Requests

support
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wiff2 file from Sciex Echo MS fails to import to skyline
(3 responses) michelle robinson 2020-10-26

Hi Brendan,
I'm unable to import MRM data generated on the Sciex Echo MS into skyline (wiff2 file format). I am using Skyline daily. The error message says, "[Experiment2ImpI::ctor()] Object reference not set to an instance of an object"
Thanks for your assistance,
Michelle

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peptides with mixtures of modified and unmodified residues
(18 responses) Keri 2018-05-09

Hi Brendan,

I have attempted to use the peptide settings > modifications feature to quantify samples that contain both fixed and variable modifications (e.g. some but not all cysteines in a peptide are isotopically labeled (+521 or +527) and all cysteines are carbamidomethylated +57). I input both the structural modification for carbamidomethylation and the variable modifications for the additional isotopic label, but when I apply those settings, I see multiple modifications, including the sums of multiple modifications (e.g. 1022, 1028, which are not correct). Can Skyline handle samples with mixed modifications and, if so, how?

Thank you

Keri

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Peaks of light and heavy peptides have unequal peak boundaries
(9 responses) fcsigloch 2020-10-22

Hi!

In an MRM experiment, I came across the problem that Skyline picks non-coeluting peaks for the light endogenous peptide and the heavy labelled reference peptide (screenshot attached). I never observed this behaviour in PRM data, where I am used to perfectly matching peak boundaries.
I could not determine a specific setting that caused this behaviour. Can I somehow force Skyline to choose the same peak boundaries for light and heavy trace?

Also, I tried to train a default or mProphet model to improve the peak picking. Training the model worked, but when I want to apply it to the data, I get the following error message:

---------------------------
Skyline
---------------------------
Failed attempting to reintegrate peaks.
The index 6 must be between 0 and 2
---------------------------
OK More Info
---------------------------
System.Reflection.TargetInvocationException: The index 6 must be between 0 and 2 ---> System.Reflection.TargetInvocationException: The index 6 must be between 0 and 2 ---> System.IndexOutOfRangeException: The index 6 must be between 0 and 2
   bei pwiz.Skyline.Model.Results.ChromatogramInfo.GetPeak(Int32 peakIndex) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromHeaderInfo.cs:Zeile 2773.
   bei pwiz.Skyline.Model.TransitionGroupDocNode.UpdateResults(SrmSettings settingsNew, SrmSettingsDiff diff, PeptideDocNode nodePep, TransitionGroupDocNode nodePrevious) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\TransitionGroupDocNode.cs:Zeile 1487.
   bei pwiz.Skyline.Model.TransitionGroupDocNode.ChangeSettings(SrmSettings settingsNew, PeptideDocNode nodePep, ExplicitMods mods, SrmSettingsDiff diff) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\TransitionGroupDocNode.cs:Zeile 1116.
   bei pwiz.Skyline.Model.PeptideDocNode.ChangeSettings(SrmSettings settingsNew, SrmSettingsDiff diff, Boolean recurse) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\PeptideDocNode.cs:Zeile 979.
   bei pwiz.Skyline.Model.SrmDocument.<>c__DisplayClass142_5.<ChangeSettingsInternal>b__7(Int32 i) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:Zeile 1080.
   bei pwiz.Common.SystemUtil.ProducerConsumerWorker`2.Consume(Object threadIndex) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProducerConsumerWorker.cs:Zeile 186.
   --- Ende der internen Ausnahmestapelüberwachung ---
   bei pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:Zeile 1944.
   bei pwiz.Skyline.Util.ParallelEx.LoopWithExceptionHandling(Action loop, Action`1 catchClause) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:Zeile 2246.
   bei pwiz.Skyline.Util.ParallelEx.For(Int32 fromInclusive, Int32 toExclusive, Action`1 body, Action`1 catchClause, Nullable`1 maxThreads) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:Zeile 2190.
   bei pwiz.Skyline.Model.SrmDocument.ChangeSettingsInternal(SrmSettings settingsNew, SrmSettingsChangeMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:Zeile 1068.
   bei pwiz.Skyline.Model.MProphetResultsHandler.ChangePeaks(IProgressMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\MProphetResultsHandler.cs:Zeile 172.
   bei pwiz.Skyline.EditUI.ReintegrateDlg.<>c__DisplayClass12_1.<OkDialog>b__1(IProgressMonitor pm) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\EditUI\ReintegrateDlg.cs:Zeile 133.
   bei pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:Zeile 254.
   --- Ende der internen Ausnahmestapelüberwachung ---
   bei pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:Zeile 1944.
   bei pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:Zeile 202.
   bei pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:Zeile 140.
   bei pwiz.Skyline.EditUI.ReintegrateDlg.OkDialog() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\EditUI\ReintegrateDlg.cs:Zeile 135.
---------------------------

Thanks for your help!

 unequalRTs.png 
view request
MRM triggered MSMS
(4 responses) heyang 2020-02-04

Dear Skyline team,

I want to compare MRM triggered MS2 with library spectra. Could I do this in skyline? if yes, how?

thanks,

Heyi

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Precursors per sample injection thinks it is transitions per sample injection
(2 responses) philip remes 2020-04-04

Hi Brendan,

This is a repost of issue 723. Sorry I didn't know to first post to Support and not Issues.

I've attached the skyline file. To reproduce what I saw,

  1. File->Export->Isolation List
  2. Instrument Type -> Thermo Fusion. Interestingly, if I use the first Instrument in the list, Agilent QTOF, and enter 216 in max precursors, the number of methods is correctly set to 9
  3. For Fusion, if 216 is set to max precursors, the number of methods is 138.

Thanks
Philip

 200404_export_list_debug.sky.zip 
view request
MS1 filtering: is manual XIC window mass tolerances possible?
(5 responses) alejandro.cohen 2020-10-23

Hi Skyline,

I'm using Skyline for a targeted metabolomics project on an QExactive (running at 70K resolution @ 200m/z according to Thermo). I inserted this value in the Transition Settings> Full-Scan>MS1- filtering>Resolving Power tab.

My standards are all showing at the expected Rt and with mass errors <2ppm. However, Skyline is also labeling other peaks between + - 2 to 9ppms. Is there any way to manually reduce the XIC mass windows, or set mass error thresholds to reduce the 'labeling clutter' in the chromatogram windows? Or do I just have to increase/fake the Resolving Power value on the Transition Settings pane.

Thanks again for you continuous support, Skyline rocks!

Alejandro

view request
Import Peptide Library from PEAKS Online from TIMS TOF data
(12 responses) ed3 2020-10-09

Hello,

I have been trying to import a peptide search from PEAKS online into Skyline but I keep getting an error and I was hoping somebody could please help me understand what is going on. The spectrum was acquired with a Bruker TIMS TOF, and when I try to import the pep.XML file in the "Peptide Search" option, I get an error saying "The .pep.xml file is not from one of the recognized sources." I have the IMS-TOF spectrum in .mzXML format in the same folder as the pep.xml file, and I am not sure why I get this error.

Thank you very much for your help.

Best,
Ed

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Problem library creation for cross-linking
(1 response) tatianibl 2020-10-22
Hi,

I am trying to build a cross-linking library using an output created by proxl from plink 2.0. However, I am having a problem. Follow bellow the error message.

Thanks,
All the best.
Tatiani.

---------------------------
Skyline-daily
---------------------------
ERROR: No spectra were found for the new library.

Command-line: C:\Users\tatiz\AppData\Local\Apps\2.0\49BTDOGX.MMZ\O1B3EWR8.PN0\skyl..tion_e4141a2a22107248_0014.0002_e354297bfe67025e\BlibBuild -s -A -H -o -c 0.95 -i teste -S "C:\Users\tatiz\AppData\Local\Temp\tmpA7B.tmp" "C:\Users\tatiz\Desktop\Arquivos_desktop\sample_input\teste.redundant.blib"
Working directory: C:\Users\tatiz\Desktop\Arquivos_desktop\sample_input
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: No spectra were found for the new library.

Command-line: C:\Users\tatiz\AppData\Local\Apps\2.0\49BTDOGX.MMZ\O1B3EWR8.PN0\skyl..tion_e4141a2a22107248_0014.0002_e354297bfe67025e\BlibBuild -s -A -H -o -c 0.95 -i teste -S "C:\Users\tatiz\AppData\Local\Temp\tmpA7B.tmp" "C:\Users\tatiz\Desktop\Arquivos_desktop\sample_input\teste.redundant.blib"
Working directory: C:\Users\tatiz\Desktop\Arquivos_desktop\sample_input
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\pwiz_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 156
view request
Confusion with Skyline and Proteome Discoverer
(3 responses) Mark Athanason 2020-10-20

Hi all,

First off thanks for providing the support that you do, hope I don't bother you all too much.

For right now I'm fine using the proteome discover software, but ultimately I want to perform DIA quant on similar data using skyline. Within PD, I perform a search using sequest and peptide validation with percolator with an FDR of 1%. Using the "import DDA search" wizard in the most recent version of skyline daily, I use the .msf file with a cut off score of 0.99. What I get in skyline doesn't seem to match what I see in PD at all. For example PD says I have 411 protein groups, but in skyline when I check remove repeat and duplicate peptides, I'm left with 222. Shouldn't these be the same since percolator is filtering for high confidence peptides? additionally, for some peptides PD does not correlate an MS1 trace where skyline does ... in my opinion incorrectly.

I'm uploading the .msf file, "Ibu1_DDA_1.raw" "Ibu1_DDA_2.raw" and "Ibu_DDA_3.raw" to https://skyline.ms/files.url

Any help would be greatly appreciated!
Mark

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Some peptides show incomplete peaks but in raw data they are okay
(17 responses) yingzhu1992 2020-10-05

Hi Skyline team,

I imported my result into the Skyline, some peptides show incomplete peaks, I checked from the raw data, they look fine. Can you please check and explain it for me and is there anything I can do to solve this?

And I also upload the raw data.

 20201001 H+L all transition.sky.zip  Capture.PNG  Raw data.PNG 
view request
HMDB-msp file import problem
(7 responses) Joerg 2020-10-14

Hi,
we tried to import the HMDB for small molecule quantification into Skyline. We downloaded the msp-file from Oliver Fiehn´s website. Skyline import part of the database, but just "forgets" the majority of the data. Is there any way to change the msp-file in a way that Skyline recognizes all entries? Or is import of the "original" xml-file from the HMDB website possible?
Many thanks in advance!
Jörg

view request
Storage location for custom PTMs
(2 responses) dkueltz 2020-10-07

Hi Nick, Brendan,
I have generated quite a few custom PTMS for one of my skyline documents (peptide settings/ modifications/ add) and would like to have them available for future documents. Where are these added PTMs stored? With only the Skyline document I entered them for or somewhere central?
Thanks,
Dietmar

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installation problem
(1 response) niyatti 2020-10-17

Hi, I tried to install Skyline (64 bit) on 64 bit Dell home basic desktop with Windows 7 through google chrome. The install.log is attached. Any idea what is wrong?

 install.log 
view request
Problem building spectral library
(3 responses) itv005 2020-10-15

Hi,

I am a farily new user of Skyline, and I am struggling to build my spectral library. Aside from some of the tutorials, my training is very limited. The source of my spectral library is a search from Proteome Discoverer 2.4 that I exported to a pep.xml file. I also have a used MSconvert to generate mzml files from my raw files.

I suspect that my issue is not due to any of the settings I made when setting up skyline, therefore I have not included those in this request. As seen in the attachment, I get an error saying the wrong directory is used, but I cannot seem to find out how it chooses this directory, or how to change it. I have put the skyline file, the raw files, and the converted spectrum files all in the same folder. The only thing missing, as I see it, is the program itself. However, I struggle to see the logic if this is the missing link, as this would be impractical for further projects.

I used the 20.2 version of Skyline and Windows 10 Home. The data I have is a non-targeted HPLC-MS/MS of CSF. I wish to use this library in a PRM project which is ready to be analyzed.

Thanks in advance! Let me know if I should add more info.
Ingrid

 skyline_error.PNG 
view request
Problems with import of peak boundaries in smallmolecule mode for .wiff data
(4 responses) Max 2020-08-24

Hi,

i am trying to import peak boundaries for a number of data sets based on .wiff data. We have set up some small molecule transitions for small molecules and peptides (due to qunatifiaction porpuses). I have used the allready discussed workarounds for small molecules (see https://skyline.ms/issues/home/issues/details.view?issueId=671) which is perfectly working for data from e.g. waters were a single filne represents a single injection. However, in the case of AbSciex 5500 the data is stored in a single .wiff file. I added therefore the sample name (alias Sample identifier) column to the peak boundaries list as stated in the FAQ for importing peak boundaries (see here: https://skyline.ms/_webdav/home/software/Skyline/@files/tutorials/ImportingIntegrationBoundaries-2_6.pdf). Nevertheless, skyline gives me an error message back stating that the sample names are not fitting to the sample names in the raw file . We have not changed the names of the single sample upon importing in Skyline thus i am looking for help to resolve this. I know i am missing something but i can not figure it out....

Looking forward to hear from you guys!

Max

PS: I have example files ready but can only send them confidentally.

view request
Skyline using questions
(5 responses) chaoxue 2020-10-05
  1. Can skyline show the same precursor and same product, but with different transition settings, such as CE and RF lens?
  2. Can skyline show different monoisotopic transitions, such as M+1, M+2?
view request
Reduction in the number of true peptide identification as the number of DIA raw files increases in the spectral library search
(13 responses) Chinmaya k 2020-09-28

Hello,

I have carried out a Spectral Library search for one of our DIA data generated with a 25m/z isolation window from 400-1000 m/z range. The spectral library search of single DIA.Raw file is able to identify 8000+ peptides with Q-Value < 0.01 from mProphet model. However, when multiple replicates of the same sample (Which includes the DIA file used for the first search) were searched against the same spectral library keeping all other parameters as the same is resulting in a drastically less number of peptides (<5000) with Q-value < 0.01 from mProphet model. And this goes on as the number of files increases.

What might be the reason for this? Why the number of true peptide identifications is decreasing as the number of DIA files are increased?

Prior to applying the mProphet model, the peptides RT was calibrated using Pierce iRT standard spiked in the sample. The screenshots of parameters used for RT prediction and calculation are attached below for your references.

Please let me whether any parameters have to be changed and if so, why? How does it will effect the DIA Spectral library search and statistical validation?

--
Chinmaya

 Peptide_Setting.PNG  Edit_iRT_Calculator.PNG  Edit_Retention_Time_Predictor.PNG 
view request
Identifying a significant protein in Skyline but it's not found in raw files.
(1 response) renad almahdali 2020-10-15

Dear Skyline team,

First, thank you loads for this amazing efficient software!
I am now working on constructing human proteomics profile from PBMC samples using Sciex TripleTOF 5600 and DDA method. I happened to come across a significant (highly expressed) protein in Skyline but when I wanted to check it in the proteins list exported from ProteinPilot raw file I didn't find it. My question is, is it possible to find a protein in Skyline software only but not from the original files?? I even tried to look it up by its protein name and accession including it synonyms.

Waiting for your support guys,
Thanks.
Renad Abdulrahman

view request
Unable to retrieve application files. Files corrupt in deployment. Skyline-64_20_2_0_286 unplugged version
(1 response) h a ebhardt 2020-10-15

Hi Brendan & team members of the Skyline team.

I tried installing Skyline-64_20_2_0_286 unplugged.

I first downloaded the zip file,
unpacked it,
ran setup.exe as administrator

Then: the setup.exe still accessed the internet and after downloading 3/4 of the program, the "Unable to retrieve application files. Files corrupt in deployment" occurred.

I attached a screenshot of the error plus the txt of Details...

There is a two problems here: first, the error itself.

Second, and more important, my IT dep't does not allow me to use programs which access the internet. Hence, the unplugged version. Is it possible to get an unplugged version which does not require the internet for installation?

Maybe the error is related to the fact that programs here are not allowed to access the outside world.

Kind regards,
Holger Alexander Ebhardt.

 skyline202error.jpg  26BB66Q3error.txt 
view request
fail to recognize spectral library from NIST
(3 responses) 851813663 2020-10-13

Dear nick
I have downloaded the Rat's spectral library from NIST, it was in msp format, and when I wanted to use the skyline to build the library, I found that the software could not recognize this format, and I was very eager for your help.
Thank you sincerely

 picture1.PNG 
view request
manual selection and change of ion mobility range for prm-PASEF
(2 responses) RBl 2020-10-14

Dear Skyline Team,
thank you very much for your constant efforts and all that you provide to the mass spec community! The software developed very much over past few years when i did not use it a lot!

I have started using Skyline in combination with the TimsTOF Pro recently. I needed to create a prm-PASEF method also with the ion mobility dimension. I noticed that ion mobility range that is showed in the Skyline was in many cases on the edge of the mobility peak/ion cloud and thereby a big proportion of the peak was not included. In some cases the ion mobility range in Skyline was in the middle of the mobility peak/ion cloud but edges were missing. I prepared some pictures so you can get the impression of what I am talking about. Please see the file attached.
I tried to set the ion mobility ranges in Skyline but I failed. Is there an option available for manual selection of ion mobility range or is it on me and I can not find it?
If there is no option for manual selection of ion mobility range, would it be possible to get something similar as manual chromatographic peak selection for Ion mobility peak/ion cloud in the future? And in the more distant future something like automatic peak peaking for ion mobility dimension if that is possible at all?

May you need more information from me, please do not hesitate to contact me.

Thank you very much for your efforts and your answer.
Best regards,
Renata

 ion mobility in skyline.pdf 
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Transition settings ion mobility "use spectral library mobility values when present" cannot be unchecked
(1 response) pierre-olivier schmit 2020-10-14

While unchecking the corresponding radio button and choosing "OK" : the radio button is automatically checked again if we re-open the transition settings ion mobility tab.

view request
ERROR: No Spectra were found for the new library
(1 response) lake n paul 2020-10-13

All,

Excited to see the DDA search implemented into Skyline. However, I tried unsuccessfully to run a search. The search goes to completion however I don't get any spectra for the detected peptides. The XIC for the peptides are present but no MS/MS spectra. An error (see attached) is given.

Help!

Lake

 ERROR.png 
view request
Skyline CMD help
(7 responses) smanda 2020-08-13

Hi Brendon/Kaipo,

I am trying to build a spectral library all using command line as we are trying to automate some of the steps at our lab. To this, I am unable to figure out, how can I choose peptides standards for RT calibration (after I import the library). My current steps are:

  1. SkylineCmd.exe --in=Empty.sky --out=New_library.sky --import-search-file=searchresults.mzid --import-fasta=20190717_Uniprot_concat_decoys.fasta
    This step creates a nr library and adds the peptides as targets.
    I do not specify RT peptides here.
    I then import a report format

  2. SkylineCmd.exe" --report-add=skyline.skyr

  3. SkylineCmd.exe --in=New_library.sky --report-name=OpenSWATH --report-file=test_report.tsv --report-format=tsv

I see that the exported reported as several NAs in the iRT column. We have around 40 endogenous peptides that are added as iRT standards in all experiments, can you please let me know, how to I add them at RT peptides and export the report with iRT values.

Regards,
Srikanth

view request
Some bugs shown in software
(4 responses) fenglinshen yzu 2020-10-13

Some peptides can't show the intact peak when we analysed MRM HR results using skyline which was the lastest released. Only three peptides were analysed with a 15 min gredient and 7.5 min-window was set. We also found the same scan data imported to the latest released skyline software showed only three transitions which was set as "full scan", and when the data was imported to earlier version, all of the transitions were shown. The details are shown in the attachments.

 新建 Microsoft PowerPoint 演示文稿.pptx 
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Unable to see any fragment ions in Skyline
(2 responses) clgunth2 2020-10-13

Hello - I created a test transition list with ions I can see in the raw mass spectra and imported into Skyline. When I imported the data, only the precursor mass is identified with the green circle next to the precursor name, even though the ions in my transition list were extracted from the raw file. The fragment ions don't have a red circle for being not detected but rather say "Chromatogram not available" if clicked on. I'm not sure if it's just not reading my data correctly or not. Again, these fragment ions were chosen directly from the raw spectra to test the workflow in Skyline, so they are present in the raw data.

My data file contains only MS2 scans for one precursor. My goal is to be able to filter through a much longer list of theoretical fragments for one complex structure. I'm following similar to what I would do if I was filtering through a transition list with MS1 data except the data file contains solely MS2 scans.

Thanks,
Crystal

 MS2_Test.sky.zip  SkylineError.PNG 
view request
Skyline 20.2 refuses to install
(5 responses) Tomas Vaisar 2020-10-13

For some reason, the manual install of 20.2 does not want to go - after Microsoft Store message - clicking Install Anyway does not do anything. It does not matter whether I run setup.exe normally or as Administrator?
My Windows 10 problem or Skyline setup.exe problem?

Thanks,

Tomas

view request
MS Amanda search result files?
(3 responses) dkueltz 2020-10-12

Hi Nick,
Are the msamanda search results stored somewhere in a format that can be imported into Scaffold or other viewers? It would be useful to have those search result files for comparison with other search engines.
Thanks,
Dietmar

view request
Customise reports
(3 responses) giulia lambiase 2020-10-09

Hello,

I happen to analyse lots of data and would like to automate the data analysis process. Is it possible to customise Skyline reports to make them run scripts for you?

Thanks,
Julia

view request
Question New Protein Abundance Report Feature
(4 responses) roman sakson 2020-10-12

Dear Skyline team,

thank you for providing us with the possibility to get numbers representing protein abundance per replicate directly from Skyline, which is also supposed to be part of the next main Skyline release. I intend to use this option rather frequently in my research and would like to understand it a bit better. I am mainly interested in the normalization case against heavy (each light signal must have a heavy counterpart to be considered).

In the release notes, you state that this protein abundance value is the same that the Group Comparison (GC) framework uses. I thought that, as default, on protein level GC sums up all transition areas that belong to the same protein (also across different peptides) that are quantitative and have a heavy counterpart and then normalizes this sum to the sum of heavy signals. This is slightly different compared with calculating normalized ratios transition by transition and then averaging those ratios, since strong signals dominate the sums of values, as previously described on several occasions. However, the explanation in the reporting window for the "Protein Abundance" feature now states that "The Protein Abundance is calculated by taking the average of the normalized areas of all of the Transitions under the Protein." This now sounds to me exactly as the latter option to do it and the question is, whether protein abundance numbers really are exactly what the GC framework uses?

Thanks a lot,

Roman

view request
Collision Energy Optimization for Precursors?
(2 responses) philip remes 2020-10-09

I've done the tutorial the does collision energy optimization by exporting transition lists: https://skyline.ms/_webdav/home/software/Skyline/@files/tutorials/SmallMoleculeMethodDevCEOpt.pdf
For small molecules, it would be useful to do the same thing for PRM experiments. But the Optimizing Dropdown box is disabled for File->Export->Isolation list, even though I see it for the Export->Transition List option. Is this a feature that could be added?

Thanks
Philip

view request
No chromatograms or spectra are reported for peptides with modified cys or met
(12 responses) caminha marcelle 2020-10-07

Hi,
I'm a new Skyline (20.1) user and I'm having difficulties analyzing peptides with cysteine carbamidomethylation and methionine oxidation. The spectra of peptides with cysteine only show up when I select carbamidomethylation as a variable modification, thus reporting them as from m/z without the expected mass differences; no chromatogram is shown in any settings. As for the peptide with methionine oxidation (as a variable modification), neither the spectra nor the chromatogram is reported. However, in both cases, the peptides are detected by the Comet search tool integrated into the PatternLab after either DDA or PRM method acquision. Could you, please, explain me how to solve this? Any advice is greatly appreciated.
Best regards,
Marcelle

view request
Crosslink search from Mascot to create library for Skyline
(8 responses) sandberg 2020-08-05

Hi,
Is it possible to use crosslinking results from the Mascot search engine to create a library to search the files for crosslinked peptides in Skyline?

Best,
Magdalena

view request
Optimizing FAIMS on new Thermo instruments
(4 responses) Martijn van Duijn 2019-09-18

Hi,

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

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

Regards,

Martijn van Duijn

view request
Peak removal from multi-injection replicate
(7 responses) Julienlab 2020-10-06

Hi,

I am analyzing PRM data collected as a multi-injection replicate (2 injections per sample) however for ~10% of my targets, skyline is quantifying the same peptide in both injections when it should only be quantified in one.

I have attached a pdf document showing an example of this where the first page is the correctly identified peak from the second injection as found in all four samples. The second page shows the chromatogram from injection 1 which should not identify this peptide, however it is still being quantified. The dotp scores in these cases are low and appear to be a combination of the two injections instead of only injection 2.

Is there any way to remove single injections from one peptide so the dotp score only reflects one injection?

Thank you,
Bridgette

 PRM-screenshots-False.pdf 
view request
Crashes for export of empty target lists for prm-PASEF
(1 response) jens decker 2020-10-05

We have observed crashes in case of most likely empty target lists. A version of the prmscheduler.dll which should be immune is in the build process. Nevertheless it would be good if Skyline will give a warning and stop the preparations for the export if there are no targets, which seems to be easy to get if certain flags are not set correctly for new users: Get aware of the fact that something is wrong.

view request
A request for support
(1 response) wangcg1119 2020-10-07
As far as I concern, Skyline is the best choice for MS data analysis. Quality data can be obtained based on Orbitrap which is equipped with FAIMS module. We wonder whether Skyline can be employed for procession on PRM data which is generated from Orbitrap Lumos equipped with FAIMS module. If so, please kindly provide relative tutorial to support our research. 
view request
Issue opening any files in Skyline-Daily
(1 response) wlstutts 2020-10-06

I just upgraded Skyline-daily today and now when I click "open file" or the folder icon to open a file, the program freezes. At this point I am unable to open any previously created Skyline documents.

view request
"Failed importing results...The calculator ... requires all of its standared peptides in order to determine a regression
(2 responses) Mark Athanason 2020-10-05

Hello,

I'm getting the following error, seen below. I'm simply trying to build a spectral library to perform DIA quant with 2 DDA runs. I've never seen this error before. I'm using the most current version of skyline daily. For RT standards, I selected automatic because when I select biognosys 10 or 11 it doesn't find any standards, for some reason. When selecting automatic, it finds 10 standards with a r^2 = 1. Going down the wizard is fine, then when it's importing the DDA data it cancels and throws this error:

At 11:25 AM:
Failed importing results file 'E:\Mark\spheroid\firstPlate\DMSO_DDA_1.mzXML'.
The calculator DMSO1 requires all of its standard peptides in order to determine a regression.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'E:\Mark\spheroid\firstPlate\DMSO_DDA_1.mzXML'.
The calculator DMSO1 requires all of its standard peptides in order to determine a regression. ---> pwiz.Skyline.Model.Irt.IncompleteStandardException: The calculator DMSO1 requires all of its standard peptides in order to determine a regression.
at pwiz.Skyline.Model.Irt.RCalcIrt.ChooseRegressionPeptides(IEnumerable`1 peptides, Int32& minCount) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Irt\RCalcIrt.cs:line 152
at pwiz.Skyline.Model.Results.ChromCacheBuilder.RetentionTimePredictor.CreateConversion() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 838
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 404
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252
--- End of inner exception stack trace ---

The search was performed with comet with .RAW files converted to .mzXML with default settings. The output file is a pep.xml. I think the error is occurring when it's trying to automatically train the mProphet model. In the search settings I performed a concatenated decoy search, with skyline being told to make 1 reverse decoy to train mProphet.
Any help would be appreciated,
Mark

view request
Exportation of peaks
(3 responses) bousahba imene 2020-10-05

Hello Skyteam :
i need to export the peaks of all my compounds in a report, I checked all the boxes in the editing list of the reports but i couldn't find it .. Is it possible ? if yes, how can i do ?

Thank you very much

view request
MS Amanda error
(4 responses) dkueltz 2020-10-03
Hi,
I am getting the following error message after specifying the PTMS (just CAM and MetOx) for a DDA search with MS Amanda within the latest build of Skyline daily. The error pops up when clicking next after specifying PTMs. I am not sure why the algorithm is looking for heavy isotope mods since I did not specify any of these in the peptide settings or ms amanda ptm options. I did report the error using the otion in the error popup and checked to include the Skyline document (since it was small) but the error report did not seem to work (at least there was no indication that anything happened). Let me know if you need a DDA raw data file for debugging. I am using bruker .d data and can upload a BSA QC file that I used to generate this error.
Thanks,
Dietmar

Skyline version: 20.1.9.268-f970ef4c5 (64-bit)
Installation ID: 62026530-da44-466f-b8a5-1c67fe40d053
Exception type: IndexOutOfRangeException
Error message: Modification type heavy not found.

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

System.IndexOutOfRangeException: Modification type heavy not found.
   at pwiz.Skyline.Model.DocSettings.PeptideModifications.ChangeModifications(IsotopeLabelType labelType, IList`1 prop) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\DocSettings\PeptideSettings.cs:line 1473
   at pwiz.Skyline.FileUI.PeptideSearch.MatchModificationsControl.AddCheckedModifications(SrmDocument document) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\PeptideSearch\MatchModificationsControl.cs:line 179
   at pwiz.Skyline.FileUI.PeptideSearch.ImportPeptideSearchDlg.UpdateModificationSettings() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\PeptideSearch\ImportPeptideSearchDlg.cs:line 0
   at pwiz.Skyline.FileUI.PeptideSearch.ImportPeptideSearchDlg.NextPage() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\PeptideSearch\ImportPeptideSearchDlg.cs:line 523
   at System.Windows.Forms.Control.OnClick(EventArgs e)
   at System.Windows.Forms.Button.OnClick(EventArgs e)
   at System.Windows.Forms.Button.OnMouseUp(MouseEventArgs mevent)
   at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.ButtonBase.WndProc(Message& m)
   at System.Windows.Forms.Button.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
Exception caught at:
   at System.Windows.Forms.Application.ThreadContext.OnThreadException(Exception t)
   at System.Windows.Forms.Control.WndProcException(Exception e)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Form.ShowDialog(IWin32Window owner)
   at pwiz.Skyline.SkylineWindow.ShowImportPeptideSearchDlg(Nullable`1 workflowType) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 3006
   at pwiz.Skyline.SkylineWindow.importPeptideSearchMenuItem_Click(Object sender, EventArgs e) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 2988
   at System.Windows.Forms.ToolStripItem.RaiseEvent(Object key, EventArgs e)
   at System.Windows.Forms.ToolStripMenuItem.OnClick(EventArgs e)
   at System.Windows.Forms.ToolStripItem.HandleClick(EventArgs e)
   at System.Windows.Forms.ToolStripItem.HandleMouseUp(MouseEventArgs e)
   at System.Windows.Forms.ToolStrip.OnMouseUp(MouseEventArgs mea)
   at System.Windows.Forms.ToolStripDropDown.OnMouseUp(MouseEventArgs mea)
   at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.ToolStrip.WndProc(Message& m)
   at System.Windows.Forms.ToolStripDropDown.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
   at pwiz.Skyline.Program.Main(String[] args) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Program.cs:line 306
view request
Error - Skyline failing to download
(1 response) claire tonry 2020-10-03

Hi,

I keep getting the error log when I attempt to download Skyline:

The following properties have been set:
Property: [AdminUser] = true {boolean}
Property: [InstallMode] = HomeSite {string}
Property: [ProcessorArchitecture] = AMD64 {string}
Property: [VersionNT] = 6.2.0 {version}
Running checks for package 'Windows Installer 3.1', phase BuildList
The following properties have been set for package 'Windows Installer 3.1':
Running checks for command 'WindowsInstaller3_1\WindowsInstaller-KB893803-v2-x86.exe'
Result of running operator 'VersionGreaterThanOrEqualTo' on property 'VersionMsi' and value '3.1': true
Result of checks for command 'WindowsInstaller3_1\WindowsInstaller-KB893803-v2-x86.exe' is 'Bypass'
'Windows Installer 3.1' RunCheck result: No Install Needed
Running checks for package 'Microsoft .NET Framework 4 (x86 and x64)', phase BuildList
Reading value 'Version' of registry key 'HKLM\Software\Microsoft\NET Framework Setup\NDP\v4\Full'
Read string value '4.5.51641'
Setting value '4.5.51641 {string}' for property 'DotNet40Full_TargetVersion'
The following properties have been set for package 'Microsoft .NET Framework 4 (x86 and x64)':
Property: [DotNet40Full_TargetVersion] = 4.5.51641 {string}
Running checks for command 'DotNetFX40\dotNetFx40_Full_x86_x64.exe'
Result of running operator 'ValueEqualTo' on property 'InstallMode' and value 'HomeSite': true
Result of checks for command 'DotNetFX40\dotNetFx40_Full_x86_x64.exe' is 'Bypass'
Running checks for command 'DotNetFX40\dotNetFx40_Full_setup.exe'
Result of running operator 'ValueNotEqualTo' on property 'InstallMode' and value 'HomeSite': false
Result of running operator 'VersionGreaterThanOrEqualTo' on property 'DotNet40Full_TargetVersion' and value '4.0.30129': true
Result of checks for command 'DotNetFX40\dotNetFx40_Full_setup.exe' is 'Bypass'
'Microsoft .NET Framework 4 (x86 and x64)' RunCheck result: No Install Needed
Running checks for package '.NET Framework 3.5 SP1', phase BuildList
Reading value 'SP' of registry key 'HKLM\Software\Microsoft\NET Framework Setup\NDP\v3.5'
Read integer value 1
Setting value '1 {int}' for property 'DotNet35SP'
The following properties have been set for package '.NET Framework 3.5 SP1':
Property: [DotNet35SP] = 1 {int}
Running checks for command 'DotNetFX35SP1\dotNetFx35setup.exe'
Result of running operator 'ValueGreaterThanEqualTo' on property 'DotNet35SP' and value '1': true
Result of checks for command 'DotNetFX35SP1\dotNetFx35setup.exe' is 'Bypass'
'.NET Framework 3.5 SP1' RunCheck result: No Install Needed
Launching Application.
Application appears to be an application manifest

view request
Error Import Skyline Daily SureQuant data
(4 responses) romeally 2020-10-02

I am developing a SureQuant assay using Skyline Daily and for the most part it is working fine, however sometimes randomly my .raw data files will not import. I can open them in Qualbrowser and they seem fine but they seem to randomly stop importing at certain scan numbers. Sometimes it fails early and sometimes it fails late in the chromatogram. I have attached the error message I get upon import. This was acquired on a Lumos instrument with software version 3.3 and the latest Skyline Daily using the triggered acquisition tab checked. Any suggestions? Appreciated!

 ImportError.docx 
view request
Failed importing results from mzML file
(2 responses) michal zawadzki 2020-10-02

Dear Skyline Team,

I am trying to upload an mzML file converted from a Thermo .raw file with MSConvert into a Skyline document where I have already imported a chromatogram library generated by EncyclopeDIA. I do not think the file is corrupted as I am able to use it in EncyclopeDIA without any issues.

I use Skyline (64bit) 20.1.0.155 (a0e7323e3) and MSConvert (Version: 3.0.20219-6a8ecb8e3 (developer build)).

Below is a full text of the error I am getting:

At 12:11:
Failed importing results file 'C:\Users\u525581\Desktop\Skyline EX4163G_DIA_maize_embryos_310720\20200731_EX4163C_001_01.mzML'.
[IO::HandlerBinaryDataArray] Unknown binary data type.

Inner exceptions:
Exception type: System.Exception
Error message: [IO::HandlerBinaryDataArray] Unknown binary data type.
[IO::HandlerBinaryDataArray] Unknown binary data type.
at pwiz.CLI.msdata.ChromatogramList.chromatogram(Int32 index, Boolean getBinaryData)
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetChromatogram(Int32 chromIndex, String& id, Single[]& timeArray, Single[]& intensityArray) in C:\proj\skyline_20_1_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 616
at pwiz.Skyline.Model.Results.GlobalChromatogramExtractor.GetChromatogram(Int32 index, Single[]& times, Single[]& intensities) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromDataProvider.cs:line 172
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.GetChromatogram(Int32 id, Target modifiedSequence, Color peptideColor, ChromExtra& extra, TimeIntensities& timeIntensities) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 599
at pwiz.Skyline.Model.Results.ChromData.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromData.cs:line 77
at pwiz.Skyline.Model.Results.ChromDataSet.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 236
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Load(ChromDataProvider provider) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 144
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 439
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252

Would you be able to advice how to solve this problem, please?

view request
Broken compatibility with Shimadzu LCMS 8050 after replacing Shimadzu DataReader with IoModule for MRM files
(3 responses) Pawel 2020-09-30
Hi Skyline Team!

Thanks for your continuous work on the software. Just wanted to make you aware that the most recent update 20.1.9.268 broke the compatibility with Shimadzu LCMS 8050. The following error appears:

"Failed importing results file 'C:\LabSolutions\Data\Chalani\20200929_QC(100x)_021.lcd'.
[ShimadzuReader::getSpectrum] GetMSSpectrumByScan: E_INVALIDARG"

Old version of Skyline still works but I am unable to use Skyline for importing Shimadzu data on any computer on which I updated the software to the above version. Would you mind having a look into this problem?

Regards,
Pawel
view request
Reprocessing of data obtained by neutral loss on a QQQ using skyline
(2 responses) julien faugere 2020-10-01

Hello,

I did analysis by neutral loss on QQQ on a lipid sample to observe triglycerides. Can we use skyline to reprocess this type of data ? I have often used skyline for reprocessing MRM data but never for neutral loss.

Thank you for your answer.

Kind regards,

Julien Faugere

view request
Retention time recognized in sec format
(1 response) Khang Huynh 2020-09-29

Hi Brendan and Team,

I have just updated to the latest version of Skyline-daily today. When I imported the LC-MS/MS data files, the retention times on the chromatograms were in "second" format instead of in "minute" as usual (photo attached). I have tried to look into the Settings but could not figure out how to change it back to the "minute" format. Could you please help?

Operating system: Windows 10.
Small molecules for Quantification.
Raw data files: Shimadzu .lcd

Thanks,
Khang Huynh

 Screenshot 2020-09-29 172658.jpg 
view request
Correct spectrum selection for non-redundant library
(1 response) Juan C. Rojas E. 2020-09-30

Dear support,

Attached you can find a few slides displaying my issue.

In multiple instances I have observed that the spectrum selected for the non-redundant library is suboptimal compared to some other spectra available (Slide 1 and 2). For DDA files the problem is easily circumvented by working with the redundant library, but for library generation for DIA, MRM, and/or PRM methods could lead to comparisons to suboptimal representative library spectrum.

The .mzXML files (exported from PEAKS) for data acquired in resolution mode are kept in resolution mode format when exported (Slide 3). Maybe this is the reason for the mismatch due to better random matching (i.e. in mass tolerance consideration) to some of the "split" peaks of some spectra compared to others even if the absolute abundance is lower.

Is it possible to manually exchange the best representative spectrum for the non-redundant library?

If not, could a peak picking step be included in the library building procedure? Or should I just perform it with MSConvert externally?

As always, thank you for your time and support.
Sincerely,
JC

 Library_msms_spectrum_selection.pptx 
view request
Broad mass range for peak extraction
(4 responses) harald schoeny 2020-09-18

Dear Skyline Team,

I have lipid data measured on an HILIC- Orbitrap QExactive HF with an all ion fragmentation (AIF) approach. I figured it out to extract single masses with the help of the forum but I would like to extract a broader mass range (m/z 530-650 ) withing one peak. It should be the sum of all lipid species within one lipid class. Unfortunately, I couldn't find a way to do that in Skyline. I have tried to set the MS/MS filtering to Isolation scheme/Use results data isolation targets/ Isolation width/fixed/120 mz but nothing really happened. Is there another workaround for that?

Thank you in advance.
Best, Harald

view request
Problem in building of spectral library
(1 response) arvindverma0078 2020-09-29

Dear Skyline team,

Recently I used Skyline to build a spectral library from .mgf files. However, I want to convert .mgf file to blib format, because skyline do not accept .mgf file. If a direct conversion is not possible, perhaps there is a way to convert the library to an intermediate format that you are aware of?
Moreover, it is very helpful to me. If you assist me to building of spectral library from my data generated from Thermo Scientific Orbitrap Fusion MS platform.

Best regards,
Arivnd

view request
Missing light product ions in SureQuant experiment
(3 responses) carmen.gonzaleztejedo 2020-09-28
Dear Team, We performed a SureQuant experiment in an Orbitrap Exploris 480. When we load the data into the Skyline, we can see the XIC for the light and heavy precursors and the XICs for the heavy fragments. However, we cannot see the fragments of the light precursors (see attached). I have checked all the peptide and transition settings but I cannot figure out what the issue is. I would really appreciate if you can help me with that. Thanks a lot in advance. Regards, Carmen.
 SkylineSupport_OE480_SureQuant.pptx 
view request
Custom iRT peptides
(3 responses) benoit fatou 2020-09-24

Dear Skyline Team,

I was wondering if it is possible to create custom iRT peptides for RT adjustment in MRM experiments. In fact, I am working on plasma samples and I would like to use some endogenous peptides from the serum albumin in my samples.
Thanks for your help,
Best,
Benoit

view request
DIA 3-plex SILAC
(1 response) trinidad martincampos 2020-09-27

Hello all,

First of all, thank you very much for the effort you put into Skyline for all of us ;)

I took a look at the post with "DIA SILAC" but my problem is still not solved... I have a 3-plex SILAC (light, medium and heavy) experiment that I am going to analyse with DIA in an orbitrap fusion.
Any hint about how could I do that? What happens with a peptide whose light and medium (for example)tags are in different windows? Any possible workflow to do this?

Thank you very much!!!
Trini

view request
How did skyline read the OpenSWATH results?
(6 responses) andyzcq 2020-09-19

I followed the tutorial, and found there is no merged.osw file in OpenSWATH results.

I have used OpenSWATH (V. 2.5) to analyze my DIA data.

So how did Skyline read OpenSWATH (V. 2.5) results?

view request
Oxonium ions for MRM glycan analysis
(3 responses) giulia lambiase 2020-09-24

Hello,
I am writing an MRM method for monitoring glycans in mAbs. I need to add oxonium ions in my transition list but cannot work out how to do it in Skyline. Could you please support me with this?
Thanks,
Julia

view request
Skyline-daily Program Won't Open
(2 responses) ehubb004 2020-09-22

Yesterday, for no clear reason, the skyline-daily program (the most current version) stopped opening on my computer. When I try to open it, my computer briefly registers that it's loading, but nothing actually happens, and task manager never registers Skyline activity. I've tried restarting my computer, as well as completely reinstalling skyline-daily and even redownloading the installer program, but nothing has affected this issue.

view request
Retention time selection
(6 responses) ojulien 2020-09-17

Hi,

We imported 4 .raw files into skyline, and used a bibliospec file to import all the sequences into skyline.

We then created an inclusion list for all the peptides of interest (~700) for PRM.

However, the RT in the inclusion list are all off if I select "average RT", since the peptides selected were not found in all datasets. The other option is to use a single dataset out of 4, but then the RT will be off for the peptides that were not found in that specific dataset.

The best way would be to use the RT from the dataset that matches the spectral library. Is there a way to do this, am I missing something? If not, is it possible to add this as an option in future release?

This would be great, as of right now, we are adjusting the RT window manually for each peptides in each datasets...

OJ

view request
Waters Method Export Failure (Masslynx 4.2 SCN986)
(1 response) Will Thompson 2020-09-23

Hi Brian et al

We have recently upgraded two of our Waters TQ-S systems to Windows 10, and purchased licenses for Masslynx 4.2 SCN986 to do this upgrade. When trying to export *.exp methods for the instrument, we are now getting errors that the method cannot be exported. I am pasting in the long text of the method below. Do you have any hints from this text on whether or not it is a masslynx version issue, or something else? We have confirmed the method export is functional on MassLynx 4.2 SCN1007, which runs on the TQ-XS. I have asked Waters if there is a more recent upgrade for the TQ-S, or if we can run SCN1007 on the TQ-S. Maybe there are other questions you guys can ask your Waters contacts?

This would mean that any Waters TQ-S users who upgrade to Windows 10 will not be able to export methods from Skyline any more... :(

Cheers,

Will

Error text below


System.IO.IOException: ERROR: Method not found: 'Boolean CompoundDatabaseClassLibrary.CompoundDatabase.InsertNewIonDetails(Int32, Int32, TransMode, Int32, Int32, Int32, Int32, Int32, Int32)'.

Command-line: Method\Waters\BuildWatersMethod -w 2 -s -m "C:\VerifyETemplate.exp"
Working directory: D:\5633
Standard input:
D:\5633~SKEB17.tmp
D:\5633\5633_TQS_v1.exp
protein.name,peptide.seq,precursor.mz,precursor.retT,product.m_z,collision_energy,cone_voltage,peptide_unmod.seq,ion_name,library_rank
1,C0.[M+],162.1,0.56,85.1,18,32,C0,Ion [85.100549/85.100549],-1
2,d3-C0.[M+],165.2,0.56,85.1,18,32,d3-C0,Ion [85.100549/85.100549],-1
3,C2.[M+],204.1,0.9,85.1,18,26,C2,Ion [85.100549/85.100549],-1
4,d3-C2.[M+],207.1,0.9,85.1,18,26,d3-C2,Ion [85.100549/85.100549],-1
13,C3.[M+],218.1,1.73,85.1,18,28,C3,Ion [85.100549/85.100549],-1
14,d3-C3.[M+],221.1,1.73,85.1,18,28,d3-C3,Ion [85.100549/85.100549],-1
15,iC4.[M+],232.1,2.19,85.1,18,28,iC4,Ion [85.100549/85.100549],-1
16,C4.[M+],232.1,2.29,85.1,18,28,C4,Ion [85.100549/85.100549],-1
12,d3-C4.[M+],235.2,2.27,85.1,18,28,d3-C4,Ion [85.100549/85.100549],-1
21,C5:1.[M+],244.1,2.66,85.1,20,32,C5:1,Ion [85.100549/85.100549],-1
22,C4:1-M.[M+],244.1,2.71,85.1,20,32,C4:1-M,Ion [85.100549/85.100549],-1
23,C5-P.[M+],246.2,2.69,85.1,18,30,C5-P,Ion [85.100549/85.100549],-1
24,C4-2M.[M+],246.2,2.81,85.1,18,30,C4-2M,Ion [85.100549/85.100549],-1
25,iC5.[M+],246.2,2.89,85.1,18,30,iC5,Ion [85.100549/85.100549],-1
26,C5.[M+],246.2,3.01,85.1,18,30,C5,Ion [85.100549/85.100549],-1
5,C3-DC.[M+],248.1,0.8,85.1,21,30,C3-DC,Ion [85.100549/85.100549],-1
6,C4-OH.[M+],248.1,1.25,85.1,21,30,C4-OH,Ion [85.100549/85.100549],-1
19,d9-C5.[M+],255.2,2.86,85.1,18,30,d9-C5,Ion [85.100549/85.100549],-1
20,C6.[M+],260.2,3.56,85.1,18,30,C6,Ion [85.100549/85.100549],-1
7,C4-DC.[M+],262.2,1.2,85.1,21,32,C4-DC,Ion [85.100549/85.100549],-1
8,C3-DC-M.[M+],262.2,1.5,85.1,21,32,C3-DC-M,Ion [85.100549/85.100549],-1
9,C5OH-I.[M+],262.2,1.78,85.1,21,32,C5OH-I,Ion [85.100549/85.100549],-1
29,d3-C6.[M+],263.2,3.55,85.1,18,30,d3-C6,Ion [85.100549/85.100549],-1
10,C5-DC.[M+],276.1,1.71,85.1,20,32,C5-DC,Ion [85.100549/85.100549],-1
11,C6-OH.[M+],276.1,2.53,85.1,20,32,C6-OH,Ion [85.100549/85.100549],-1
31,C8:1.[M+],286.2,4.16,85.1,22,34,C8:1,Ion [85.100549/85.100549],-1
32,C8.[M+],288.2,4.28,85.1,22,34,C8,Ion [85.100549/85.100549],-1
17,C6-DC.[M+],290.2,2.01,85.1,20,34,C6-DC,Ion [85.100549/85.100549],-1
18,C5-M-DC.[M+],290.2,2.08,85.1,20,34,C5-M-DC,Ion [85.100549/85.100549],-1
33,d3-C8.[M+],291.2,4.27,85.1,22,34,d3-C8,Ion [85.100549/85.100549],-1
28,C10:1.[M+],314.2,4.76,85.1,22,37,C10:1,Ion [85.100549/85.100549],-1
34,C10.[M+],316.2,4.86,85.1,22,38,C10,Ion [85.100549/85.100549],-1
27,C8-DC.[M+],318.2,2.94,85.1,22,35,C8-DC,Ion [85.100549/85.100549],-1
35,d3-C10.[M+],318.9,4.85,85.1,22,38,d3-C10,Ion [85.100549/85.100549],-1
37,C12:1.[M+],342.3,5.29,85.1,24,40,C12:1,Ion [85.100549/85.100549],-1
39,C12.[M+],344.2,5.37,85.1,22,38,C12,Ion [85.100549/85.100549],-1
30,C10-DC.[M+],346.2,3.62,85.1,22,36,C10-DC,Ion [85.100549/85.100549],-1
40,d3-C12.[M+],347.2,5.37,85.1,22,38,d3-C12,Ion [85.100549/85.100549],-1
36,C12-OH.[M+],360.3,4.87,85.1,23,38,C12-OH,Ion [85.100549/85.100549],-1
38,C14:2.[M+],368.3,5.33,85.1,23,39,C14:2,Ion [85.100549/85.100549],-1
42,C14:1.[M+],370.2,5.76,85.1,23,39,C14:1,Ion [85.100549/85.100549],-1
43,C14.[M+],372.2,5.85,85.1,24,40,C14,Ion [85.100549/85.100549],-1
44,d3-C14.[M+],375,5.85,85.1,24,40,d3-C14,Ion [85.100549/85.100549],-1
49,cis-9-C16:1.[M+],398.3,5.98,85.1,25,42,cis-9-C16:1,Ion [85.100549/85.100549],-1
50,trans-2-C16:1.[M+],398.3,6.2,85.1,25,42,trans-2-C16:1,Ion [85.100549/85.100549],-1
51,C16.[M+],400.4,6.27,85.1,24,44,C16,Ion [85.100549/85.100549],-1
52,d3-C16.[M+],403.4,6.27,85.1,24,44,d3-C16,Ion [85.100549/85.100549],-1
41,C16:1-OH.[M+],414.3,5.51,85.1,26,44,C16:1-OH,Ion [85.100549/85.100549],-1
45,C16-OH.[M+],416.3,5.85,85.1,26,44,C16-OH,Ion [85.100549/85.100549],-1
48,C18:2.[M+],424.4,6.09,85.1,26,45,C18:2,Ion [85.100549/85.100549],-1
54,C18:1.[M+],426.4,6.34,85.1,27,45,C18:1,Ion [85.100549/85.100549],-1
55,C18.[M+],428.4,6.62,85.1,28,50,C18,Ion [85.100549/85.100549],-1
56,d3-C18.[M+],431.4,6.61,85.1,28,50,d3-C18,Ion [85.100549/85.100549],-1
46,C18:1-OH.[M+],442.4,5.97,85.1,28,46,C18:1-OH,Ion [85.100549/85.100549],-1
53,C18-OH.[M+],444.3,6.27,85.1,28,46,C18-OH,Ion [85.100549/85.100549],-1
47,C20:4.[M+],448.3,6.08,85.1,28,48,C20:4,Ion [85.100549/85.100549],-1
---> System.IO.IOException: ERROR: Method not found: 'Boolean CompoundDatabaseClassLibrary.CompoundDatabase.InsertNewIonDetails(Int32, Int32, TransMode, Int32, Int32, Int32, Int32, Int32, Int32)'.

Command-line: Method\Waters\BuildWatersMethod -w 2 -s -m "C:\VerifyETemplate.exp"
Working directory: D:\5633
Standard input:
D:\5633~SKEB17.tmp
D:\5633\5633_TQS_v1.exp
protein.name,peptide.seq,precursor.mz,precursor.retT,product.m_z,collision_energy,cone_voltage,peptide_unmod.seq,ion_name,library_rank
1,C0.[M+],162.1,0.56,85.1,18,32,C0,Ion [85.100549/85.100549],-1
2,d3-C0.[M+],165.2,0.56,85.1,18,32,d3-C0,Ion [85.100549/85.100549],-1
3,C2.[M+],204.1,0.9,85.1,18,26,C2,Ion [85.100549/85.100549],-1
4,d3-C2.[M+],207.1,0.9,85.1,18,26,d3-C2,Ion [85.100549/85.100549],-1
13,C3.[M+],218.1,1.73,85.1,18,28,C3,Ion [85.100549/85.100549],-1
14,d3-C3.[M+],221.1,1.73,85.1,18,28,d3-C3,Ion [85.100549/85.100549],-1
15,iC4.[M+],232.1,2.19,85.1,18,28,iC4,Ion [85.100549/85.100549],-1
16,C4.[M+],232.1,2.29,85.1,18,28,C4,Ion [85.100549/85.100549],-1
12,d3-C4.[M+],235.2,2.27,85.1,18,28,d3-C4,Ion [85.100549/85.100549],-1
21,C5:1.[M+],244.1,2.66,85.1,20,32,C5:1,Ion [85.100549/85.100549],-1
22,C4:1-M.[M+],244.1,2.71,85.1,20,32,C4:1-M,Ion [85.100549/85.100549],-1
23,C5-P.[M+],246.2,2.69,85.1,18,30,C5-P,Ion [85.100549/85.100549],-1
24,C4-2M.[M+],246.2,2.81,85.1,18,30,C4-2M,Ion [85.100549/85.100549],-1
25,iC5.[M+],246.2,2.89,85.1,18,30,iC5,Ion [85.100549/85.100549],-1
26,C5.[M+],246.2,3.01,85.1,18,30,C5,Ion [85.100549/85.100549],-1
5,C3-DC.[M+],248.1,0.8,85.1,21,30,C3-DC,Ion [85.100549/85.100549],-1
6,C4-OH.[M+],248.1,1.25,85.1,21,30,C4-OH,Ion [85.100549/85.100549],-1
19,d9-C5.[M+],255.2,2.86,85.1,18,30,d9-C5,Ion [85.100549/85.100549],-1
20,C6.[M+],260.2,3.56,85.1,18,30,C6,Ion [85.100549/85.100549],-1
7,C4-DC.[M+],262.2,1.2,85.1,21,32,C4-DC,Ion [85.100549/85.100549],-1
8,C3-DC-M.[M+],262.2,1.5,85.1,21,32,C3-DC-M,Ion [85.100549/85.100549],-1
9,C5OH-I.[M+],262.2,1.78,85.1,21,32,C5OH-I,Ion [85.100549/85.100549],-1
29,d3-C6.[M+],263.2,3.55,85.1,18,30,d3-C6,Ion [85.100549/85.100549],-1
10,C5-DC.[M+],276.1,1.71,85.1,20,32,C5-DC,Ion [85.100549/85.100549],-1
11,C6-OH.[M+],276.1,2.53,85.1,20,32,C6-OH,Ion [85.100549/85.100549],-1
31,C8:1.[M+],286.2,4.16,85.1,22,34,C8:1,Ion [85.100549/85.100549],-1
32,C8.[M+],288.2,4.28,85.1,22,34,C8,Ion [85.100549/85.100549],-1
17,C6-DC.[M+],290.2,2.01,85.1,20,34,C6-DC,Ion [85.100549/85.100549],-1
18,C5-M-DC.[M+],290.2,2.08,85.1,20,34,C5-M-DC,Ion [85.100549/85.100549],-1
33,d3-C8.[M+],291.2,4.27,85.1,22,34,d3-C8,Ion [85.100549/85.100549],-1
28,C10:1.[M+],314.2,4.76,85.1,22,37,C10:1,Ion [85.100549/85.100549],-1
34,C10.[M+],316.2,4.86,85.1,22,38,C10,Ion [85.100549/85.100549],-1
27,C8-DC.[M+],318.2,2.94,85.1,22,35,C8-DC,Ion [85.100549/85.100549],-1
35,d3-C10.[M+],318.9,4.85,85.1,22,38,d3-C10,Ion [85.100549/85.100549],-1
37,C12:1.[M+],342.3,5.29,85.1,24,40,C12:1,Ion [85.100549/85.100549],-1
39,C12.[M+],344.2,5.37,85.1,22,38,C12,Ion [85.100549/85.100549],-1
30,C10-DC.[M+],346.2,3.62,85.1,22,36,C10-DC,Ion [85.100549/85.100549],-1
40,d3-C12.[M+],347.2,5.37,85.1,22,38,d3-C12,Ion [85.100549/85.100549],-1
36,C12-OH.[M+],360.3,4.87,85.1,23,38,C12-OH,Ion [85.100549/85.100549],-1
38,C14:2.[M+],368.3,5.33,85.1,23,39,C14:2,Ion [85.100549/85.100549],-1
42,C14:1.[M+],370.2,5.76,85.1,23,39,C14:1,Ion [85.100549/85.100549],-1
43,C14.[M+],372.2,5.85,85.1,24,40,C14,Ion [85.100549/85.100549],-1
44,d3-C14.[M+],375,5.85,85.1,24,40,d3-C14,Ion [85.100549/85.100549],-1
49,cis-9-C16:1.[M+],398.3,5.98,85.1,25,42,cis-9-C16:1,Ion [85.100549/85.100549],-1
50,trans-2-C16:1.[M+],398.3,6.2,85.1,25,42,trans-2-C16:1,Ion [85.100549/85.100549],-1
51,C16.[M+],400.4,6.27,85.1,24,44,C16,Ion [85.100549/85.100549],-1
52,d3-C16.[M+],403.4,6.27,85.1,24,44,d3-C16,Ion [85.100549/85.100549],-1
41,C16:1-OH.[M+],414.3,5.51,85.1,26,44,C16:1-OH,Ion [85.100549/85.100549],-1
45,C16-OH.[M+],416.3,5.85,85.1,26,44,C16-OH,Ion [85.100549/85.100549],-1
48,C18:2.[M+],424.4,6.09,85.1,26,45,C18:2,Ion [85.100549/85.100549],-1
54,C18:1.[M+],426.4,6.34,85.1,27,45,C18:1,Ion [85.100549/85.100549],-1
55,C18.[M+],428.4,6.62,85.1,28,50,C18,Ion [85.100549/85.100549],-1
56,d3-C18.[M+],431.4,6.61,85.1,28,50,d3-C18,Ion [85.100549/85.100549],-1
46,C18:1-OH.[M+],442.4,5.97,85.1,28,46,C18:1-OH,Ion [85.100549/85.100549],-1
53,C18-OH.[M+],444.3,6.27,85.1,28,46,C18-OH,Ion [85.100549/85.100549],-1
47,C20:4.[M+],448.3,6.08,85.1,28,48,C20:4,Ion [85.100549/85.100549],-1

at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
at pwiz.Skyline.Util.Extensions.UtilProcess.RunProcess(ProcessStartInfo psi, String stdin, String messagePrefix, IProgressMonitor progress, IProgressStatus& status) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Extensions\UtilProcess.cs:line 45
at pwiz.Skyline.Model.MethodExporter.ExportMethod(String exeName, List1 argv, String fileName, String templateName, Dictionary2 dictTranLists, IProgressMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Export.cs:line 4162
at pwiz.Skyline.Model.WatersMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Export.cs:line 3959
at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254 --- End of inner exception stack trace --- at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1942 at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 204
at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140 at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action1 performExport) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2095

view request
Export method to MassLynx 4.2 version
(6 responses) danielacgranato 2018-11-27

Dear,

I was wondering if the new version of Skyline (v.4.2) allows to export SRM methods to MassLynx version 4.2. Last year I was unable to do so and I received a response that Waters was working on making it possible. As a solution to the problem I have been exporting the method to a computer not connected to the equipment (Xevo TQ-XS) and to an older version of MassLynx (v.4.1). Do you know if it is possible now to export directly to MassLynx 4.2?

Thank you very much. Best, Daniela

view request
Cannot Roundtrip Small Molecule Transition List
(3 responses) damien ready 2020-09-17

Greetings,
We are looking into using Skyline for some of our small molecule processing, and I ran into a rough edge I thought could be improved around managing transition lists. If I graphically build a small molecule transition list in Skyline and export it, Skyline fails to import the list into a new document.

If you follow the below, I was able to manipulate the exported document into a format that Skyline would import, but the workflow is cumbersome for something that feels like it should be natively handled by the GUI.

If you do intend to investigate the issue, I should be clear that my actual interest is in automating the creation of Skyline documents + transition lists through the Skyline Commandline runner. This work through the GUI was just to establish POC.

Skyline Daily 20.1.9.234
To reproduce:

  • Start Skyline
  • Select Small Molecule workflow
  • Edit->Insert-Transition List
    • Molecule List Name: aspirin
    • Precursor Name: aspirin
    • Precursor Formula: C9H8O4
    • Precursor Adduct: [M+H]
    • Precursor m/z: 181.0495348
    • Precursor Charge: 1
  • Insert molecule
  • File->Export->Transition List
  • File->New
  • File->Import->Transition List
    Results in following error message:
System.IO.InvalidDataException: Failed to find peptide column., line 1. ---> System.IO.InvalidDataException: Failed to find peptide column., line 1. ---> pwiz.Skyline.Model.LineColNumberedIoException: Failed to find peptide column., line 1.
   at pwiz.Skyline.Model.MassListImporter.Import(IProgressMonitor progressMonitor, String sourceFile, ColumnIndices indices, IDictionary`2 dictNameSeq, List`1& irtPeptides, List`1& librarySpectra, List`1& errorList) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Import.cs:line 485
   at pwiz.Skyline.Model.MassListImporter.Import(IProgressMonitor progressMonitor, String sourceFile, List`1& irtPeptides, List`1& librarySpectra, List`1& errorList) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Import.cs:line 423
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Model.MassListImporter.Import(IProgressMonitor progressMonitor, String sourceFile, List`1& irtPeptides, List`1& librarySpectra, List`1& errorList) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Import.cs:line 427
   at pwiz.Skyline.Model.SrmDocument.ImportMassList(MassListInputs inputs, IProgressMonitor progressMonitor, IdentityPath to, IdentityPath& firstAdded, List`1& irtPeptides, List`1& librarySpectra, List`1& errorList, List`1& peptideGroups) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1454
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass1079_0.<ImportMassList>b__2(IProgressMonitor longWaitBroker) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1925
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1940
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 202
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.SkylineWindow.ImportMassList(MassListInputs inputs, String description, Boolean assayLibrary) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1923
   at pwiz.Skyline.SkylineWindow.ImportMassList(String fileName) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1902

Consulting the Help on the "Transition List" window, I attempted to hand-edit the exported CSV to include the columns (MoleculeGroup, PrecursorName, ProductName, PrecursorFormula, ProductFormula, PrecursorMz, ProductMz, PrecursorCharge, ProductCharge, PrecursorAdduct, ProductAdduct, PrecursorRT, PrecursorRTWindow, PrecursorCE, PrecursorDT, HighEnergyDTOffset, PrecursorIM, HighEnergyIMOffset, IMUnits, PrecursorCCS, SLens, ConeVoltage, CompensationVoltage, DeclusteringPotential, Note, LabelType, InChiKey, CAS, HMDB, InChi, SMILES, KEGG, ProductNeutralLoss), added the required data, but this too failed to import. Lastly, I added the suggested columns, filled in the extra data, and deleted the originally exported columns - this worked.

Attached:

  • small_molecule_ts_export.csv - failed to import - Skyline small molecule built and exported transition list
  • small_molecule_ts_export_extracolumns.csv - failed to import - Skyline small molecule export + manually added suggested columns
  • small_molecule_ts_export_extracolumns_only.csv - successful import - Skyline small molecule export + manually added suggested columns only

Thanks for your attention. Happy to give any more details that you require.

 small_molecule_ts_export.csv  small_molecule_ts_export_extracolumns.csv  small_molecule_ts_export_extracolumns_only.csv 
view request
Comments need to be deleted
(5 responses) mattkarasu 2020-09-11

When exporting an MS Method for our Waters Xevo TQ from Skyline it seems to be creating an .exp file with comments added to every line of the MRM table. Oddly, there seem to be two different comments though? One mentioning Quanpedia and the other mentioning VerifyESkylinLibrary.

It is also worth pointing out that the output .exp file has these comments present even when opened as a raw text file, so I know it is not something our MS File Editor is adding or misinterpreting.

view request
Extracting MS1 peak area for peptides of known mass from DDA/bbCID experiments
(3 responses) Mus 2020-09-14
Hi folks, I have 6 datafiles from DDA runs on a Bruker QTOF. I collected MS/MS fragmentation data in bbCID mode (similar to all ion fragmentation), - only to assign MS1 peaks confidently. Each of the 6 samples are varying but defined mixtures of the following, in a sea of trypsin digested BSA: - 35 synthetically made LIGHT peptides (with K or R C-terminus to mimic a trypsin digest) - 35 synthetically made HEAVY (deuterium labelled) peptides (with K or R C-terminus to mimic a trypsin digest) The peptide sequences are known and I have precurser masses for +2/+3 charge states of all the 70 peptides. The samples contain varying ratio's of LIGHT:HEAVY peptides and I am trying to determine these ratio's by extracting peak area for MS1 peaks for each of 70 peptides. What's the best way to do this in Skyline? I have tried MS1 filtering workflow without success. Thanks in advanvce.
view request
support in Shimadzu LabSOlutions LCMS 5.99 SP2
(4 responses) Stephane MOREAU 2020-08-28

Hi,

it seems that Skyline cannot read data coming from the latest version of SHimadzu LabSolutions LCMS 5.99 SP2 ?

sincerely

view request
Prosit Server Unavailibility
(3 responses) Chinmaya k 2020-09-14

Hello,

I am not able to connect to the Prosit server (131.159.152.7:8500) through Skyline. What is the issue?

--
Chinmaya

 Skyline_Prosit_Server_Unavailability.PNG 
view request
error building Prosit library
(1 response) bin fang 2020-08-28
pwiz.Skyline.Model.Prosit.PrositException: Status(StatusCode=Unknown, Detail="CUDNN_STATUS_BAD_PARAM
in external/org_tensorflow/tensorflow/stream_executor/cuda/cuda_dnn.cc(1249): 'cudnnSetTensorNdDescriptor( tensor_desc.get(), data_type, sizeof(dims) / sizeof(dims[0]), dims, strides)'
     [[{{node encoder1/CudnnRNN}} = CudnnRNN[T=DT_FLOAT, direction="unidirectional", dropout=0, input_mode="linear_input", is_training=true, rnn_mode="gru", seed=87654321, seed2=0, _device="/job:localhost/replica:0/task:0/device:GPU:0"](encoder1/transpose, encoder1/ExpandDims_1, encoder1/Const, encoder1/concat)]]
     [[{{node out/Reshape/_55}} = _Recv[client_terminated=false, recv_device="/job:localhost/replica:0/task:0/device:CPU:0", send_device="/job:localhost/replica:0/task:0/device:GPU:0", send_device_incarnation=1, tensor_name="edge_605_out/Reshape", tensor_type=DT_FLOAT, _device="/job:localhost/replica:0/task:0/device:CPU:0"]()]]") ---> Grpc.Core.RpcException: Status(StatusCode=Unknown, Detail="CUDNN_STATUS_BAD_PARAM
in external/org_tensorflow/tensorflow/stream_executor/cuda/cuda_dnn.cc(1249): 'cudnnSetTensorNdDescriptor( tensor_desc.get(), data_type, sizeof(dims) / sizeof(dims[0]), dims, strides)'
     [[{{node encoder1/CudnnRNN}} = CudnnRNN[T=DT_FLOAT, direction="unidirectional", dropout=0, input_mode="linear_input", is_training=true, rnn_mode="gru", seed=87654321, seed2=0, _device="/job:localhost/replica:0/task:0/device:GPU:0"](encoder1/transpose, encoder1/ExpandDims_1, encoder1/Const, encoder1/concat)]]
     [[{{node out/Reshape/_55}} = _Recv[client_terminated=false, recv_device="/job:localhost/replica:0/task:0/device:CPU:0", send_device="/job:localhost/replica:0/task:0/device:GPU:0", send_device_incarnation=1, tensor_name="edge_605_out/Reshape", tensor_type=DT_FLOAT, _device="/job:localhost/replica:0/task:0/device:CPU:0"]()]]")
   at System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
   at System.Runtime.CompilerServices.TaskAwaiter.HandleNonSuccessAndDebuggerNotification(Task task)
   at Grpc.Core.Internal.AsyncCall`2.UnaryCall(TRequest msg)
   at Grpc.Core.DefaultCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   at Grpc.Core.Interceptors.InterceptingCallInvoker.<BlockingUnaryCall>b__3_0[TRequest,TResponse](TRequest req, ClientInterceptorContext`2 ctx)
   at Grpc.Core.ClientBase.ClientBaseConfiguration.ClientBaseConfigurationInterceptor.BlockingUnaryCall[TRequest,TResponse](TRequest request, ClientInterceptorContext`2 context, BlockingUnaryCallContinuation`2 continuation)
   at Grpc.Core.Interceptors.InterceptingCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   at Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, CallOptions options) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:line 96
   at Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, Metadata headers, Nullable`1 deadline, CancellationToken cancellationToken) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:line 86
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 230
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 234
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.PredictBatches(PredictionServiceClient predictionClient, IProgressMonitor progressMonitor, IProgressStatus& progressStatus, SrmSettings settings, IList`1 inputs, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 314
   at pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibraryOrThrow(IProgressMonitor progress, IProgressStatus& progressStatus) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:line 113
   at pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:line 69
view request
Cannot fill in Protein Description field in 'Edit>Insert>Peptides'
(2 responses) shaoen 2020-09-09

Hi,

I would like to fill all three fields (peptide sequence, protein name, protein description) when adding a list of peptides but it seems I can't enter anything in the protein description field. Data in the first two columns work fine. What am I missing?

Skyline version: Skyline-Daily 20.1.9.234

view request
System.IO.IOException: ERROR: Error parsing modifications file: modifications.xml(line 5662): Missing required attribute 'composition'.
(2 responses) m kuppusamy 2020-09-09

I am trying to load some DDA data on skyline using the import DDA peptide search option. I get an error "System.IO.IOException: ERROR: Error parsing modifications file: modifications.xml(line 5662): Missing required attribute 'composition'." when I try to load the msms.txt file from the Maxquant search. I already have modifications.xml file from maxquant in the same directory as well. Could you please tell me what could be the problem here?

view request
issue when importing peptide search from a MaxQuant msms.txt file - ignored modifications
(2 responses) marie locard-paulet21061 2020-09-08

Hello,

I would like to use Skyline to generate a spectral library compatible with a library-based DIA search in DIA-NN.
My issue is that when I import an output from MaxQuant (1.6.14) using the "import peptide search" pipeline (msms.txt file in the same folder as the raw files, the mqpar.xml and the modifications.xml containing the list of variable modifications -I added the Ammonium and Ammonia loss in the bottom-), the only modifications that I find in the output are oxidation (M) and carbamydomethylation (C). So several modifications are missing.
I am pretty sure that I miss something but I cannot figure out what.

The idea is to then export a custom report from Skyline that would be in a format that I can make compatible with a DIA-NN input library. This bit works.

I work with Skyline (64-bit) 20.1.0.155 (a0e7323e3).
I have uploaded the MaxQuant outputs I work with on the skyline upload site, with the name "Ticket_MLocardPaulet.zip".
Please let me know if you need more information.
Thank you for your help.
Kind regards,
Marie.

view request
Bibliospec for spectral library searching
(2 responses) david morgenstern 2020-09-06

Hi,

I'm considering moving to using spectral library searching for glycoprotoemics due to the prohibitive search times (especially for large datasets). apart from the old manuscript - is there any tutorial I can use for it? there are multiple questions I've got regarding the use of the software:

  1. how does FDR performed after the search?
  2. how does it deal with very small database (50 entries) for FDR calculations?
  3. how does the scoring algorithm (as well as the annotator) deal with c/z B/Y spectra from glycopeptides and EThcD/sHCD?
  4. can it look for modifications on top of what comes out of the library?
  5. Can you convert the .blib libraries to .msp?
  6. can it deal with PRM data for library preparation? PRM with separate MS1?

I'd appreciate any resource you can point out to me.

Thanks!
David.

view request
bibliospec error
(4 responses) dkueltz 2020-09-05

Hi Nick, Brendan,
I have used the integrated bibliospec for many years and never had a problem but now for some files the error below pops up when trying to build a new spectral library. I am using pep.xml + corresponding mzxml files generated in PEAKS Xplus for building libraries. DDA data from two consecutive runs that look similar in terms of peptide and protein coverage and file size give different results. One file works fine for library generation but the other throws this error. I tried this with Skyline v19, v20, and the latest daily. I also tried windows admin and regular user accounts - same result. There is no disk space issue either. I can upload an example pep.xml + mzxml file pair that works and another one that does not work if that would help troubleshoot the issue - let me know.
Thanks,
Dietmar
System.IO.IOException: Error occurred running process.

Command-line: C:\Users\dkueltz\AppData\Local\Apps\2.0\CHO9N7RC.HQC\PCZXTA8K.NOQ\skyl..tion_e4141a2a22107248_0013.0001_e50c2c68eab478d7\BlibBuild -s -A -H -o -c 0.95 -i t -S "C:\Users\dkueltz\AppData\Local\Temp\tmpA47.tmp" "D:\Spectral-Libraries\t.redundant.blib"
Working directory: D:\LR0013_Oremo-Kidney\PEAKS_mzID-mzXml_Export\PEAKS10P__LR0013_Oremo-Kidney_gradual2weeks_PEAKS PTM_42
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_19_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in C:\proj\skyline_19_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 186
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 144

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Peaks not found
(2 responses) jessica medina 2020-08-27

Dear Skyline team,

I am trying to process my lipidomics data in positive mode, but some peaks are not found (mostly the ammonium adducts).
I have tried many options but nothing works yet.

I have processed already the same samples from negative mode and everything worked well.

I am using the paramenters and files in the attachment.

Thank you very much for your help

 OPtPOSSCIEX.wiff  OPtPOSSCIEX.wiff.scan  positive.sky  Lipidomics data positive mode.pptx 
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Question about calculating standard errors of mean and CVs for group comparison
(2 responses) roman sakson 2020-09-06

Dear Skyline team,

I did an experiment where I monitored four peptides for a particular protein and afterwards decided to include all transitions for my first peptide but only one quantitative transition for the other three peptides, respectively, when performing the group comparison. I noticed that the peptide with several transitions had a higher significance in the volcano plot compared to others (see volcano_plot.jpg) and I investigated further. When I had a look at the respective CVs for all transitions for that peptide across the two conditions, CV values were terrible (around 90 %, see CVs_Transitions.jpg), which did not agree with the significance in the volcano plot nor with my expectations based on "okayish" to good XICs. However, once I summarized transitions from "all" to "total", overall CVs dropped below 10 %, which would correspond to the volcano plot (see CVs_All.jpg). For the other peptides, where there are no transitions to sum up, I do not see this pattern. This effect is present in the latest daily version as well as in 20.1. I assume that CVs for individual transitions are somehow off. Do you have an idea what the reason might be?

Thank you,

Roman

 Volcano_Plot.jpg  CVs_Transitions.jpg  CVs_All.jpg  200906_Support_Case.sky.zip 
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Array dimensions exceeded supported range error on DIA .raw file import - ELIB from EncyclopeDIA
(1 response) cabarnescabarnes 2020-09-05

Hi all,

I have been doing the gas phase fractionation workflow with EncyclopeDIA to generate an ELIB from the "Save Quant Reports" in EnclyclopeDIA. Then, I import the ELIB generated after that step into Skyline and usually can import .raw DIA files cleanly and look at all the data in Skyline. I'm working on an experiment now, however, which has a lot of transitions (238,774 transitions / 58,099 peptides / 6310 proteins) and I'm importing 36 injections. I've tried the import multiple times with this library and it fails with the below error. However, if I delete all of the peptides down to a single peptide, I can get it to import just fine. When it fails, it looks like it gets all the way to the end and then fails after the visual import status graph looks finished. Is this a memory problem? Or something else?

At 11:47 AM:
Failed importing results file 'C:\TPP\data\smORF_discovery_Experiements_BAT_scWAT_huWAT_C2C12\2020_Beige_BAT_WAT_secretome_proteome\Proteome\20200731_LUM1_CPBA_EASY04_085_30_SA_90mingrad_80B_DIA_400_1000_8mzol_15k_20IIT_4e5agc_1850_02_01.raw'.
Array dimensions exceeded supported range.

Thank you so much!!

Best,

chris

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MS/MS Spectrum screen
(1 response) jfarrera 2020-09-04

Dear Skyline team,

I'm new on Skyline, I have started working with Skyline recently doing peptide sequencing. Till now I have created some MRM methods with Skyline (v. 20.1.0.155, a0e7323e3) which I could run in a triple quadrupole LCMS8040 (LabSolutions, Shimadzu). I was able to open and process the acquired MRM data in the Skyline platform, but I was not able to open a MS/MS spectrum with Skyline. Is it possible to open in Skyline a MS/MS spectrum from a triple quadrupole LCMS8040 (Shimadzu)? When I open the View menu, the MS/MS Spectra option is not available (is not in the menu). Any idea?

Thank you very much for your support!! Best regards,

Josep

 Skyline screen.odp 
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Spectral library export
(3 responses) Juergen 2020-09-02

Dear Skyline team,

recently I used Skyline to build a spectral library from mascot search results (.dat files). However, I want to use the very same library also in spectraST later. Is there any way to convert the blib format to splib? I remember, that some years (~10) ago it was possible to import these libraraies into spectraST directly. But it seems that this support was stopped in the meantime. If a direct conversion is not possible, perhaps there is a way to convert the library to an intermediate format that you are aware of?

Best regards,
Juergen

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Wrong position of amino acids?
(3 responses) fcsigloch 2020-01-13

Dear Skyline team,

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

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

Is this a bug or a feature of Skyline?

Greetings,
Florian

 skyline_aa_numbering.JPG 
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Small molecules Bracketed calibration and data consolidation
(1 response) anders honore 2020-09-02

Hi,

Hoping you could point me to webinars/tutorials to assist me in solving the following questions

  • How to (conveniently) import concentration data for calibration for multiple components?

  • How to perform bracketed calibration , i.e. applying the repeated inj. of calibration solutions surrounding a given set of unknowns in sequence?

Finally:

  • How to consolidate technical replicates after calculations, i.e such calculated amt/conc in unknowns replicates A, B, C are reported as either mean or median and stdev?

Fingers crossed that your advise will help me cut some corners in a complex method implementation

BR,
Anders

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Spectrum missing
(7 responses) cunain1gx 2020-08-31

I am using Mascot DAT file to build a library in skyline. I can find a MS/MS spectrum for one peptide in mascot search result, but cant find this peptide spectrum in the skyline library. As I am trying to extract transition for this peptide from skyline, now it just not showed in the library. Should I change any parameters or anything? Because the spectrum can be observed with mascot search result. Thank you

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Small molecule (non-global) standard concentrations
(2 responses) jrenders 2020-09-01

Hi there,
Thanks so much for adding the ion ratio features to skyline - we are currently rebuilding all of our small molecule methods in skyline and have unfortunately hit a snag. I think you previously made mention of (in the future) adding the ability to assign calibration curve regression and weighting on a per-analyte basis. Right now, via the "Document grid --> Reports --> Replicates" report we can assign analyte concentrations, but only globally. However, we have methods where our calibrators do not have all analytes at the same concentration. Is there plans to allow for a per-analyte concentration assignment as well in the future? Or perhaps there is already a way to handle this via the document grid or pivot table? Thanks for any guidance you can suggest.

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Isotope labels on some residue
(2 responses) Vincent Lacasse 2020-08-31

Hello Skyline team,

I'm currently preparing an MRM assay using an internal calibration curve with two internal standards spiked-in a different concentrations. Although I was able to add my different labels to my skyline file, some of the residue of the same amino acid are labelled and not others. For instance:

SQPGQDCR
The first "Q" is not labelled, but the second "Q" is labelled. I have 28 different peptides and there are multiple instances where this problem shows up.

How can I tell Skyline to label only some of the amino acids? I've also tried to put it as a variable modification on the protein, however it does the same thing, it labels both amino acids.

Thank you and have a nice day!

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Error when copy + pasting a peptide list into Skyline daily
(5 responses) roberthardt 2020-08-28

Dear Skyline-Team,

if I want to insert peptides via "Edit - Insert - Peptides" in Skyline Daily (20.1.9.234, 77dodc4a4) the software always freezes. In contrast, if I do exactly the same in the current public Skyline release (20.1.0.155, a0e7323e3) it works without a glitch.

Regards

Robert

 pep_import_freeze_daily.pptx 
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Stable isotope tracing experiments: Is is possible to change the order of isotopologues in the peak area replicate comparison?
(2 responses) Michi 2020-08-27

Dear Skyline team,

Thank you so much for all your effort, I don't know what I would do without Skyline anymore. I recently discovered that you also implemented new plots for the peak area replicate comparison that allows you to directly see the labeling patterns when doing stable isotope tracing experiments in the format I'm used to. It works very well for small metabolites. However, when looking at compounds with more than 10 carbons, the ordering of isotopologues is not by mass, but alphanumeric?, i.e. it shows the M-H, then the M10C13-H, M11C13-H,...., and only at the end the M1C13-H, M2C13-H, etc.

Is there an option to sort these differently? I need to export the data to do natural isotope abundance correction and plot the different isotopologues as fractions afterwards anyway, but it would be very helpful to see them in the right order in Skyline already. It is currently confusing if you don't look very closely and realize how they are ordered.

I attached an example.

Thanks,
Michi

 Skyline_isotopologue_order.PNG  Isotopologues_order_example.sky 
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peak integration
(6 responses) monasharar 2020-08-26

Good evening,

I could see that for samples if they have 4 min peak width for example, some of them all the peak appears
while for others there is a cut, so first min appears then the line stops in the middle.
Manual peak integration cannot do anything other than adjusting boards, while if I view TIC for the run I cannot do manual integration.
Can you please advice ?

Thank you

 Skyline.png 
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DDA Spectral Library building from Thermo Lumos ,
(1 response) sray 2020-08-24

Hello All,

I'm building a spectral library from DDA peptide search results generated on Thermo Lumos.

In "Configure MS1 Full-Scan Settings" section, under "precursor mass analyzer" , which option is better, "Centroided" or "Orbitrap" (with corresponding resolution settings) ?

Thanks

view request
DIA overlap with margin demultiplex in MSConvert
raghuram 2020-08-24

Dear Skyline team,

I had issues demultiplexing overlap with margin in MS convert. Is there any other open-source code that I can use to demultiplex overlap with margin files. And also is there a way to control demultiplexing by providing the isolation scheme?

cheers,
Bharath

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IMS Predictor questions
(2 responses) Juan C. Rojas E. 2020-08-21

Dear Skyline team,

I have a few questions I hope you can help me clarifying.

  1. How does Skyline determine the CCS area from WATERS HD files? What equation is implemented?

I checked at some point if the CCS areas reported in Skyline matched the ones calculated with Driftscope and I noticed slight differences. Maybe due to small differences in the application of the calibration equation stored in the files?

  1. When the IMS predictor is run automatically with "Use Results" it will attempt to only use the areas inside of the RT boundaries?

I import my DIA data with small retention time windows to limit false integrations and then run the predictor. However, after running the predictor I noticed that the EICs show the full chromatogram and not only the small retention windows imported in beginning.

  1. Is there a bias for optimizing the IMS window of the fragments (i.e. high energy scan) above the one for the precursors (i.e. low energy scan)? Or is it some sort of weighed contribution?

I have tried using the automatic predictor and very often its determining incorrect offset estimations. In the image attached you can see that when automatic is ran it usually ends up with lots of positive offsets which is the complete opposite of what is expected from associated fragment ions in the HDMSE high energy scans. This happened because it chose same fragment ions that came from other co-eluting or closely eluting molecules.

Of course, with my manual corrections I still had some mistakes due to badly curated peaks, but at least the bias is towards negative offsets for most of them. I should mention that I use a low resolving power of 15 since is the value the encompasses all area for most of my molecules with my current instrument settings and analytes relative abundance.

Sorry if these were too many questions for one post, but I thought they were all related.

Sincerely,
JC

 Offset estimations.png 
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Export of Transition List and Method for Sciex 6500+ broken
(13 responses) Will Thompson 2020-08-19

Hi Brendan et al

Export of transition lists and of methods for Sciex 6500+ is not currently working in Skyline -daily or 20.x build, meaning method development is not possible on this instrument. Specifically this is the case for negative ion small molecule data. Some problems may be specific to that use case, but others seem generic. Not sure how long this has been going on, we have not done any routine method development on this system in a while. There are a number of issues. Recognized so far:

  1. Skyline will not allow a negative collision energy or declustering potential. These are required for negative ion acquisition for Sciex. When you paste in a negative CE or DxP using the import or Ctrl+V method, Skyline does not give an error, but it just puts nothing in the appropriate fields.
  2. Even if you change the polarity of the CE in Skyline to (the incorrect) positive value, then you export an acquisition method, the method contains a random negative number instead of the CE that was programmed.
  3. Even if you change the polarity of the CE in Skyline to (the incorrect) positive value, then you export a transition list, the TL is the incorrect format and Analyst cannot import it.

I hope you can reproduce these errors with the attached documents but if not we can set up a Zoom call for me to walk through in more detail. Please let me know if you need more information.

Cheers

Will

 forSkylineSupport.sky.zip  forSkylineSupport.xlsx 
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How can I select endogenous peptides not spiked-in iRT peptides for RT nomalization in Skyline?
(1 response) andyzcq 2020-07-23

I have acquired diaPASEF data, but I forgot to spike iRT peptide into the samples.

So how can I select endogenous peptides for RT normalization?

CiRT peptides in the CiRT calculator are not applicable.

view request
Applying manual integration windows when adding new transitions/fragments for an existing compound in Targets
(6 responses) Emmanuel 2020-07-27

Dear Skyline team,

I've manually adjusted the integration of a targeted small molecule across a dozen of samples and I'd like to add few more transitions/fragments for that compound (additional fragments found in another database than the first ones).

Is there a simple way to apply the manual integration boundaries to the new fragments without using the workaround of exporting/re-importing the peak boundaries as a report (as suggested by Tobi some years ago)?

When re-importing the results, it seems that the new transitions are integrated according to the default integration settings without taking into account the manual integration from the previous transitions. Therefore, I need to manually re-integrate all the samples ....

Thanks in advance for your help.

Emmanuel

view request
MS Amanda DDA search
sstoychev 2020-08-21

Hi Skyline team,

I'm trying to do a DDA search but a following error pops up during method set-up:

Skyline version: 20.1.9.233-4850f5574 (64-bit)
Installation ID: ed56f5a5-04ae-49bc-bfc9-021aa0ce9225
Exception type: Exception
Error message: Obo files (psi-ms.obo and unimod.obo) not found


System.Exception: Obo files (psi-ms.obo and unimod.obo) not found
at pwiz.Skyline.Model.DdaSearch.MSAmandaSearchWrapper..ctor() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\DdaSearch\MSAmandaSearchWrapper.cs:line 88
at pwiz.Skyline.FileUI.PeptideSearch.ImportPeptideSearchDlg.NextPage() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\PeptideSearch\ImportPeptideSearchDlg.cs:line 565
at System.Windows.Forms.Control.OnClick(EventArgs e)
at System.Windows.Forms.Button.OnClick(EventArgs e)
at System.Windows.Forms.Button.OnMouseUp(MouseEventArgs mevent)
at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
at System.Windows.Forms.Control.WndProc(Message& m)
at System.Windows.Forms.ButtonBase.WndProc(Message& m)
at System.Windows.Forms.Button.WndProc(Message& m)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
Exception caught at:
at System.Windows.Forms.Application.ThreadContext.OnThreadException(Exception t)
at System.Windows.Forms.Control.WndProcException(Exception e)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
at System.Windows.Forms.Form.ShowDialog(IWin32Window owner)
at pwiz.Skyline.SkylineWindow.ShowImportPeptideSearchDlg(Nullable`1 workflowType) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 3006
at pwiz.Skyline.SkylineWindow.importPeptideSearchMenuItem_Click(Object sender, EventArgs e) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 2988
at System.Windows.Forms.ToolStripItem.RaiseEvent(Object key, EventArgs e)
at System.Windows.Forms.ToolStripMenuItem.OnClick(EventArgs e)
at System.Windows.Forms.ToolStripItem.HandleClick(EventArgs e)
at System.Windows.Forms.ToolStripItem.HandleMouseUp(MouseEventArgs e)
at System.Windows.Forms.ToolStrip.OnMouseUp(MouseEventArgs mea)
at System.Windows.Forms.ToolStripDropDown.OnMouseUp(MouseEventArgs mea)
at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
at System.Windows.Forms.Control.WndProc(Message& m)
at System.Windows.Forms.ToolStrip.WndProc(Message& m)
at System.Windows.Forms.ToolStripDropDown.WndProc(Message& m)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
at pwiz.Skyline.Program.Main(String[] args) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Program.cs:line 306

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Skyline and QTRA6500-CE and DP optimization method export
(1 response) qin fu 2020-08-19

Dear Skyline team,
when I tried to export CE or DP optimization method using QTRAP6500 setting, I have encountered the error messages, enclosed here. My guess is the choice of the instrument in the transition setting, I must have gotten some details wrong in the setting. What did I do wrong?
Thanks
Best
Qin Fu

 6500QTRAP_CV optimizationwitherrormessage.pptx 
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Retention time shift
(15 responses) yulun 2017-04-28
Hi all,
I run some standards (metabolite) in different runs and would like to analyze by Skyline. Please see the attachment. The most right panel is the correct peak identified (~13.5 min) by the Skyline. However, the most left (1.5 min) and the middle (22.8 min) ones return the wrong retention time. I know the Skyline did the good job to pick the highest peak. My question is: Is there any possible way from setting to get rid of these 2 peaks identification since it is out of rt windows (0.2 min for rt window)?
Thank you
Yulun
 RT_shift.png 
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DIA TIC does not match DDA
(4 responses) Mark Athanason 2020-08-14

Hi again Brendan,

I know this is not a skyline question, but since I followed your tutorial for setting up the DDA and DIA methods in Xcalibur I figured I would ask here. In the past this worked perfectly well with a 2 hour gradient, but now I'm running the two methods as a one hour gradient and something funky is going on with the DIA acquisition. The DIA and DDA chromatography parameters are exactly the same, and the DIA instrument method is as shown in your tutorial. I have attached the two method files along with a screen shot of the TICs to demonstrate what I am talking about. If you can see something I'm obviously doing wrong, I would be very appreciative. This is being run on a nanoAquity LC with a picochip spray source coupled to a Thermo QE HF mass spec.

Best
Mark

 ms_DDAvsDIA.PNG  DIA_400to1260by20_60min_trapping.meth  DDA_top20_60min_trapping.meth 
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Calibration curve X axis Analyte Concentration/Light:Heavy con ratio
(3 responses) Zac 2020-08-14

I have calibartion curve data with heavy labelled peptides for normalization. For most peptides the x-axis of the calibration curve is Analyte Concentration but for one peptide this changes to Light:Heavy concentration ratio. Not sure how to standardize all peptides to show Analyte concentration, the X-axis values are also different.

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Skyline not recognizing in house Labkey server as 'Panorama server'
(5 responses) mlane 2020-08-13

Hi,
We're using Skyline-daily 20.1.1.175 and just set up new Community edition of Labkey so not sure if this is Labkey or Skyline issue. I can directly connect to Labkey server through browser, log-in fine and create Panorama folders, etc. However, when I try to add server to be recognized in Skyline, an error comes back on the url that it is not recognized as Panorama server.
See attached for url and error.

Thanks!
Monica

 Labkey_error_08132020.pdf 
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An error occurred attempting to install Skyline
(1 response) shobhabr20 2020-08-12

Hi Skyline team,

I get an error when trying to install skyline daily and I've included the install.log file.

Skyline-daily 20.1.1.213 (https://skyline.ms/project/home/software/Skyline/daily/begin.view?)

I am installing skyline daily onto our lab server. If the error is stemming from our end, please let me know so I can tell my IT person.

Thanks,
Shobha

 Installation_Error.png  install.log 
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Feature detection capability for small molecules
(1 response) FC 2020-08-13

Hello Skyline team,

I am new to Skyline and would like to know if Skyline has the capability to detect features/peaks from DIA-MS/MS data for small molecules without first going through a compound identification process. I am interested in using these features/peaks to build a spectral library. Thank you!

Kind regards,
FC

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Peaks integration with different boundaries between light and heavy
(3 responses) Anne Incamps 2013-11-06
Hi Brendan

I don't manage to integrate manually light and corresponding heavy peptides with different boundaries.
If I clic on the light peptide and integrate the right peak, it automatically shift the same boundaries for the heavy one. I cannot set different boundaries for light and heavy. How can I sort this out ?

Thanks for your answer,

Anne
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Exclude counting of decoys from prot, pept, precurs, transitions numbers in target list
(2 responses) dkueltz 2020-08-06

Hi Brendan et al.,
When constructing DIA assay libraries from raw spectral libraries by sequentially applying QC filter criteria my lab is routinely recording the numbers of proteins, peptides, precursors, and transitions remaining after each filter step. We get these numbers from the display on the lower right hand corner of the skyline app. However, these numbers currently include decoys and we have to delete the decoys and then paste them back tp the target list after every filter step. It would be nice to not have to do that. So I am asking if you could program Skyline such that it excludes decoys automatically from the display in the lower right hand corner of the screen.
Thanks,
Dietmar

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(Loss of) resolving power when importing mzXML files from HRAM QTOF
(8 responses) anders honore 2020-08-04

Hi
Working with small molcules in MS1 from Bruker QTOF instrument. Data is stored as centroided and converted to mzXML (by Bruker converter) prior to upload. Resolution is typically +40,000.
After import resolving power is reduced to <10,000 and otherwise obvious peaks when reviewed e.g. in MZmine2 becomes severely distorted. Appears as though resolution is reduced to <<10,000
Transition Settings: precursor as monotopic, full scan count, peaks 3, precursor mass analyzer TOF, resolving power 50,000
Include all matching scans

Probably error-40 by a novice user - but please advise...

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