DiGly_Skyline.sky 
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not possible to open old skyline files
(1 response) io 2024-04-02 03:51

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

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

Dear Skyline Support Team,

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

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

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

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

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

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

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

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

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

[Generated by ChatGPT]

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

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

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

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

I also tried to perform a workaround as per https://skyline.ms/announcements/home/support/thread.view?rowId=55778, but when I checked the registry, it was already a full path (as provided in the attachment). I also checked the destination folder, and the "Thermo.TNG.MethodXMLFactory.dll " file was placed correctly.

Regards

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

Dear Skyline team,

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

Thanks,
Philipp

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

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

Hi,

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

Thank you so much.
-Nitha

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

Hello,

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

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

Thank you.

 20240315_MRM_10.d.zip  240325_Skyline_MRM.zip  Skyline_transitionlist.xlsx 
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